The expression pattern of Notch is not well documented. One excellent paper (Kooh, 1993) compares the expression of Delta and Notch in individual tissues and finds small but potentially very important differences, for example, the instar antennal disc. Delta expression is elevated in the antennal anlage and the peripodial membrane. Notch is expressed apically throughout the eye antennal disc. In the retina, to take a second example, Delta and Notch are coincident in vesicles near the apical surfaces of photoreceptor clusters. Delta, but not Notch, accumulates within photoreceptors in subapical regions (Kooh, 1993). Since the Delta-Notch interaction functions in cell adhesion as well as for signalling of Notch, these different distributions regulate cell migration, for example, axon pathfinding (Giniger, 1993).

Notch is involved in several developmental processes outlined below:

A summary of how Notch carries out these processes is found in in the biological overview (above) and in the Notch pathway section. For information about the role of Notch in the development of specific tissues see also:

Notch is required for the formation of the stomatogastric nervous system (SNS). The SNS consists of several peripheral ganglia which receive input from the brain and in turn innervate the muscles and the pharynx and gut. Precursors originate from the primordium of the foregut or stomodeum [Image]. Several subsets of precursors delaminate from the stomodeal epithelium as individual cells early in development. At later stages these precursors migrate to various locations where they differentiate as neurons. The function of achaete-scute and neurogenic genes is identical here to their function in the development of the ventral nerve cord (Hartenstein, 1996).

The gut-innervating stomatogastric nervous system of Drosophila, unlike the central and the peripheral nervous system, derives from a compact, single layered epithelial anlage. This anlage is initially defined during embryogenesis by the expression of proneural genes of the achaete-scute complex in response to the maternal terminal pattern forming system. Within the stomatogastric nervous system anlage, the wingless-dependent intercellular communication system adjusts the cellular range of Notch-dependent lateral inhibition to single-out three achaete-expressing cells. Those cells define distinct invagination centers which orchestrate the behavior of neighboring cells to form epithelial infoldings, each headed by an achaete-expressing tip cell. These results suggest that the wingless pathway acts not as an instructive signal, but as a permissive factor which coordinates the spatial activity of morphoregulatory signals within the stomatogastric nervous system anlage (Gonzalez-Gaitan, 1995).

Mutant Presenilin proteins cause early-onset familial Alzheimer's disease in humans. Caenorhabditis elegans Presenilins may facilitate Notch receptor signaling. A Drosophila Presenilin homolog has been isolated and the spatial and temporal distribution of the encoded protein as well as its localization relative to the fly Notch protein has been determined. Presenilin is widely expressed throughout oogenesis, embryogenesis, and imaginal development, and generally accumulates at comparable levels in neuronal and nonneuronal tissues. Double immunolabeling with Notch antibodies reveals that Presenilin and Notch are coexpressed in many tissues throughout Drosophila development and display partially overlapping subcellular localizations, supporting a possible functional link between Presenilin and Notch (Ye, 1998).

Notch and gliogenesis

During Drosophila neurogenesis, glial differentiation depends on the expression of glial cells missing. Understanding how glial fate is achieved thus requires knowledge of the temporal and spatial control mechanisms directing gcm expression. In the adult bristle lineage, gcm expression is negatively regulated by Notch signaling. The effect of Notch activation on gliogenesis is context-dependent. In the dorsal bipolar dendritic (dbd) sensory lineage in the embryonic peripheral nervous system (PNS), asymmetric cell division of the dbd precursor produces a neuron and a glial cell, where gcm expression is activated in the glial daughter. Within the dbd lineage, Notch is specifically activated in one of the daughter cells and is required for gcm expression and a glial fate. Thus Notch activity has opposite consequences on gcm expression in two PNS lineages. Ectopic Notch activation can direct gliogenesis in a subset of embryonic PNS lineages, suggesting that Notch-dependent gliogenesis is supported in certain developmental contexts. Evidence is presented that POU-domain protein Nubbin/PDM-1 is one of the factors that provides such context (Umesono, 2002).

Notch signaling promotes glial fate during asymmetric division in the embryonic dbd lineage. Notch is specifically activated in the presumptive DBD support glia cell (DBDG) owing to the negative regulation by Numb in the sibling cell, and provides instructive information to induce gcm transcription and glial development. Expression of gcm occurs quickly after the artificial activation of Notch, even in cells that have initiated neuronal development. In gcm mutants, DBDG are transformed into neurons, although the activation of Notch, visualized by the Su(H)-reporter, is normal in the presumptive glia. Likewise, ectopic expression of gcm in presumptive dbd neurons causes neuron-to-glia transformation without affecting Notch activity. These findings suggest that gcm expression appears to be the sole target of Notch activation in establishing glial fate in the dbd lineage. Within the 3.5 kb region upstream of the gcm gene, two sequences have been identified that perfectly match the consensus core sites for Su(H). Thus, gcm could be a direct target of Su(H), downstream of the Notch signaling pathway (Umesono, 2002).

While the present data demonstrate a positive role for Notch in gliogenesis in the dbd lineage, other embryonic PNS glial cells do not require Notch activity for their formation. For example, in the adult bristle lineage Notch has an opposite function on gliogenesis; that of repressing gcm expression and glial development. Thus the role of Notch in the regulation of gcm expression is context-dependent. Notch has recently been shown to be a component of combinatorial signaling in cell fate determination in the Drosophila eye. It is possible that Notch signaling has different consequences depending on other factors that act on the same regulatory element (Umesono, 2002).

The context-dependent effect of Notch suggests that the gcm promoter may have a modular structure where each unit integrates different developmental signals. However, given the large diversity of glial subtypes in the nervous system, it is unlikely that each glial subtype has its own regulatory sequences and a unique mode of regulation. A model is favored in which gcm has a limited number of regulatory elements that respond to developmental signals that are present in multiple environments. Indeed a subset of glial subtypes respond in a similar way to Notch signals: in addition to the dbd lineage, the dda lineage can also induce gcm transcription upon Notch activation. Comparison of these two lineages offers hints on the nature of the developmental context in which Notch activation causes gcm transcription (Umesono, 2002).

One common feature that distinguishes dbd and dda lineages from other PNS lineages is the cell division pattern of their SOP. In dbd and dda lineages, SOPs divide to generate a neuron and a glial cell through an asymmetric division. In other gliogenic PNS lineages, the sibling cells of glial cells are not postmitotic neurons, but tertiary precursors that undergo further division to generate neurons and associated cells. These observations suggest that an interaction with the neuronal sibling may play a crucial part in promoting the Notch-dependent gcm activation during asymmetric cell division. Recently, Notch was shown to positively regulate gcm expression in the Neuroblast 1-1A lineage of the CNS, where the sibling pattern is identical to that of the dbd lineage. This also supports the idea that the cell division pattern provides a context that determines the effect of Notch activity (Umesono, 2002).

Coexpression of constitutively active Notch with Nubbin also generates ectopic glia outside dbd and dda lineages. This raises the possibility that Nubbin may be a part of the developmental context that allows Notch to promote gliogenesis. Within the embryonic PNS, dbd and dda neurons are the only two neurons that express Nubbin. In both lineages, Nubbin is present in both SOP daughter cells, at the time of glia versus neuron cell fate choice. Furthermore, temporal activation of Nubbin has been detected in presumptive glial cells derived from the NB1-1A lineage. Nubbin thus might create a permissive environment for the activation of gcm expression by the Notch signal. Since coexpression of Nubbin and constitutively active Notch does not cause glial transformation of all neurons, additional factors must exist that create a Notch-dependent gliogenic context (Umesono, 2002).

Nubbin is a POU-domain transcription factor with sequence-specific DNA-binding activity. The contextual role of Nubbin in Notch-dependent expression of gcm could employ a similar mechanism to the modulation of Notch activity in wing development, where Nubbin and Su(H) bind on the same enhancer element of Notch target genes. It will be interesting to further analyze the role of Nubbin in gliogenic lineages (Umesono, 2002).

Notch and Numb are required for normal migration of peripheral glia in Drosophila

A prominent feature of glial cells is their ability to migrate along axons to finally wrap and insulate them. In the embryonic Drosophila PNS, most glial cells are born in the CNS and have to migrate to reach their final destinations. To understand how migration of the peripheral glia is regulated, a genetic screen was conducted, looking for mutants that disrupt the normal glial pattern. Analysis of two of these mutants is described: Notch and numb. Complete loss of Notch function leads to an increase in the number of glial cells. Embryos hemizygous for the weak NotchB-8X allele display an irregular migration phenotype and mutant glial cells show an increased formation of filopodia-like structures. A similar phenotype occurs in embryos carrying the Notchts1 allele when shifted to the restrictive temperature during the glial cell migration phase, suggesting that Notch must be activated during glial migration. This is corroborated by the fact that cell-specific reduction of Notch activity in glial cells by directed numb expression also results in similar migration phenotypes. Since the glial migration phenotypes of Notch and numb mutants resemble each other, these data support a model where the precise temporal and quantitative regulation of Numb and Notch activity is not only required during fate decisions but also later during glial differentiation and migration (Edenfeld, 2007).

Within the peripheral nervous system of Drosophila axons are insulated from the surrounding hemolymph by only a few identified glial cells. Most of the peripheral glial cells are born in the CNS and migrate to stereotypic positions along the segmental nerves. To understand the genetic programs that govern the selection of migrating cells, the control of the normal migratory path and the final conversion into sessile cells, a genetic screen was conducted. Most of the mutants identified affected the fidelity of glial cell migration along the segmental nerves. Among this class of alleles Notch and numb were identified as being required for normal PNS glia migration (Edenfeld, 2007).

Notch was first extensively studied in Drosophila where it was found to be required for mediating lateral inhibition during the selection of neural progenitor cells. Since then, Notch has been shown to be involved in a large variety of cellular processes controlling cell proliferation, fate, survival and differentiation in organisms ranging from worms to man. Regulation of Notch activity appears to be an example for an archetype of signaling system. Upon binding of one of its most important ligands, Delta or Serrate (Jagged in vertebrates), the intracellular domain of Notch (Notchintra) is liberated by the large Presenilin protein complex that is equipped with transmembrane protease activity. Subsequently, Notchintra is able to access the nucleus where it interacts with Su(H) to activate the transcription of target genes. However, genetic evidence supports the notion that Notch can also activate signaling pathways independent of Su(H) (Edenfeld, 2007).

Notch signaling has frequently been implicated in different aspects of gliogenesis not only in Drosophila but also in different vertebrate glial lineages including Müller glia, radial glia, astrocytes, Schwann cells, and oligodendrocytes. Developing oligodendrocytes express the Notch1 receptor that is activated by neuronally expressed Jagged. Jagged-induced Notch activation conveys an inhibitory function in regulating the timing of axonal myelination. In contrast, the GPI-linked neuronal adhesion protein F3/contactin is capable of promoting Notch-dependent oligodendrocyte differentiation. Both receptor-ligand interactions lead to the generation of Notchintra but nevertheless cause different cellular responses, suggesting that the tight temporal and spatial regulation of Notch activity is of extreme importance (Edenfeld, 2007).

In vertebrates, axonally derived Notch ligands such as Jagged or F3/contactin appear in an excellent position to regulate glial growth and myelination. This study shows that in Drosophila Notch becomes activated in migrating peripheral glial cells. Numb generally inhibits Notch function. This study found Numb expression at early stages among the glial cells close to the nerve exits of the CNS. These cells represent the prospective subpopulation of glial cells that subsequently will migrate along the growing nerves out into the periphery. During the migration (from stage 12 onwards) Numb expression becomes down-regulated and thus correlates with the activation of Notch. Loss- or gain-of-function of numb leads to a disruption of peripheral glial cell migration comparable to the Notch mutant phenotype. This suggests that regulation of Notch activity in migrating glial cells depends on the precise control of the amounts and timing of Numb expression. Thus, this cell-autonomous mechanism appears to be responsible for the temporal control of glial migration (Edenfeld, 2007).

In previous reports, Notch expression has been found on neuronal membranes. However, since Notch is generally involved in direct cell-cell contact, it may be difficult to resolve Notch expression to axonal or glial cell membranes by standard light microscopic techniques. A function of Notch during cell migration was first implicated in the analysis of Delta-1 mutant mice, where neural crest migration phenotypes where found. In Drosophila, a disruption of Notch function impairs morphogenetic movements that lead to the formation of the proventriculus. This phenotype, however, may be due to fate changes, but the finding that the expression of short stop, which encodes a cytoskeletal linker protein of the Spectraplakin family, is regulated by Notch activity provides some evidence that Notch activity can influence cell structure and motility (Edenfeld, 2007 and references therein).

The Notch mutant phenotype is characterized by an increase in the number of filopodia on glial cells. This suggests that Notch activity can - directly or indirectly - influence the dynamics of the cytoskeleton. In the context of the dorsal closure of the Drosophila embryo, it has been shown that Notch may regulate JNK and RhoA signaling, which act on controlling actin dynamics. A direct function of Notch in influencing actin dynamics has also recently been described for a NIH 3T3 tissue culture model, where soluble forms of Delta and Jagged antagonize Notch signaling and in addition affect different cellular functions. Whereas soluble Jagged expression leads to an impaired cell motility, reduced F-actin stress fibers and focal adhesion site formation, the expression of soluble Delta does not interfere with the normal motility but reduces the amount of contactin phosphorylation (Edenfeld, 2007 and references therein).

Taken together, these data indicate that Notch acts in a cell-autonomous manner to instruct peripheral glial cell migration along the peripheral nerves. The precise temporal control and the quantitative adjustment of Notch activity is crucial for this migration. Neuronally expressed Delta presumably initiates Notch activation, however, the fine-tuning of Notch activity is mediated through regulating the levels or localization of Numb expression (Edenfeld, 2007).

Robustness and flexibility in the neurogenic network

Many gene networks used by developing organisms have been conserved over long periods of evolutionary time. Why is that? A model is presented of the core neurogenic network in Drosophila. This model exhibits at least three related pattern-resolving behaviors that the real neurogenic network accomplishes during embryogenesis in Drosophila. Furthermore, the model exhibits these behaviors across a wide range of parameter values, with most of its parameters able to vary more than an order of magnitude while it still successfully forms these test patterns. With a single set of parameters, different initial conditions (prepatterns) can select between different behaviors in the network's repertoire. Two new measures are introduced for quantifying network robustness that mimic recombination and allelic divergence and these were used to reveal the shape of the domain in the parameter space in which the model functions. Lateral inhibition yields robustness to changes in prepatterns and a reconciliation of two divergent sets of experimental results is suggested. Finally, it is shown that, for this model, robustness confers functional flexibility. It is concluded that the neurogenic network is robust to changes in parameter values, which gives it the flexibility to make new patterns. The model also offers a possible resolution of a debate on the role of lateral inhibition in cell fate specification (Meir, 2002).

The experimental literature includes both support for, and refutation of, an important role for lateral inhibition in neural determination. The results of this analysis can account for both sets of experiments. If the prepattern that initiates neuroblast selection is well tuned, the prepattern plus a constant level of inhibition could select the winner, absent lateral inhibition. But lateral inhibition buffers the patterning against perturbations in the initial prepatterning (e.g., due to genetic or environmental variation, or 'developmental noise'). Seugnet (1997) reported that, with only constant production of Dl, 80% of proneural clusters developed normally, but 20% produced an extra NB. These experiments are interpreted to say that the prepattern is well tuned in most proneural clusters, but in 20%, either a poorly tuned prepattern or noise causes errors in the absence of lateral inhibition. This is a testable idea. One could remove lateral inhibition as Seugnet did. It would then be predicted that the embryo would be much more sensitive to hyper- and hypo-morphs in prepatterning genes such as extramachrochaete and hairy. It would also be predicted that such embryos would be more sensitive to mutations in genes within the network itself, such as missing or extra copies of Dl or N. The latter prediction is made because those mutations should change the threshold to which the prepattern is tuned. In the absence of lateral inhibition, a prepattern that was well tuned to the former threshold could not also be well tuned to the new threshold (Meir, 2002).

From these results, it is deduced that E(spl) greatly reduces the percentage of random parameter sets that enable lateral inhibition. It is believed this is because E(spl) acts as a homeostat. As the expression levels of the proneural genes (ac and sc) rise, their products activate E(spl). E(spl) then downregulates the proneural genes. As with the thermostat in a house, this negative feedback loop tends to keep the proneural genes at an intermediate level rather than allowing them to switch to either a high or low state. Both E(spl) autoinhibition, and to a lesser extent cis-Dl inhibition of N activation, help overcome this homeostat. On the face of it, this seems a strange design. The ac/sc network itself is a bistable switch that tends to go in the direction it is pushed and remain there. The switch and homeostat mechanisms are exact opposites. Removing the homeostat (the reduced model) makes it easier to find parameter sets that pass various tests (which all involve throwing the switch). Why incorporate counteracting mechanisms in the same circuit (Meir, 2002)?

It is, of course, possible that this is simply a vestige of the network's evolutionary history, with no design rationale. But electrical engineering suggests one possible advantage. An op-amp is a famous circuit that amplifies the difference between two inputs. Good op-amps can amplify a voltage difference more than a million-fold. Usually, though, engineers add a negative feedback circuit (that is, a homeostat). This greatly attenuates the gain but makes the amplifier much more stable; noise generated internally inside the op-amp will not affect the output signal. Reducing function to gain stability is common in other electrical circuits as well. These electrical circuits do not make good direct analogies to genetic networks, but the concept of adding negative feedback to increase stability might still apply. Perhaps the E(spl) homeostat reduces the network's sensitivity to developmental noise such as stochastic changes in transcription or translation rates, in the prepattern, or in the concentrations of modulators such as Da and Emc (Meir, 2002).

A related design benefit might be that the E(spl) homeostat prevents the network from switching individual cells on or off before the prepattern has a chance to decree the winner. A simple bistable switch consisting of ac and sc alone could not help but be thrown in one direction or the other by noise (as apparently takes place in C. elegans anchor cell specification). Adding E(spl) leads to a new, neither-on-nor-off steady state, which could enable the proneural switch to procrastinate until some extrinsic cue forces the system to choose one or the other switched state (Meir, 2002).

Notch and the stomatogastric nervous system

The stomatogastric nervous system (SNS) of Drosophila is a simply organized neural circuitry that innervates the anterior enteric system. Unlike the central and the peripheral nervous systems, the SNS derives from a compact epithelial anlage in which three invagination centers, each giving rise to an invagination fold headed by a tip cell, are generated. Tip cell selection involves lateral inhibition, a process in which Wingless (Wg) activity adjusts the range of Notch signaling. RTK signaling mediated by the Epidermal growth factor receptor plays a key role in two consecutive steps during early SNS development. Like Wg, Egfr signaling participates in adjusting the range of Notch-dependent lateral inhibition during tip cell selection. Subsequently, tip cells secrete the Egfr ligand Spitz and trigger local RTK signaling, which initiates morphogenetic movements resulting in the tip cell-directed invaginations within the SNS anlage (González-Gaitán, 2000).

In order to investigate the role of RTK signaling in SNS development, lack-of-function mutants of the Egfr ligand Spitz were examined. In spitz mutants, the formation of the four SNS ganglia is strongly impaired. The SNS anlage, however, forms normally. In addition, the expression domain of wg and proneural AS-C genes is indistinguishable from a wild-type SNS anlage. At the stage when the three ac-expressing cells were singled-out within the wild-type SNS anlage, only one ac positive cell is found in spitz mutants. The same phenotype has been observed in wg mutants or mutants lacking an integral component of the wg pathway. Since no altered wg pattern was found in the spitz mutant SNS anlage, Spitz-dependent RTK signaling may act in parallel or in combination with wg to adjust the proper range of Notch-dependent lateral inhibition. In contrast to wg mutants, however, no invagination fold is observed. This observation indicates that the singled-out ac-expressing cell of spitz mutants has lost the ability to function as a tip cell and possibly fails to induce morphogenetic movements within the SNS anlage (González-Gaitán, 2000).

spitz, like other genes encoding components of the Egfr signaling pathway such as Egfr, Ras, Raf and the cascade of MAP kinases, is ubiquitously expressed. Local activation of Egfr signaling requires the transmembrane protein Star, which is necessary for the secretion of Spitz. Star is expressed in restricted patterns corresponding to the Spitz secreting cells. In the SNS anlage, it was noted that Star becomes restricted to the three tip cells and is maintained in these cells when invagination takes place. As in spitz mutants, the Star mutant SNS anlage is established normally; only one ac-expressing cell is selected and no invagination occurs. Consistently, Star mutants fail to develop the proper set of SNS ganglia and the associated nerves. These observations suggest that tip cells are a Star-dependent source of Spitz activity that triggers Egfr-dependent RTK signaling in the neighboring cells within the SNS anlage. This conclusion is supported by the finding that phosphorylated MAPK, a cellular marker for RTK signaling activity, is indeed activated in cells of the invagination folds, whereas phosphorylated MAPK does not appear in the Star mutant or in the spitz mutant SNS anlage (González-Gaitán, 2000).

To examine whether activated Spitz is sufficient to induce cell movements within the SNS anlage, use was made of the GAL4/UAS system to misexpress secreted Spitz in an ectopic pattern. This was achieved through the expression of activated Spitz from a UAS promotor driven transgenethat was activated by Gal4 under the control of the actin promotor. Under the conditions applied, scattered UAS-dependent transgene expression is observed throughout the early embryo, including the SNS anlage. When activated Spitz is expressed in such a pattern, a variable number of supernumerary infoldings within the SNS anlagen are observed, indicating that activated Spitz is sufficient to initiate cell movements. This result, in conjunction with the observation that the invaginated cells express phosphorylated MAPK, provides evidence that tip cell-derived activated Spitz triggers RTK signaling to initiate the invagination process. This proposal was tested by blocking Egfr signaling in the anterior most region of the SNS anlage that gives rise to the first invagination fold. For this, a GAL4 driver (SNS1-Gal4) was used that causes UAS-dependent gene expression in the corresponding region of the SNS anlage. SNS1-Gal4-mediated expression of a dominant-negative Egfr mutant form from a UAS-controlled transgene causes a specific suppression of the anterior most invagination fold without affecting the others (González-Gaitán, 2000).

The results demonstrate that RTK signaling participates in the selection of tip-cell-dependent invagination centers in the SNS anlage and is subsequently required to initiate morphogenetic movements resulting in invagination folds. This study does not focus on how RTK signaling ties into the wg-modulated Notch signaling process previously shown to be necessary for the selection of the three SNS invagination centers. The data indicate, however, that RTK signaling acts either in parallel or in combination with wg signaling to adjust the proper range of Notch-dependent lateral inhibition. Although in both wg and Egfr signaling mutants, only one ac-expressing cell is singled-out, the selected cells differ with respect to whether they function as tip cells or not. In wg mutants, the single cell causes an invagination, whereas in Egfr signaling mutants, the selected cell fails to provide this feature of SNS invagination centers. The results, therefore, consistently argue that tip cell-derived Spitz triggers local RTK signaling and thereby initiates the formation of invagination folds each headed by the Spitz-secreting tip cell. Thus, Egfr-dependent RTK signaling in Drosophila does not only participate in cell fate decisions and cell proliferation, but also triggers morphogenetic movements within an epithelium, as has been recently demonstrated for fibroblast growth factor (FGF) signaling. It will be interesting to see whether the role of the EGF pathway in cell migration differs at the cellular level from cell migration events triggered by activated FGF receptors (González-Gaitán, 2000).

Cell movements controlled by the Notch signalling cascade during foregut development in Drosophila

Notch signalling is an evolutionarily conserved cell interaction mechanism; its role in controlling cell fate choices has been studied extensively. Recent studies in both vertebrates and invertebrates have revealed additional functions of Notch in proliferation and apoptotic events. Evidence suggests an essential role of the Notch signalling pathway during morphogenetic cell movements required for the formation of the foregut-associated proventriculus organ in the Drosophila embryo. The activation of the Notch receptor occurs in two rows of boundary cells in the proventriculus primordium. The boundary cells delimit a population of foregut epithelial cells that invaginate into the endodermal midgut layer during proventriculus morphogenesis. Notch receptor activation requires the expression of its ligand Delta in the invaginating cells and apical Notch receptor localization in the boundary cells. The movement of the proventricular cells is dependent on the short stop gene that encodes the Drosophila plectin homolog of vertebrates and is a cytoskeletal linker protein of the spectraplakin superfamily. short stop is transcriptionally activated in response to the Notch signalling pathway in boundary cells, and it has been shown that the localization of the Notch receptor and Notch signalling activity depend on short stop activity. These results provide a novel link between the Notch signalling pathway and cytoskeletal reorganization controlling cell movement during the development of foregut-associated organs (Fuss, 2004).

The proventriculus is a multiply folded, cardia-shaped organ that functions as a valve to regulate food passage from the foregut into the midgut of Drosophila larvae. It is derived from the stomodeum, which gives rise to the foregut tube and to parts of the anterior midgut in the early embryo. Cell shape changes are initiated at stage 12 when cell proliferation has been completed within the proventriculus primordium. Anti-Forkhead (Fkh)/anti-Defective proventriculus (Dve) double immunostainings which specifically visualize ectodermal and endodermal cells, respectively, reveal that the first step of proventriculus morphogenesis involves the formation of a ball-like evagination at the ectoderm/endoderm boundary of the posterior foregut tube. The formation of this evagination is initiated by a local constriction of apical membranes at the ectoderm/endoderm boundary leading to an accumulation of membrane-associated markers such as Arm towards the luminal (apical) side. It is notable that the ectodermal part of the ball-like evagination localizes in a mesoderm-free region, whereas the surrounding cells of the developing foregut and the midgut are covered by visceral mesoderm. At stage 14, a constriction forms at the boundary of the ectodermal and the endodermal cells. This results in the formation of the 'keyhole' structure. From stage 14 onward, cells from the anterior portion of the ectodermal keyhole part (in the mesoderm-free area) begin to move inward into the endodermal part of the keyhole and a heart-like structure is formed. The ectodermal keyhole cells continue to move inward until late stage 17 and give rise to the recurrent layer of the proventriculus; it links the outer endodermal layer (derived from the endodermal keyhole cells) and the inner layer of the proventriculus which is a continuation of the esophagus. The cells at the tip of the invaginating ectodermal keyhole cells thatderive from the most anterior region of the keyhole, are not covered by visceral mesoderm. It has been observed before that these cells assume a stretched appearance with long cytoplasmic extensions (Fuss, 2004 and references therein).

Immunohistochemical analysis demonstrates that the ligands of the Notch receptor, Delta and Serrate are expressed in the ectodermal keyhole cells that invaginate into the endodermal cell layer during proventriculus development. Their expression becomes downregulated in the anterior and posterior boundary cells in which the Notch receptor is elevated and in which the Notch signalling pathway is activated, as demonstrated by the Notch-dependent Gbe-Su(H)m8-lacZ reporter construct. Whereas there is no proventricular phenotype in Ser mutants, the invagination movement of the ectodermal keyhole cells is defective in mutants of other components of the Notch signalling pathway, such as Notch, Delta, fng or Su(H). This strongly suggests that the boundary cells play a crucial role for cell movement during proventriculus development. It is not known whether the cell movements are driven by the anterior boundary cells, dragging the esophageal cells behind or whether the major force for the inward movement is contributed by the ectodermal foregut cells changing their shapes from a cuboidal to a more stretched appearance. The latter is known to occur during mid and late stages of embryogenesis when the foregut and the hindgut elongate dramatically increasing their size by two- to threefold. It has been shown for dorsal closure that multiple forces contribute to cell sheet morphogenesis. A similar scenario may apply for proventriculus morphogenesis. Genetic mosaic studies have revealed that the activity of the Notch receptor occurs in cells that are adjacent to the ligand-expressing cells. Therefore, the downregulation of Delta in the boundary cells may be a prerequisite for Notch signalling and cell movement, which would be consistent with the observation that a Notch-like proventriculus phenotype is induced when Delta expression is maintained in the anterior and posterior boundary cells (Fuss, 2004).

Recent studies on neural crest cells in the mouse also have suggested a role for the Notch signalling pathway during cell migration. The neural crest cells in vertebrates give rise to a wide range of cell types, including nerve cells, pigment cells, as well as skeletal and connective tissue. These cells constitute a migratory cell population that leaves the dorsal neural tube to migrate along specific tracks to their final destinations in the periphery of the body. In Delta1 knockout mice, the local expression of Ephrin receptors and ligands, which are guiding molecules, is reduced in the caudal region of the sclerotome, as well as in neural crest-derived peripheral ganglia. A connection of Notch signalling with the modulation of cytoskeletal architecture has not been shown in these mutants. From loss-of-function experiments in the case of the proventriculus cell migration, the alternative view that Notch signalling may determine the fate of the boundary cells rather than directly controlling cell movement cannot be excluded. However, when the Notch pathway was ectopically activated by misexpressing NICD in the proventricular endoderm, this did not result in a change of cell fates of the endodermal cells towards ectodermal boundary cell fates. Furthermore, the link between Notch signalling and the activation of shot, a known cytoskeletal regulator, provides good evidence of a more direct role for Notch in controlling cell movements rather than determining cell fates (Fuss, 2004).

The results further demonstrate that the shot gene is directly or indirectly transcriptionally regulated by the Notch signalling pathway. Members of the spectraplakin superfamily such as Shot in flies or dystonin/BPAG1 or MACF1 in mammals share features of both the spectrin and plakin superfamilies and produce a large variety of giant proteins of up to almost 9000 amino acids in length. These proteins contain motifs interacting with all three elements of the cytoskeleton (the actin, the microtubules and the intermediate filaments), and they contribute to the linkage between membrane receptors and the cytoskeletal elements. shot is strongly expressed during embryogenesis at the muscle attachment sites, which are the most prominent sites of position-dependent integrin adhesion. An essential role for Shot has been shown for muscle-dependent tendon cell differentiation. In the shot mutant tendon cells, Vein, a neuregulin-like factor that activates the EGF-Receptor signalling pathway, fails to be localized properly at the muscle-tendon junctional site; Vein is dispersed and its level is reduced. In these cells, Shot is concentrated at the apical and basal sides. Similarly, the results place shot both upstream and downstream of Notch signalling during proventricular development. In the posterior boundary cells, shot transcription is activated in response to Notch signalling; Shot protein, in turn, is required in the posterior boundary cells for Notch receptor localisation and/or stability as receptor expression and Notch signalling activity in the posterior boundary cells are affected in shot mutants. This indicates a feedback loop, as has been suggested for Crumbs-dependent localization of the Notch receptor in the boundary cells of the hindgut. It is not clear how shot expression is regulated in the endodermal part of the keyhole, in which it may require additional inputs from other yet unknown signalling pathways. Further molecular and biochemical experiments will have to demonstrate whether there exists a direct interaction between the Notch receptor and the cytoskeletal Shot protein (Fuss, 2004).

In the tracheal system, Shot is required for the formation of the RhoA-dependent F-actin cytoskeleton in the fusion cells and to form the lumenal connections between tracheal branches. It has been suggested that Shot may function downstream of RhoA to form E-cadherin-associated cytoskeletal structures that are necessary for apical determinant localization. The analysis of the actin cytoskeleton using phalloidin staining reveals a strong apical localization of F-actin filaments in the posterior boundary cells in which Shot also accumulates apically to a high level. By contrast, the density of the actin cytoskeleton is reduced in the anterior boundary cells that move inward and in which the contribution of Shot for Notch signalling activity seems minor. A stabilized cytoskeletal architecture in the posterior boundary cells may be required to provide stiffness/tension that may enable the inward movement of the anterior boundary cells. Lack- and gain-of-function results suggest that the small GTPase Cdc42, is one of several known cytoskeletal regulators, may play a major role to control cytoskeletal architecture during the inward movement of the proventricular cells. These results are consistent with the idea that Notch signalling controls cytoskeletal organization via the cytoskeletal linker protein Shot and they suggest a role for Cdc42 in this process, the specific involvement of which, however, has to be studied in more detail (Fuss, 2004).

The endoderm specifies the mesodermal niche for the germline in Drosophila via Delta-Notch signaling

Interactions between niche cells and stem cells are vital for proper control over stem cell self-renewal and differentiation. However, there are few tissues where the initial establishment of a niche has been studied. The Drosophila testis houses two stem cell populations, which each lie adjacent to somatic niche cells. Although these niche cells sustain spermatogenesis throughout life, it is not understood how their fate is established. This study shows that Notch signaling is necessary to specify niche cell fate in the developing gonad. Surprisingly, these results indicate that adjacent endoderm is the source of the Notch-activating ligand Delta. Niche cell specification occurs earlier than anticipated, well before the expression of extant markers for niche cell fate. This work further suggests that endoderm plays a dual role in germline development. The endoderm assists both in delivering germ cells to the somatic gonadal mesoderm, and in specifying the niche where these cells will subsequently develop as stem cells. Because in mammals primordial germ cells also track through endoderm on their way to the genital ridge, this work raises the possibility that conserved mechanisms are employed to regulate germline niche formation (Okegbe, 2011).

The data reveal that Notch signaling is necessary to specify hub cell fate. A similar conclusion has recently been reached by Kitadate (2010). It is interesting to note that in three well-characterized stem cell-niche systems in Drosophila, including the transient niche for adult midgut progenitors, the female gonad and now the developing male gonad, Notch signaling is directly responsible for niche cell specification. Moreover, Notch has been found to play a role in the maintenance of various mammalian stem cell populations, including neural stem cells, HSCs and hair follicle stem cells. However, owing to difficulty in performing lineage-specific knockouts in these systems, it remains unclear which cells require Notch activity. As the various cases in Drosophila all require direct Notch activation for niche cell specification, perhaps this reveals a conserved role for Notch signaling in other, more complex stem cell systems (Okegbe, 2011).

Notch signaling specifies niche cells in both the male and female Drosophila gonad; however, it is important to note that there are still some differences. For the ovary, only Delta is required to activate the Notch receptor for proper niche cell specification. For the testis, both ligands contribute to the process, although, here too, it appears that Delta is the dominant ligand employed. Interestingly, depleting Delta or (genetically) separating the endoderm from SGPs (somatic gonadal precursors) both led to a 70% reduction in hub cell number, while depleting Serrate yielded a 30% reduction. Perhaps Delta-Notch signaling from the endoderm accounts for two-thirds of hub cell specification, while Serrate-Notch signaling accounts for only one-third of this process. Although the source of Serrate could not be identifed in this study, Kitadate (2010) has shown that Serrate mRNA is expressed from SGPs after gonad coalescence. Perhaps, this late expression accounts for the modest role Serrate plays in hub specification. That study did not explore in detail a potential role for Delta in hub specification, and the current data suggests that that role is carried out at earlier stages, and from outside the gonad proper (Okegbe, 2011).

In the ovary, cells within the developing gonad appear to present the Notch-activating ligand, although it is unclear whether germ cells or somatic cells are the source of Delta. The current data suggests that cells from a distinct germ layer, the endoderm, present Delta to SGPs in the male gonad. These differences may indicate distinct evolutionary control over gonadal niche development between the sexes (Okegbe, 2011).

Although the gonad first forms during mid-embryogenesis, hub cells only become identifiable just prior to hatching of the larvae, some 6 hours later. At that time, hub cells begin to tightly pack at the anterior of the gonad, upregulate several cell adhesion and cytoskeletal molecules (Fascilin 3, Filamin, DN-Cadherin, DE-Cadherin) as well as induce Upd expression and other markers of hub fate. Surprisingly, the current data reveal that most hub cells are specified well before these overt signs of hub cell differentiation, as judged by Notch reporter activation and Notch rescue. Although it was previously thought that SGPs were equivalent at the time of gonad coalescence it is now clear that due to Notch activity, the SGPs are parsed into a group of either hub cells or cyst cells before gonad coalescence occurs (Okegbe, 2011).

Thus, it is believed that a series of steps must occur before the hub can function as a niche. First, the PMG (posterior midgut) presents Delta, leading to Notch activation in some SGPs as they are carried over these endodermal cells during germ band retraction. Activation might be dependent on, for example, length of time in contact with passing PMG cells. At the present time, it is unclear whether all SGPs are activated for Notch (Kitadate, 2010), or only some of them (this study). After gonad coalescence, activated SGPs must then migrate anteriorly. Although it is known that integrin-mediated adhesion is required to maintain the hub at the anterior (Tanentzapf, 2007), no cues have been identified that could guide the migration of the Notch-activated SGPs. Next, as the cells reach the anterior of the gonad they must execute a mesenchymal-to-epithelial transition, as evidenced by the upregulation of cell-adhesion molecules and preferential associations between hub cells. This step occurs independently of the integrin-mediated anchoring at the anterior. Finally, the hub cells must induce Upd expression and recruit neighboring cells to adopt stem cell fate. The apparent delay between the activation of the Notch pathway and the initiation of the hub cell gene expression program might suggest that initiating that hub program first requires that the cells coalesce into an epithelium. Such a mechanism would prevent precocious or erroneous stem cell specification within the gonad (Okegbe, 2011).

Although these data reveal Notch-activated SGPs at all positions within the gonad and that some of these become hub cells, it is unclear how hub cell number is tightly regulated. Potentially, SGP migration over endodermal cells could induce Notch activation among SGPs throughout the forming gonad, potentiating these cells to become hub cells. However, solely relying on that mechanism could lead to the specification of too many hub cells. It appears, though, that specification is regulated by EGFR pathway activation (Kitadate, 2010). EGFR protein is observed on most SGPs throughout the embryonic gonad, beginning at gonad coalescence (stage 13). The EGFR ligand Spitz is expressed from all germ cells during gonad coalescence and activates EGFR among posterior SGPs. This activity antagonizes Notch and that appears to regulate final hub cell number. How EGFR activation is restricted or enhanced only among posterior SGPs is at present unclear (Okegbe, 2011).

Given that this study found that hub cell specification occurs prior to gonad coalescence, it is also possible that Notch and EGFR act in a temporal sequence. In this case, early Notch-activated SGPs, perhaps even those in the posterior will adopt hub cell fate. But, as EGFR becomes activated, further induction of the Notch pathway in the posterior is antagonized, prohibiting the specification of too many hub cells. Such a temporal inhibition might be important, as Serrate is expressed on the SGPs (Kitadate, 2010) and both Delta and Serrate are robustly expressed on tracheal cells, the activity of which might otherwise lead to excess hub cell induction. Perhaps during later stages of gonadogenesis (stages 14-16) a small number of anterior SGPs become Notch activated due to the activity of Serrate-Notch signaling from other SGPs, supplementing the hub cells previously specified by Delta-Notch signaling (Okegbe, 2011).

Given that niche cells in the Drosophila ovary become activated via Delta-Notch signaling by neighboring somatic cells, it was initially expected that Notch would be activated in a subset of SGPs by ligand presented from other SGPs. However, this study could not detect Delta nor Serrate expression among SGPs. Furthermore, although nearby tracheal cells expressed both ligands robustly, that expression appears later than Notch rescue suggests would be necessary, and genetic ablation of tracheal cells did not influence hub cell number (Okegbe, 2011).

Instead, this study found that a crucial signal for niche cell specification is presented from the endoderm, as Delta is expressed robustly on posterior midgut cells, at a time consistent with the requirement for Notch function. Furthermore, these endodermal cells are close enough to SGPs for productive Delta-Notch signaling to occur. Although visceral mesodermal cells are also close to the PMG and the SGPs, this tissue does not affect hub specification, since this study found that brachyenteron mutants exhibited normal hub cell number. By contrast, in mutants that do not internalize the gut (fog), and thus would not present Delta to SGPs, a drastic reduction was seen in hub cell number (Okegbe, 2011).

Additionally, it is noted that absolute hub cell number varies among animals and according to genetic background. This is attributed to normal biological variation, just as germline stem cell number varies. Potentially, this variation could be caused by the robustness with which the Notch pathway is activated in SGPs, as they are carried over the midgut cells. It will be interesting to test this hypothesis by genetically manipulating the number of midgut cells or the time of contact between endoderm and SGPs. Additionally, the antagonistic effects of EGFR signaling might account for some of the observed variation. In fact, gonads heterozygous for Star, a component of the EGFR pathway, exhibit increased hub cell number (Okegbe, 2011).

Finally, it is interesting to consider why the endoderm would be crucial for the proper specification of the GSC niche. In Drosophila, as in many animals, there is a special relationship between the gut and the germ cells. Primordial germ cells in mammals and in Drosophila must migrate through the endoderm to reach the gonadal mesoderm. In fact, in Drosophila, the gut exercises elaborate control over germ cell migration. As the germ cells begin their transepithelial migration and exit from the midgut pocket, tight connections between midgut cells are dissolved, allowing for easy germ cell passage. Germ cells then migrate on the basal surface of endodermal cells and midgut expression of wunens (which encodes lipid phosphate phosphatases) repels germ cells, driving them into the mesoderm. Thus, the endoderm not only delivers germ cells to the somatic mesoderm, but the same endoderm specifies niche cells from among the somatic mesoderm wherein germ cells can subsequently develop into stem cells. In mammals, although the exact make-up of the spermatogonial stem cell niche has not been determined, it must (in part) derive from cells of the genital ridge. It will be interesting to determine whether proximity to the gut endoderm is important for the specification of this niche (Okegbe, 2011).

Notch and Myogenesis

The visceral muscles of the Drosophila midgut consist of syncytia and arise by fusion of founder myoblasts with fusion-competent myoblasts (fcms), as described for the somatic muscles. A single-step fusion results in the formation of binucleate circular midgut muscles, whereas a multiple-step fusion process produces the longitudinal muscles. A prerequisite for muscle fusion is the establishment of myoblast diversity in the mesoderm prior to the fusion process itself. Evidence is provided for a role of Notch signalling during establishment of the different cell types in the visceral mesoderm, demonstrating that the basic mechanism underlying the segregation of somatic muscle founder cells is also conserved during visceral founder cell determination. Searching for genes involved in the determination and differentiation of the different visceral cell types, two independent mutations were identified causing loss of visceral midgut muscles. In both of these mutants, visceral muscle founder cells are missing and the visceral mesoderm consists of fusion-competent myoblasts only. Thus, no fusion occurs resulting in a complete disruption of visceral myogenesis. Subsequent characterization of the mutations revealed that they are novel alleles of jelly belly (jeb) and the Drosophila Alk homolog named milliways (miliAlk or just plain Alk). The process of founder cell determination in the visceral mesoderm depends on Jeb signalling via the Milliways/Alk receptor. Moreover, it has been demonstrated that in the somatic mesoderm determination of the opposite cell type, the fusion-competent myoblasts, also depends on Jeb and Alk, revealing different roles for Jeb signalling in specifying myoblast diversity. This novel mechanism uncovers a crosstalk between somatic and visceral mesoderm leading not only to the determination of different cell types but also maintains the separation of mesodermal tissues, the somatic and splanchnic mesoderm (Stute, 2004).

The process of lateral inhibition involving Notch and its ligand Delta plays a role in determining the founder myoblasts and fusion-competent myoblasts (fcms) of the somatic musculature. Since many of the processes involved in the development of the somatic musculature also seem to affect the development of the visceral muscles, whether the mechanism of determination of founder cells and fcms is also conserved was examined. In Notch mutant embryos more founder cells appear to be present in the visceral mesoderm. The visceral fcms seem to be reduced compared with the wild-type expression of sticks and stones (sns) as a marker for these cells. This reduction is not as severe as in the somatic mesoderm but still quite obvious. In Delta mutants, the number of founder cells also seems to be increased in comparison with the wild type and the fcms are reduced in mutant embryos (Stute, 2004).

These observations cannot exclude the possibility that the observed phenotypes are induced by secondary effects from defects in other tissues, among others the lack of fcms in the somatic mesoderm. Therefore overexpression studies were undertaken using the UAS-GAL4 system. The GAL4 and UAS lines employed in this study also carry rP298-lacZ, which serves to mark the founder cells. As a driver line bap-GAL4 was used to drive expression in the entire trunk visceral mesoderm. Expression of UAS-Notch+Delta, which contains the entire coding regions of both genes or UAS-Notchintra, which represents a constitutively active form of Notch, in the visceral mesoderm, both result in a distinct phenotype. In midgut preparations of these embryos the founder cells of the circular visceral mesoderm are strongly reduced and later on, no functional visceral mesoderm can be observed. By contrast, the founder cells of the longitudinal visceral muscles, which have a different origin at the posterior tip of the embryo, are still present. Interestingly,bap-GAL4-driven expression of the Notch ligand Delta does not result in fewer founder cells in the visceral mesoderm (Stute, 2004).

To exclude the possibility that the described defects are due to non-endogenous effects induced by the overexpression of the examined genes in the wrong tissue, wild-type Notch expression was analyzed and found to be expressed in the visceral mesoderm. Notch is localized at cell membranes in the entire visceral mesoderm during stage 11, with expression becoming weaker in the fcms of the visceral mesoderm, that continue to express bap-lacZ after the determination process is finished. This reduction of Notch expression in the fcms after the establishment of the founder cells is similar to its expression in the somatic mesoderm, where Notch expression is also highest in the progenitor cell after the determination process is completed. Surprisingly, the analysis of Delta expression exhibits that this Notch ligand is not expressed in the visceral mesoderm during founder cell formation. Delta expression was found in adjacent, probably somatic cells and might be needed there to participate in the visceral determination process, as indicated by the increased number of founder cells and reduced number of fcms in Delta mutants. Even though Dl is expressed in the cells surrounding the visceral mesoderm, ectopic expression of UAS-Dl in these cells with a twi-GAL4 driver line does not result in an obvious phenotype, which might be due to the fact that the amount of Delta in this tissue is not the limiting factor that restricts Notch signalling. Another explanation for a missing Delta expression in the visceral mesoderm might be that a different factor acts as a ligand for Notch in the visceral mesoderm and that the observed phenotype in Delta mutants is due to secondary effects (Stute, 2004).

Since the ectopic expression causes such a severe phenotype, the lethality of these embryos was tested. Most of the progeny of the cross between the bap-GAL4 driver line and UAS-N+Dl or UASNintra develop and hatch but die as first larvae (78% or 70%), presumably owing to the fact that they cannot ingest any food. Ectopic expression of UAS-Dl alone also increased lethality compared with the UAS and GAL4 lines alone, but still ~65% of the larvae survive (Stute, 2004).

To confirm these results, a dominant-negative form of Notch (UAS-dnN) was overexpressed specifically in the visceral mesoderm with a bap-GAL4 driver. The embryos exhibit an obvious duplication of most visceral founder cells but still some fcms remain (Stute, 2004).

From these results, it is concluded that Notch plays a role in the determination of the founder cells and fcms in the visceral mesoderm. Delta, which is expressed in the cells surrounding the visceral mesoderm, might serve as the ligand in this process but it is also possible that another factor takes over this role. Hence, not only is the fusion mechanism between the founder cells and the fcms in the somatic and visceral mesoderm conserved, but so is the initial mechanism of determination of these two cell types (Stute, 2004).

Identification of a new stem cell population which generates Drosophila flight muscles

How myoblast populations are regulated for the formation of muscles of different sizes is an essentially unanswered question. The large flight muscles of Drosophila develop from adult muscle progenitor (AMP) cells set-aside embryonically. The thoracic segments are all allotted the same small AMP number, while those associated with the wing-disc proliferate extensively to give rise to over 2500 myoblasts. An initial amplification occurs through symmetric divisions and is followed by a switch to asymmetric divisions in which the AMPs self-renew and generate post-mitotic myoblasts. Notch signaling controls the initial amplification of AMPs, while the switch to asymmetric division additionally requires Wingless, which regulates Numb expression in the AMP lineage. In both cases, the epidermal tissue of the wing imaginal disc acts as a niche expressing the ligands Serrate and Wingless. The disc-associated AMPs are a novel muscle stem cell population that orchestrates the early phases of adult flight muscle development (Gunage, 2014. PubMed).

Evidence for a fruit fly hemangioblast and similarities between lymph-gland hematopoiesis in fruit fly and mammal aorta-gonadal-mesonephros mesoderm

The Drosophila lymph gland is a hematopoietic organ and, together with prospective vascular cells (cardioblasts) and excretory cells (pericardial nephrocytes), arises from the cardiogenic mesoderm. Clonal analysis provided evidence for a hemangioblast that can give rise to two daughter cells: one that differentiates into heart or aorta and another that differentiates into blood. In addition, the GATA factor gene pannier (pnr) and the homeobox gene tinman (tin), which are controlled by the convergence of Decapentaplegic (Dpp), fibroblast growth factor (FGF), Wingless (Wg) and Notch signaling, are required for the development of all cardiogenic mesoderm, including the lymph gland. An essential genetic switch differentiates between the blood or nephrocyte and vascular lineages involves the Notch pathway. Further specification occurs through specific expression of the GATA factor Serpent (Srp) in the lymph-gland primordium. These findings suggest that there is a close parallel between the molecular mechanisms functioning in the Drosophila cardiogenic mesoderm and those functioning in the mammalian aorta-gonadal-mesonephros mesoderm (Mandal, 2004).

Blood and vascular cells in the vertebrate embryo are thought to derive from oligopotent progenitor cells, called hemangioblasts, that arise in the yolk sac and in the aorta-gonadal-mesonephros (AGM) mesenchyme. A close relationship between blood and vascular progenitors is well established, but in vivo evidence that a single cell can divide to produce a blood cell and an endothelial cell is lacking in vertebrate systems. Similarly, the molecular mechanism that distinguishes between the two lineages is not well understood. To address these issues in a simple, genetically amenable system, the genetic control of hematopoiesis was analyzed in Drosophila. The results show that there are close lineage relationships between hematopoietic and vascular cells, similar to those present in the AGM of mammalian systems. Evidence is provided for conserved cassettes of transcription factors and signaling cascades that limit the pool of hemangioblastic cells and promote the blood versus vascular fate (Mandal, 2004).

In the mature Drosophila embryo, the lymph gland is formed by a paired cluster of ~20 cells flanking the aorta. The aorta and heart represent a contractile tube lined by a layer of myoepithelial vascular cells called cardioblasts. The cells flanking the aorta and heart posterior to the lymph gland are the pericardial cells, which function as excretory cells (nephrocytes). Lymph gland progenitors express the prohemocyte marker Srp and ultrastructurally resemble prohemocytes that develop at an earlier stage from the head mesoderm. Monitoring expression of the zinc-finger protein Odd-skipped (Odd) shows that the lymph gland originates from the dorsal thoracic mesoderm. Odd is expressed in segmental clusters in the dorsal mesoderm of segments T1-A6. The three thoracic Odd-positive clusters coalesce to form the lymph gland, whereas the abdominal clusters formed the pericardial nephrocytes (Mandal, 2004).

Lymph-gland progenitors, cardioblasts and pericardial cells are closely related by lineage. Labeled 'flipout' (FLP/FRT) clones were induced in embryos aged 3-4 h such that the clones contained only 2-4 cells. Of the two-cell clones, ~50% contained cardioblast and lymph-gland cells; the other clones comprised either cardioblasts or lymph-gland cells alone. Mixed clones were recovered at the late third larval stage. The finding of mixed clones indicates that the cardiogenic mesoderm of D. melanogaster contains oligopotent progenitors that, up to the final division, can give rise both to Srp-positive blood-cell progenitors that form the lymph gland and to vascular cells (Mandal, 2004).

The cardiogenic mesoderm forms part of the dorsal mesoderm, which requires the homeobox protein Tin and the GATA factor Pnr. In embryos with mutations in tin or pnr, the lymph gland was absent. Maintenance of Tin expression in the dorsal mesoderm requires the activity of at least two signaling pathways regulated by Dpp (the Drosophila homolog of transforming growth factor-ß) and Heartless (Htl; one of the D. melanogaster homologs of the FGF receptor); the dependence of cardioblast and pericardial nephrocyte development on these signaling pathways has been documented. Lymph-gland progenitors did not develop in loss-of-function dpp and htl mutants (Mandal, 2004).

Between 6 h and 8 h of development, the dorsal mesoderm splits into the cardiogenic mesoderm and the visceral mesoderm. The cardiogenic mesoderm is regulated positively by Wg and negatively by Notch. Lack of Wg signaling results in the absence of all cardiogenic lineages including lymph gland. Notch signaling has the opposite effect and restricts cardiogenic mesodermal fate. Notch is active in the dorsal mesoderm from 6 h to 10 h of development. Eliminating Notch during the first half of this interval by raising embryos homozygous with respect to the temperature-sensitive allele Nts1 at the restrictive temperature resulted in substantially more cardioblasts, pericardial cells and lymph-gland progenitors (Mandal, 2004).

Lymph-gland progenitors, cardioblasts and pericardial nephrocytes are specified in the cardiogenic mesoderm around the phase of germband retraction 8-10 h after fertilization. At this stage, Tin, which was initially expressed in the whole cardiogenic mesoderm, becomes restricted to a narrow medial compartment containing the cardioblasts. Pnr follows the same restriction. Cells located at a more lateral level in the cardiogenic mesoderm give rise to lymph-gland progenitors (in the thoracic domain) and pericardial nephrocytes (in the abdominal domain) and activate the gene odd. Slightly later, Srp is expressed in lymph-gland progenitors. As reported for the early hemocytes derived from the embryonic head, srp is centrally involved in lymph-gland specification. In srp-null embryos, Odd-expressing cells still formed a lymph gland-shaped cluster flanking the aorta, but these cells also express the pericardial marker pericardin (Prc), suggesting that they lose some aspects of hemocyte precursor identity or gain properties of nephrocytes. As a countercorrelate, ectopic expression of Srp in the whole cardiogenic mesoderm directed by mef2-Gal4 induces pericardial cells to adopt lymph-gland fate (Mandal, 2004).

Downregulation of tin and pnr in cells in the lateral domain of the cardiogenic mesoderm is essential for lymph-gland specification. Ectopic expression of tin or pnr by twist-Gal4 (or mef2-Gal4) causes a marked reduction in the number of lymph-gland and pericardial cells. The antagonistic effect of tin on lymph-gland progenitors resembles its earlier role in the head mesoderm that gives rise to the larval blood cells; here too, ectopic expression of tin causes a reduction in the number of hemocytes (Mandal, 2004).

Inhibiting tin and upregulating odd and srp requires input from the Notch signaling pathway. A function of Notch at 6-8 h in specification of the cardiogenic mesoderm is described. Reducing Notch function between 8 h and 10 h causes an increase in the number of cardioblasts and a concomitant loss of pericardial and lymph-gland cells. Overexpressing an activated Notch construct causes a marked increase in lymph-gland size. This late requirement for Notch signaling is separable from the earlier role of Notch in restricting the overall size of the cardiogenic mesoderm. Thus, the sum total of cardioblasts and pericardial or lymph-gland cells in Nts1 embryos shifts between 8 h and 10 h and does not differ substantially from that in wild type, whereas a combined effect on cell number and cell fate is seen in embryos with a Notch deletion. In these embryos, the cardiogenic mesoderm is hyperplasic and develops as cardioblasts at the expense of lymph-gland progenitors and pericardial nephrocytes. The dual role of Notch in restricting the numbers of a pluripotent progenitor pool and in distinguishing between the progeny of these progenitors is reminiscent of the function of Notch in sense-organ development (Mandal, 2004).

Lymph-gland formation is restricted to the thoracic region by positional cues that are provided by expression of the homeobox proteins of the Antennapedia and Bithorax complex. Specifically, Ultrabithorax (Ubx), which is expressed in segments A2-A5 of the cardiogenic mesoderm, inhibits lymph-gland formation. Loss of Ubx results in the expansion of the lymph-gland fate into the abdominal segments. Conversely, overexpression of Ubx driven by mef2-Gal4 causes the transformation of lymph-gland progenitors into pericardial nephrocytes (Mandal, 2004).

These findings are suggestive of a model of lymph-gland development in Drosophila that is similar to mammalian hematopoiesis. Lymph-gland progenitors develop as part of the cardiogenic mesoderm that also gives rise to the vascular cells (aorta and heart) and to excretory cells. Similarly, progenitor cells of the blood, aorta and excretory system are closely related both molecularly and developmentally in mammals, where they form part of the AGM. Specification of the cardiogenic mesoderm requires the input of FGF and Wg signaling, as in vertebrate hematopoiesis, where the AGM region is induced in response to several converging signaling pathways including FGF, BMP and Wnt (Mandal, 2004).

The cardiogenic mesoderm in Drosophila evolves from the dorsal mesoderm and requires input from the Htl, Dpp, Wg and Notch (N) signaling pathways. The cardiogenic mesoderm then differentiates into lymph gland, vascular cells (cardioblasts) and excretory cells (pericardial nephrocytes). A subpopulation of cardioblasts and lymph-gland cells is derived from one progenitor (hemangioblast; HB). Essential for the differentiation of the cardiogenic mesoderm is the Notch-Delta (Dl)-dependent restriction of Tin and Pnr to cardioblasts and the expression of Srp in the lymph gland. In vertebrates, similar cell types are derived from a mesodermal domain called the AGM, which also requires the input of FGF, BMP and Wnt signaling. A subset of AGM-derived cells has been proposed to constitute hemangioblasts, which produce blood progenitors and endothelial cells (Mandal, 2004).

These findings show that in Drosophila, the cardiovascular and blood-cell lineages are differentiated by an antagonistic relationship between Tin or Pnr expression in the cardioblasts and Srp expression in the lymph-gland progenitors. In vertebrates, GATA factors also have a pivotal role in specifying different lineages among blood-cell progenitors, although not much is known about what differentiates between blood progenitors as a group and endothelial progenitors. The results indicate that this step is driven by input from the Notch signaling pathway. In the thoracic cardiogenic mesoderm, Notch antagonizes tin and pnr expression and aortic cardioblast formation, and promotes srp expression and the development of lymph-gland progenitors. In vertebrates, Notch signaling is also involved in both blood and vascular development. The role of Notch during AGM morphogenesis remains to be investigated (Mandal, 2004).

Cardioblasts and lymph-gland cells can arise from the division of a single cardiogenic mesodermal cell, which should be called a hemangioblast. A previous study induced clones in the cardiogenic mesoderm but used only Tin as a marker. This study also yielded mixed two-cell clones comprising a cardioblast and a nonlabeled cell, which, in light of the current findings, must be interpreted as a lymph-gland cell. Hemangioblasts have been proposed in vertebrates, although the definitive experiment in which a precursor is marked and its lineage is tracked has not been done. Blast colony-forming cells that give rise to both lineages in vitro and common markers that belong to both cell types in vivo have been identified, but direct evidence for the existence of a common precursor has not yet been found. This study, using genetic analysis of two-cell clones, establishes the existence of such a population in Drosophila. On the basis of these results, and given the conservation of the signaling and transcriptional components described here, the prediction is that many cells of the AGM in vertebrates may give rise to only blood or only vascular cells, but a number of intermixed hemangioblasts may give rise to mixed lineages. Future genetic screens aimed at finding components in early lymph-gland development will probably identify additional pathways and strategies important for vertebrate hematopoiesis (Mandal, 2004).

Coordinated sequential action of EGFR and Notch signaling pathways regulates proneural wave progression in the Drosophila optic lobe.

During neurogenesis in the medulla of the Drosophila optic lobe, neuroepithelial cells are programmed to differentiate into neuroblasts at the medial edge of the developing optic lobe. The wave of differentiation progresses synchronously in a row of cells from medial to the lateral regions of the optic lobe, sweeping across the entire neuroepithelial sheet; it is preceded by the transient expression of the proneural gene lethal of scute [l(1)sc] and is thus called the proneural wave. This study found that the epidermal growth factor receptor (EGFR) signaling pathway promotes proneural wave progression. EGFR signaling is activated in neuroepithelial cells and induces l(1)sc expression. EGFR activation is regulated by transient expression of Rhomboid (Rho), which is required for the maturation of the EGF ligand Spitz. Rho expression is also regulated by the EGFR signal. The transient and spatially restricted expression of Rho generates sequential activation of EGFR signaling and assures the directional progression of the differentiation wave. This study also provides new insights into the role of Notch signaling. Expression of the Notch ligand Delta is induced by EGFR, and Notch signaling prolongs the proneural state. Notch signaling activity is downregulated by its own feedback mechanism that permits cells at proneural states to subsequently develop into neuroblasts. Thus, coordinated sequential action of the EGFR and Notch signaling pathways causes the proneural wave to progress and induce neuroblast formation in a precisely ordered manner (Yasugi, 2010).

Loss of EGFR function in progenitor cells caused failure of L(1)sc expression and differentiation into neuroblasts (see A model of progression of the proneural wave). In addition, elevated EGFR signaling resulted in faster proneural wave progression and induced earlier neuroblast differentiation. The activation of the EGFR signal is regulated by a transient expression of Rho, which cleaves membrane-associated Spi to generate secreted active Spi. This study also demonstrated that Rho expression itself depends on EGFR function, and thus the sequential induction of the EGFR signal progresses the proneural wave. Clones of cells mutant for pnt were not recovered unless Minute was employed, suggesting that the EGFR pathway is required for the proliferation of neuroepithelial cells. However, the progression of the proneural wave is not regulated by the proliferation rate per se (Yasugi, 2010).

The function of the Notch signaling pathway in neurogenesis is known as the lateral inhibition. A revision of this notion has recently been proposed for mouse neurogenesis, in which levels of the Notch signal oscillate in neural progenitor cells during early stages of embryogenesis, and thus no cell maintains a constant level of the signal. The oscillation depends mainly on a short lifetime and negative-feedback regulation of the Notch effecter protein Hes1, a homolog of Drosophila E(spl). This prevents precocious neuronal fate determination. The biggest difficulty in analysis of Notch signaling is the random distribution of different stages of cells in the developing ventricular zone, which is thus called a salt-and-pepper pattern. In medulla neurogenesis, however, cell differentiation is well organized spatiotemporally and the developmental process of medulla neurons can be viewed as a medial-lateral array of progressively aged cells across the optic lobe. Such features allowed the functions of Notch to be precisely analyzed. Cells are classified into four types according to their developmental stages: neuroepithelial cells expressing PatJ, neuronal progenitor I expressing a low level of Dpn, neuronal progenitor II expressing L(1)sc and neuroblasts expressing high levels of Dpn. The Notch signal is activated in neuronal progenitor I and II. The EGFR signal turns on in the neuronal progenitor II stage and progresses the stage by activating L(1)sc expression. Cells become neuroblasts when the Notch and EGFR signals are shut off. Cells stay as neuronal progenitor I when Notch signal alone is activated, whereas cells stay as neuronal progenitor II when the Notch signal is activated in conjunction with the EGFR signal. Although the Notch signal is once activated, it must be turned off to let cells differentiate into neuroblasts. In neuronal progenitor II, E(spl)-C expression is induced by Notch signaling, and the increased E(spl)-C next downregulates Dl expression and subsequent activation of the Notch signal (Yasugi, 2010).

What does Notch do in medulla neurogenesis? It is infered that the Notch signal sustains cell fates, whereas the EGFR signal progresses the transitions of cell fate. This was well documented when a constitutively active form of each signal component was induced. EGFR, or its downstream Ras, induces expression of L(1)sc but does not fix its state, even though the constitutively active form is employed. At the same time, a constitutively active Notch sustains cell fates in a cell-autonomous manner. Constitutively active N receptors, by contrast, autonomously determine cell fates depending on the context: cells become neuronal progenitor I in the absence of EGFR and neuronal progenitor II in the presence of EGFR. The precocious neurogenesis caused by the impairment of Notch signaling suggests that Notch keeps cells in the progenitor state for a certain length of time in order to allow neuroepithelial cells to grow into a sufficient population. In the prospective spinal cord of chick embryo (Hammerle, 2007), the development from neural stem cells to neurons progresses rostrocaudally, during which the transition from proliferating progenitors to neurogenic progenitors is regulated by Notch signaling (Yasugi, 2010).

Although Notch plays a pivotal role in determining cell fate between neural and non-neural cells, the function may be context dependent and can be classified into three categories. (1) Classical lateral inhibition is seen in CNS formation in embryogenesis and SOP formation in Drosophila. Cells that once expressed a higher level of the Notch ligand maintain their cell states and become neuroblasts. (2) Oscillatory activations are found in early development of the mouse brain (Shimojo, 2008). Progenitor cells are not destined to either cell types. (3) An association with the proneural wave found in Drosophila medulla neurogenesis as is described in this study. The Notch signal is transiently activated only once and then shuts off in a synchronized manner. The notable difference in the outcome is the ratio of neural to non-neural cells; a small number of cells from the entire population become neuroblasts or neural stem cells in the former cases (1 and 2), whereas most of the cells become neuroblasts in the latter case (3). The differences between (1) and (2) can be ascribed at least in part to the duration of development. Hes1 expression has been shown to oscillate within a period of 2 hours in the mouse, whereas in Drosophila embryogenesis, selection of neuroblasts from neuroectodermal cells takes place within a few hours. Thus, even if Drosophila E(spl) has a half-life time equivalent to Hes1, the selection process during embryogenesis probably finishes within a cycle of the oscillation. The process of medulla neuroblast formation continues for more than 1 day, but Notch signaling is activated for a much shorter period in any given cell. This raises the possibility that E(spl)/Hes1 may have a similarly short half-life but outcome would depend on the developmental context (Yasugi, 2010).

The functions of EGFR and Notch described in this study resemble their roles in SOP formation of adult chordotonal organ development; the EGFR signal provides an inductive cue, whereas the Notch signal prevents premature SOP formation. In addition, restricted expression of rho and activation of the EGFR signal assure reiterative SOP commitment. Several neuroblasts are also sequentially differentiated from epidermal cells in adult chordotonal organs (Yasugi, 2010).

Unpaired, a ligand of the JAK/STAT pathway is expressed in lateral neuroepithelial cells and shapes an activity gradient that is higher in lateral and lower in the medial neuroepithelium. The JAK/STAT signal acts as a negative regulator of the progression of the proneural wave (Yasugi, 2008). This report has shown that activation of both EGFR and Notch signaling pathways depends on the activity of the JAK/STAT signal. The JAK/STAT signal probably acts upstream of EGFR and Notch signals in a non-autonomous fashion. These three signals coordinate and precisely regulate the formation of neuroblasts (Yasugi, 2010).

Notch regulates the switch from symmetric to asymmetric neural stem cell division in the Drosophila optic lobe

The proper balance between symmetric and asymmetric stem cell division is crucial both to maintain a population of stem cells and to prevent tumorous overgrowth. Neural stem cells in the Drosophila optic lobe originate within a polarised neuroepithelium, where they divide symmetrically. Neuroepithelial cells are transformed into asymmetrically dividing neuroblasts in a precisely regulated fashion. This cell fate transition is highly reminiscent of the switch from neuroepithelial cells to radial glial cells in the developing mammalian cerebral cortex. To identify the molecules that mediate the transition, neuroepithelial cells were microdissected, and their transcriptional profile was compared with similarly obtained optic lobe neuroblasts. Genes encoding members of the Notch pathway were found expressed in neuroepithelial cells. Notch mutant clones are extruded from the neuroepithelium and undergo premature neurogenesis. A wave of proneural gene expression is thought to regulate the timing of the transition from neuroepithelium to neuroblast. The proneural wave transiently suppresses Notch activity in neuroepithelial cells, and inhibition of Notch triggers the switch from symmetric, proliferative division, to asymmetric, differentiative division (Egger, 2010).

In the developing mammalian cortex, neural stem cells initially divide symmetrically to produce two neural stem cells, thereby increasing the neural precursor pool. The radial glial cells subsequently divide asymmetrically to produce a neural stem cell and either a basal progenitor cell or an immature neuron. Most basal progenitor cells divide once more to generate two postmitotic neuron. The Notch signalling pathway is thought to play a role in maintaining the undifferentiated state of neuroepithelial cells, radial glia and basal progenitors, but the downstream signalling cascades activated in these cells might be differentially regulated. Neurogenesis is initiated by proneural genes, such as Mash1 and Neurogenin2 (Ngn2) (Egger, 2010).

This study shows that this sequence of neurogenic events is remarkably similar to that seen in the development of the optic lobe in Drosophila. Notch is activated in the neuroepithelial cells, which remain undifferentiated. The proneural gene l'sc is expressed within the transition zone, and levels of Delta are increased, while Notch activity is decreased. Thus neuroepithelial cells ultimately give rise to a variety of differentiated neurons, but only after they have passed through the transition zone (Egger, 2010).

Low levels of Delta expression were found thoughout the optic lobe neuroepithelium, with increased expression in the transition zone. Several Brd genes were found within the neuroepithelium. Negative regulation of Delta activity by the Brd proteins would be expected to further reduce the level of Delta signalling. This situation might be analogous to the oscillations in Delta and Ngn2 levels observed in vertebrates, and it will be interesting to assess whether the expression of Delta, HLHm5 or proneural genes also oscillate in flies. Strikingly, when Delta activity is inhibited throughout the epithelium, the premature transformation of the entire neuroepithelium into neuroblasts is observed. This suggests that neuroepithelial cells might both send and receive the Notch signal (Egger, 2010).

Higher levels of Delta were observed in the L'sc positive transition zone. High levels of Delta or Serrate can inhibit Notch signalling through cis-inhibition, suggesting one possible mechanism for the downregulation of Notch signalling at the transition zone. Interestingly, very recent results suggest that cis-inhibition can create sharp boundaries and this could be the role of the high levels of Delta that were observe in the transition zone (Egger, 2010).

Epithelial integrity might be important to maintain proliferative cell division. This study shows that Notch mutant clones are extruded from the neuroepithelium. Furthermore, expression in the optic lobe neuroepithelial cells was found of a number of genes involved in cell adhesion. Notch could regulate cell adhesion molecules at the transcriptional level, or might itself form a complex with adhesion molecules. In either case, Notch loss of function would disrupt cell adhesion and lead to the extrusion of epithelial cells. Subtypes of cadherins, such as DE-Cad, Cad99C, Fat, which were found preferentially expressed in the neuroepithelium, might be activated by Notch to maintain the neuroepithelium, and repressed by L'sc to promote neurogenesis. Notch mutant clones also upregulate expression of the neuroblast transcription factor Dpn (but not of L'sc), and divide asymmetrically only once they have delaminated from the epithelium. In contrast to Notch mutant clones, L'sc misexpression clones upregulate Dpn and switch to asymmetric division whilst still embedded within the neuroepithelium. L'sc acts, at least in part, through repression of Notch signalling, but might also induce neuroblast-specific genes directly (Egger, 2010).

JAK/STAT signalling negatively regulates the progression of the proneural wave and neurogenesis in the optic lobe. Interestingly, the ability of Notch to maintain radial glial cell fate appears to be largely dependent on functional JAK/STAT signalling. It remains to be seen whether the Notch pathway interacts with JAK/STAT in the Drosophila optic lobe (Egger, 2010).

This study has shown that the development of the Drosophila optic lobe parallels that of the vertebrate cerebral cortex, suggesting that the pathways regulating the transition from symmetric to asymmetric division might be conserved from flies to mammals. Identifying the effector genes that are regulated by Notch and L'sc, and the links between JAK/STAT and Notch signalling, will yield further insights into the molecular mechanisms that maintain an expanding neural stem cell pool and regulate the timely transition to differentiation (Egger, 2010).

Function of Nerfin-1 in preventing medulla neurons dedifferentiation requires its inhibition of Notch activity

Drosophila larval central nervous system comprises the central brain, ventral nerve cord and optic lobe. In these regions, neuroblasts divide asymmetrically to self-renew and generate differentiated neurons or glia. To date, mechanisms of preventing neuron dedifferentiation are still unclear, especially in the optic lobe. This study shows that the zinc finger transcription factor Nerfin-1 is expressed in early stage of medulla neurons and essential for maintaining their differentiation. Loss of Nerfin-1 activates Notch signaling, which promotes neuron-to-NB reversion. Repressing Notch signaling largely rescues dedifferentiation in nerfin-1 mutant clones. Thus, it is concluded that Nerfin-1 represses Notch activity in medulla neurons and prevents them from dedifferentiation (Xu, 2017).

Stem cells generate progeny that undergo terminal differentiation. In Drosophila CNS, the balance of self-renewal and differentiation of neural stem and progenitor cells is a central issue during development. On the other hand, the maintenance of differentiated status of post-mitotic neurons is also crucial for tissue function and homeostasis. It is obvious that mechanisms must exist to prevent the cells from dedifferentiation. Although proteins that function to keep differentiation have been well studied in other cell types, few have been implicated in post-mitotic neuronal maintenance. In the central brain, loss of Midlife crisis (Mdlc), a CCCH zinc-finger protein, results in a decrease in Pros, thus derepressing NB genes in neurons. However, it is insufficient to make neurons revert to proliferating NBs. Furthermore, as Pros is not expressed in medulla neurons, it is unclear whether Mdlc has the same function in the optic lobe. On the other hand, absence of Lola leads to neuron-to-NB reversion and tumorigenesis , but it is crucial for neuronal maintenance only in the optic lobe. Recently, a paper reported that Nerfin-1 loss induces neuron dedifferentiation in both central brain and VNC (Froldi, 2015). This paper demonstrates a conserved function for Nerfin-1 in medulla neurons in the optic lobe. These findings indicate that Nerfin-1 is expressed mainly in early-stage medulla neurons and functions to maintain their differentiated state (Xu, 2017).

Interestingly, it was noticed that ectopic NB induced by Nerfin-1 depletion in the optic lobe appeared much earlier than that in the central brain. Considering that Lola loss causes dedifferentiation just in the optic lobe, it is speculated that the differentiated state of medulla neurons is less stable, possibly owing to absence of Pros. Furthermore, different from the mechanism in the central brain, the function of Nerfin-1 in the optic lobe requires the silencing of Notch signaling. Neither Myc knockdown nor Tor-DN misexpression inhibits dedifferentiation caused by Nerfin-1 loss in the medulla neurons. Thus, these findings identify a distinct regulatory mechanism in medulla neurons and validate different regulatory modes between the optic lobe and the rest of the CNS (Xu, 2017).

On the other hand, cell cycle genes play important roles in cell differentiation. Among them, Cyclin E (CycE) is reported to be regulated directly by Lola-N and is involved in the neuron dedifferentiation caused by loss of Mdlc. Thus, this study also examined whether CycE is regulated directly by Nerfin-1 and controls cell differentiation independently of Notch and neuroblast genes. Interestingly, CycE expression levels were upregulated dramatically in nerfin-1159 clones, but such upregulation was mostly blocked by Notch repression. These results suggest that CycE is not a direct target of Nerfin-1 for maintaining medulla neuron differentiation. CycE acts downstream of Notch signaling or it is subsequently upregulated after cell type change (Xu, 2017).

As Notch signaling is hyper-activated in nerfin-1 mutant clones, it was of interest to discover how it is regulated. One possibility is that Notch signaling becomes constitutively activated without the inhibition by Nerfin-1. To investigate this, Delta was knocked down upon Nerfin-1 loss and it was found that dedifferentiation was suppressed. These results indicate that Notch signaling is not constitutively activated and that it needs a ligand. Furthermore, Notch signal is both produced and received by medulla neurons. At the same time, the results show that Nerfin-1 loss induces dramatic upregulation of the expression level of Notch receptor. Thus, it is hypothesized that Nerfin-1 suppresses the expression of the Notch receptor in normal medulla neurons and inhibits Notch pathway activity. When Nerfin-1 is absent, expression levels of the Notch receptor increase strikingly. The receptors then bind to Delta from the adjacent cells and activate Notch signaling in its own. However, it is still unclear whether Notch receptor is a direct target of Nerfin-1. Therefore, subsequent studies on Nerfin-1 may help to clarify the underlying mechanisms and provide better understanding about neuronal maintenance (Xu, 2017).

Temporal patterning of neuroblasts controls Notch-mediated cell survival through regulation of Hid or Reaper

Temporal patterning of neural progenitors is one of the core mechanisms generating neuronal diversity in the central nervous system. This study shows that, in the tips of the outer proliferation center (tOPC) of the developing Drosophila optic lobes, a unique temporal series of transcription factors not only governs the sequential production of distinct neuronal subtypes but also controls the mode of progenitor division, as well as the selective apoptosis of NotchOFF or NotchON neurons during binary cell fate decisions. Within a single lineage, intermediate precursors initially do not divide and generate only one neuron; subsequently, precursors divide, but their NotchON progeny systematically die through Reaper activity, whereas later, their NotchOFF progeny die through Hid activity. These mechanisms dictate how the tOPC produces neurons for three different optic ganglia. It is concluded that temporal patterning generates neuronal diversity by specifying both the identity and survival/death of each unique neuronal subtype (Bertet, 2014).

Although apoptosis is a common feature of neurogenesis in both vertebrates and Drosophila, the mechanisms controlling this process are still poorly understood. For instance, several studies in Drosophila have shown that, depending on the context, Notch can either induce neurons to die or allow them to survive during binary cell fate decisions. This is the case in the antennal lobes where Notch induces apoptosis in the antero-dorsal projecting neurons lineage (adpn), whereas it promotes survival in the ventral projecting neurons lineage (vPN). In both of these cases, the entire lineage makes the same decision whether the NotchON or NotchOFF cells survive or die. This suggests that, in this system, Notch integrates spatial signals to specify neuronal survival or apoptosis (Bertet, 2014).

This study shows that, during tOPC neurogenesis, neuronal survival is determined by the interplay between Notch and temporal patterning of progenitors. Indeed, within the same lineage, Notch signaling leads to two different fates: it first induces neurons to die, whereas later, it allows them to survive. This switch is due to the sequential expression of three highly conserved transcription factors-Dll/Dlx, Ey/Pax-6, and Slp/Fkh-in neural progenitors. These three factors have distinct functions, with Dll promoting survival of NotchOFF neurons, Ey inducing apoptosis of NotchOFF neurons, and Slp promoting survival of NotchON neurons. These data suggest that Ey induces death of NotchOFF neurons by activating the proapoptotic factor hid. Thus, Dll probably antagonizes Ey activity by preventing Ey from activating hid. The data also suggest that Notch signaling induces neuronal death by activating the proapoptotic gene rpr. Thus, Slp might promote survival of NotchON neurons by directly repressing rpr expression or by preventing Notch from activating it. In both cases, the interplay between Notch and Slp modifies the default fate of NotchON neurons, allowing them to survive. Further investigations will test these hypotheses and determine how Dll, Ey, Slp, and Notch differentially activate/repress hid and rpr (Bertet, 2014).

Although the tOPC and the main OPC have related temporal sequences, their neurogenesis is very different. This difference is in part due to the fact that newly specified tOPC neuroblasts express Dll, which controls neuronal survival, instead of Hth. Why do tOPC neuroblasts express Dll? The tOPC, which is defined by Wg expression in the neuroepithelium, is flanked by a region expressing Dpp. Previous studies have shown that high levels of Wg and Dpp activate Dll expression in the distal cells of the Drosophila leg disc. Wg and Dpp could therefore also activate Dll in the neuroepithelium and at the beginning of the temporal series in tOPC progenitors. Another difference between the main OPC and tOPC neurogenesis is that Ey and Slp have completely different functions in these regions. Indeed, unlike in the main OPC, Ey and Slp control the survival of tOPC neurons. This suggests that autonomous and/or nonautonomous signals interact with these temporal factors and modify their function in the tOPC (Bertet, 2014).

Finally, tOPC neuroblasts produce neurons for three different neuropils of the adult visual system, the medulla, the lobula, and the lobula plate. This ability could be due to the particular location of this region in the larval optic lobes. Indeed, the tOPC is very close to the two larval structures giving rise to the lobula and lobula plate neuropils-Dll-expressing neuroblasts are located next to the lobula plug, whereas D-expressing neuroblasts are close to the IPC. Interestingly, Dll and D neuroblasts specifically produce lobula plate neurons. This raises the possibility that these neuroblasts and/or the neurons produced by these neuroblasts receive signals from the lobula plug and the IPC, which instruct them to specifically produce lobula plate neurons. These nonautonomous signals could also modify the function of Ey and Slp in the tOPC (Bertet, 2014).

In summary, this study demonstrates that temporal patterning of progenitors, a well-conserved mechanism from Drosophila to vertebrates, generates neural cell diversity by controlling multiple aspects of neurogenesis, including neuronal identity, Notch-mediated cell survival decisions, and the mode of intermediate precursor division. In the tOPC temporal series, some factors control two of these aspects (Ey), whereas others have a specialized function (Dll, Slp, and D). This suggests that temporal patterning does not consist of a unique series of transcription factors controlling all aspects of neurogenesis but instead consists of multiple superimposed series, each with distinct functions (Bertet, 2014).

Integration of temporal and spatial patterning generates neural diversity

In the Drosophila optic lobes, 800 retinotopically organized columns in the medulla act as functional units for processing visual information. The medulla contains over 80 types of neuron, which belong to two classes: uni-columnar neurons have a stoichiometry of one per column, while multi-columnar neurons contact multiple columns. This study shows that combinatorial inputs from temporal and spatial axes generate this neuronal diversity: all neuroblasts switch fates over time to produce different neurons; the neuroepithelium that generates neuroblasts is also subdivided into six compartments by the expression of specific factors (see The OPC neuroepithelium is patterned along its dorsal-ventral axis). Uni-columnar neurons are produced in all spatial compartments independently of spatial input; they innervate the neuropil where they are generated. Multi-columnar neurons are generated in smaller numbers in restricted compartments and require spatial input; the majority of their cell bodies subsequently move to cover the entire medulla. The selective integration of spatial inputs by a fixed temporal neuroblast cascade thus acts as a powerful mechanism for generating neural diversity, regulating stoichiometry and the formation of retinotopy (Erclik, 2017).

The optic lobes, composed of the lamina, medulla and the lobula complex, are the visual processing centres of the Drosophila brain. The lamina and medulla receive input from photoreceptors in the compound eye, process information and relay it to the lobula complex and central brain. The medulla, composed of ~40,000 cells, is the largest compartment in the optic lobe and is responsible for processing both motion and colour information. It receives direct synaptic input from the two colour-detecting photoreceptors, R7 and R8. It also receives input from five types of lamina neuron that are contacted directly or indirectly by the outer photoreceptors involved in motion detection (Erclik, 2017).

Associated with each of the ~800 sets of R7/R8 and lamina neuron projections are 800 medulla columns defined as fixed cassettes of cells that process information from one point in space. Columns represent the functional units in the medulla and propagate the retinotopic map established in the compound eye. Each column is contributed to by more than 80 neuronal types, which can be categorized into two broad classes. Uni-columnar neurons have arborizations principally limited to one medulla column and there are thus 800 cells of each uni-columnar type. Multi-columnar neurons possess wider arborizations, spreading over multiple columns. They compare information covering larger receptor fields. Although they are fewer in number, their arborizations cover the entire visual field (Erclik, 2017).

The medulla develops from a crescent-shaped neuroepithelium, the outer proliferation centre (OPC). During the third larval instar, the OPC neuroepithelium is converted into lamina on its lateral side and into medulla neuroblasts on its medial side. A wave of neurogenesis moves through the neuroepithelial cells, transforming them into neuroblasts; the youngest neuroblasts are closest to the neuroepithelium while the oldest are adjacent to the central brain. Neuroblasts divide asymmetrically multiple times to regenerate themselves and produce a ganglion mother cell that divides once more to generate medulla neurons. Recent studies have shown that six transcription factors are expressed sequentially in neuroblasts as they age: neuroblasts first express Homothorax (Hth), then Klumpfuss (Klu), Eyeless (Ey), Sloppy-paired 1 (Slp1), Dichaete (D) and Tailless (Tll). This temporal series is reminiscent of the Hb --> Kr --> Pdm --> Cas --> Grh series observed in Drosophila ventral nerve cord neuroblasts that generates neuronal diversity in the embryo. Indeed, distinct neurons are generated by medulla neuroblasts in each temporal window. Further neuronal diversification occurs through Notch-based asymmetric division of ganglion mother cells. In total, over 20 neuronal types can theoretically be generated using combinations of temporal factors and Notch patterning mechanisms. However, little is known about how the OPC specifies the additional ~60 neuronal cell types that constitute the medulla (Erclik, 2017).

To understand the logic underlying medulla development, late larval brains were stained with 215 antibodies generated against transcription factors and 35 genes were identifiied that are expressed in subsets of medulla progenitors and neurons. The OPC neuroepithelial crescent can be subdivided along the dorsal-ventral axis by the mutually exclusive expression of three homeodomain-containing transcription factors: Vsx1 is expressed in the central OPC (cOPC), Rx in the dorsal and ventral posterior arms of the crescent (pOPC), and Optix in the two intervening 'main arms' (mOPC). These three proteins are regionally expressed as early as the embryonic optic anlage and together mark the entire OPC neuroepithelium with sharp, non-overlapping boundaries. Indeed, these three regions grow as classic compartments: lineage trace experiments show that cells permanently marked in the early larva in one OPC region do not intermingle at later stages with cells from adjacent compartments. Of note, Vsx1 is expressed in cOPC progenitor cells and is maintained in a subset of their neuronal progeny whereas Optix and Rx are not expressed in post-mitotic medulla neurons. The OPC can be further subdivided into dorsal (D) and ventral (V) halves: a lineage trace with hedgehog-Gal4 (hh-Gal4) marks only the ventral half of the OPC, bisecting the cOPC compartment. As hedgehog is not expressed in the larval OPC, this dorsal-ventral boundary is set up in the embryo. Thus, six compartments (ventral cOPC, mOPC and pOPC and their dorsal counterparts) exist in the OPC. The pOPC compartment can be further subdivided by the expression of the wingless and dpp signalling genes. Cells in the wingless domain behave in a very distinct manner from the rest of the OPC, and have been described elsewhere (Erclik, 2017).

The Hth --> Klu --> Ey --> Slp1 --> D --> Tll temporal progression is not affected by the compartmentalization of the OPC epithelium; the same neuroblast progression throughout the entire OPC. Thus, in the developing medulla, neuroblasts expressing the same temporal factors are generated by developmentally distinct epithelial compartments (Erclik, 2017).

To test whether the intersection of the dorsal-ventral and temporal neuroblast axes leads to the production of distinct neural cell types, focus was placed on the progeny of Hth neuroblasts, which maintain Hth expression. In the late third instar, Hth neurons are found in a crescent that mirrors the OPC (see Distinct neuronal cell types are generated along the dorsal-ventral axis of the OPC). The NotchON (NON) progeny of Hth+ ganglion mother cells express Bsh and Ap, and they are distributed throughout the entire medulla crescent. In contrast, the NotchOFF (NOFF) progeny, which are BshHth+ neurons, express different combinations of transcription factors, and can be subdivided into three domains along the dorsal-ventral axis: (1) in the cOPC, NOFFHth+ neurons express Vsx1, Seven-Up (Svp) and Lim3; (2) in the pOPC, these neurons also express Svp and Lim3, but not Vsx1; (3) in the ventral pOPC exclusively, these neurons additionally express Teashirt (Tsh). NOFFHth+ cells are not observed in the mOPC. Rather, Cleaved-Caspase-3+ cells are intermingled with Bsh+ neurons. When cell death is prevented, Bsh+Hth+ cells become intermingled with neurons that express the NOFF marker Lim3, confirming that the NOFFHth+ progeny undergo apoptosis in the mOPC (Erclik, 2017).

It was therefore possible to distinguish three regional populations of Hth neurons (plus one that is eliminated by apoptosis) and a fourth population that is generated throughout the OPC. The neuronal identity of each of these populations was identified, as follows. (1) Bsh is a specific marker of Mi1 uni-columnar interneurons that are generated in all regions of the OPC. (2) To determine the identity of Hth+NOFF cOPC-derived neurons, Hth+ single cell flip-out clones were generated (using hth-Gal4) in the adult medulla. The only Hth+ neurons that are also Vsx1+Svp+ are Pm3 multi-columnar local neurons. (3) For Hth+NOFF pOPC-derived neurons, 27b-Gal4 was used; it drives expression in larval pOPC Hth+NOFF neurons and is maintained to adulthood. Flip-out clones with 27b-Gal4 mark Pm1 and Pm2 neurons, as well as Hth- Tm1 uni-columnar neurons that come from a different temporal window. Both Pm1 and Pm2 neurons (but not Tm1) express Hth and Svp. Pm1 neurons also express Tsh, which only labels larval ventral pOPC neurons (Erclik, 2017).

Thus, in addition to uni-columnar Mi1 neurons generated throughout the OPC, Hth neuroblasts generate three region-specific neuronal types: multi-columnar Pm3 neurons in the cOPC; multi-columnar Pm1 neurons in the ventral pOPC; and multi-columnar Pm2 neurons in the dorsal pOPC (Erclik, 2017).

To determine the contribution of the temporal and spatial factors to the generation of the different neuronal fates, the factors were mutated them and whether neuronal identity was lost was examined. To test the temporal axis, hth was mutated. As previously reported, Bsh expression is lost in hth mutant clones. Loss of hth in clones also leads to the loss of the Pm3 marker Svp without affecting expression of Vsx1, indicating that Vsx1 is not sufficient to activate Svp and can only do so in the context of an Hth+ neuroblast. Hth is also required for the specification of Pm1 and Pm2 in the pOPC as Svp and Tsh expression is lost in hth mutant larval clones. Ectopic expression of Hth in older neuroblasts is not able to expand Pm1, 2 or 3 fates (on the basis of the expression of Svp) into later born neurons, although it is able to expand Bsh expression. Thus, temporal input is necessary for the specification of all Hth+ neuronal fates but only sufficient for the generation of Mi1 neurons (see Temporal and spatial inputs are required for neuronal specification in the medulla. ) (Erclik, 2017).

Next, whether regional inputs are necessary and/or sufficient to specify neuronal fates in the progeny of Hth+ neuroblasts was determined. In Vsx1 RNA interference (RNAi) clones, Svp expression is lost in the cOPC but Bsh is unaffected. Additionally, Hth+Lim3+ cells are absent, suggesting that NOFF cells undergo apoptosis in these clones. Conversely, ectopic expression of Vsx1 leads to the expression of Svp in mOPC Hth+ neurons but does not affect Bsh expression. Therefore, Vsx1 is both necessary and sufficient for the specification of Pm3 fates in the larva. However, unlike the temporal factor Hth, Vsx1 does not affect the generation of Mi1 neurons (Erclik, 2017).

In Rx whole mutant larvae and in mutant clones, Svp+Lim3+Hth+ larval neurons (that is, Pm1 and Pm2 neurons) in the pOPC are lost. Additionally, the Pm1 marker Tsh is lost in ventral pOPC Hth+ cells. Consistent with the Vsx1 mutant data, larval Bsh expression is not affected by the loss of Rx. In adults, the Pm1/Pm2 markers (Svp, Tsh and 27b-Gal4) are lost in the medulla (Erclik, 2017).

Ectopic expression of Rx leads to the activation of Svp in mOPC Hth+ neurons, but does not affect the expression of Bsh. It also leads to the activation of Tsh, but only in the ventral half of mOPC Hth+ neurons, suggesting that a ventral factor acts together with Rx to specify ventral fates. Taken together, the above data show that Rx is both necessary and sufficient for the specification of Pm1/2 neurons but (like Vsx1) does not affect the generation of Mi1 neurons (Erclik, 2017).

Finally, the role of the mOPC marker Optix in neuronal specification was examined. In Optix mutant clones, Svp is ectopically expressed in the mOPC, but Bsh expression is not affected. Of note, these ectopic Svp+ neurons fail to express the region-specific Pm markers Vsx1 or Tsh (in ventral clones), which suggests that they assume a generic Pm fate. Conversely, ectopic expression of Optix leads to the loss of Svp expressing neurons in both the cOPC and pOPC but does not affect Bsh. These NOFF neurons die by apoptosis as no Lim3+ neurons are found intermingled with Bsh+Hth+NON neurons. When apoptosis is prevented in mOPC-derived neurons, Svp is not derepressed in the persisting Hth+NOFF neurons, which suggests that Optix both represses Svp expression and promotes cell death in Hth+NOFF neurons (Erclik, 2017).

The above data demonstrate that input from both the temporal and regional axes is required to specify neuronal fates. The temporal factor Hth is required for both Mi1 and Pm1/2/3 specification. The spatial genes are not required for the specification of NON Mi1 neurons, consistent with the observation that Mi1 is generated in all OPC compartments. The spatial genes, however, are both necessary and sufficient for the activation (Vsx1 and Rx) or repression (Optix) of the NOFF Pm1/2/3 neurons. Thus, Hth+ neuroblasts generate two types of progeny: NOFF neurons that are sensitive to spatial input (Pm1/2/3) and NON neurons that are refractory to spatial input (Mi1). Vsx1 expression in the cOPC is only maintained in Hth+NOFF neurons, suggesting that spatial information may be 'erased' in Mi1, thus allowing the same neural type to be produced throughout the OPC (Erclik, 2017).

Do spatial genes regulate each other in the neuroepithelium? In Vsx1 mutant clones, Optix (but not Rx) is derepressed in the cOPC epithelium. Conversely, ectopic Vsx1 is sufficient to repress Optix in the mOPC and Rx in the pOPC. Similarly, Optix, but not Vsx1, is derepressed in Rx mutant clones in the pOPC epithelium and ectopic Rx is sufficient to repress Optix in the mOPC (but not Vsx1 in the cOPC). In Optix mutant clones, neither Vsx1 nor Rx are derepressed in the mOPC epithelium, but ectopic Optix is sufficient to repress both Vsx1 in the cOPC and Rx in the pOPC. The observation that Optix is not necessary to suppress Vsx1 or Rx in the mOPC neuroepithelium is surprising because Svp is activated in a subset of Hth+ neurons in the mOPC in Optix mutant clones. Nevertheless, when cell death in the mOPC is abolished, the ectopic undead NOFF neurons express Lim3 but not Svp, which confirms that Optix represses Svp expression in mOPC neurons. Taken together, these results support a model in which Optix is sufficient to repress Vsx1 and Rx, to promote the death of Hth+NOFF neurons and to repress Pm1/2/3 fates (see Spatial genes cross-regulate each other in the OPC neuroepithelium). Vsx1 and Rx act to promote Pm3 (Vsx1) or Pm1/2 (Rx) fates but can only do so in the absence of Optix (Erclik, 2017).

These results suggest that multi-columnar neurons are generated at specific locations in the medulla crescent. However, since these neurons are required to process visual information from the entire retina in the adult medulla, how does the doral-ventral position of neuronal birth in the larval crescent correlate with their final position in the adult? Lineage-tracing experiments were performed with Vsx1-Gal4 to permanently mark neurons generated in the cOPC and with Optix-Gal4 for mOPC neurons, and the position of the cell bodies of these neurons was analyzed. In larvae, neurons from the cOPC or from the mOPC remain located in the same dorsal-ventral position where they were born. However, in adults, both populations have moved to populate the entire medulla cortex along the dorsal-ventral axis (see Neuronal movement during medulla development is restricted to multi-columnar cell types). The kinetics of cell movement during development was analyzed by following cOPC neurons. Neurons born in the cOPC remain tightly clustered until 20 h after puparium formation (P20), after which point the cell bodies spread throughout the medulla cortex. By P30 the neurons are distributed over the entire dorsal-ventral axis of the medulla cortex. In the adult, most neurons derived from the cOPC neuroepithelium are located throughout the cortex although there is an enrichment of neurons in the central region of the cortex (Erclik, 2017).

To determine whether these observed movements involve the entire neuron or just the cell body, the initial targeting of cOPC or mOPC-derived neurons in larvae was examined before the onset of cell movement. In larvae, both populations send processes that target the entire dorsal-ventral axis of the medulla neuropi. Therefore, medulla neurons first send projections to reach their target columns throughout the entire medulla. Later, remodelling of the medulla results in extensive movement of cell bodies along the dorsal-ventral axis, leading to their even distribution in the cortex (Erclik, 2017).

What is the underlying logic behind why some neurons move while others do not? Markers were studied for the Mi1 (Bsh), Pm2 (Hth+Svp+), Pm1 (Hth+Svp+Tsh+), and Pm3 (Vsx1+Svp+Hth+) populations of neurons through pupal stages and up to the adult. Mi1 neurons are generated evenly throughout the larval OPC and remain regularly distributed across the dorsal-ventral axis at all stages. The lineage-tracing experiment was repeated with Vsx1-Gal4 to follow Mi1 neurons produced by the cOPC. These Mi1 neurons remain exclusively in the centre of the adult medulla cortex, demonstrating that they do not move. In contrast, Pm3 neurons remain tightly clustered in the central region until P20, at which point they move to occupy the entire cortex (Erclik, 2017).

However, not all multi-columnar neurons have cell bodies that move to occupy the entire medulla cortex. Unlike Mi1 and Pm3, adult Pm1 and Pm2 cell bodies are not located in the adult medulla cortex but instead in the medulla rim, at the edges of the cortex. Pm1 and Pm2 markers remain clustered at the ventral (Pm1) or dorsal (Pm2) posterior edges of the medulla cortex throughout all pupal stages. In the adult, both populations occupy the medulla rim from where they send long horizontal projections that reach the entire dorsal-ventral axis of the medulla neuropil. The pOPC may be a specialized region where many of the medulla rim cell types are generated. Even though most of cOPC-derived neurons move during development, a cOPC-derived multi-columnar neuron (TmY14) was identified that sends processes targeting the entire dorsal-ventral length of the medulla neuropil but whose cell bodies remain in the central medulla cortex in the adult (Erclik, 2017).

Thus, the four populations of Hth neurons follow different kinetics: Mi1 neurons are born throughout the OPC and do not move; Pm3 neurons are born centrally and then move to distribute throughout the entire cortex; and Pm1/Pm2 neurons are born at the ventral or dorsal posterior edges of the OPC and occupy the medulla rim in adults (Erclik, 2017).

It is noted that uni-columnar Mi1 neurons, whose cell bodies do not move, reside in the distal cortex whereas multi-columnar Pm3 neurons, which move, reside in the proximal cortex. The hypothesis was thus tested that neurons whose cell bodies are located distally in the medulla cortex represent uni-columnar neurons generated homogeneously throughout the OPC that do not move. In contrast, proximal neurons, which are fewer in number and are generated in specific subregions of the medulla OPC, would be multi-columnar and move to their final position (Erclik, 2017).

It was first confirmed that neurons that move have their cell bodies predominantly in the proximal medulla cortex. The cell body position of neurons born ventrally that have moved dorsally was analyzed using the hh-Gal4 lineage trace: in the adult, the cell bodies found dorsally are mostly in the proximal medulla cortex, whereas the cell bodies in the ventral region are evenly distributed throughout the distal-proximal axis of the ventral cortex. They probably represent both distal uni-columnar neurons that did not move as well as proximal multi-columnar neurons that remained in the ventral region (Erclik, 2017).

Next the pattern of movement of Tm2 uni-columnar neurons from the ventral and dorsal halves of the OPC was analyzed using the hh-based lineage-trace. The cell bodies of Tm2 neurons are located throughout the dorsal-ventral axis in the adult medulla cortex but are co-labelled with the hh lineage marker only in the ventral half. Thus, like Mi1, Tm2 uni-columnar neurons do not move. Furthermore, uni-columnar Tm1 neurons, labelled by 27b-Gal4, are born throughout the dorsal-ventral axis of the OPC crescent with distal cell bodies in the adult, suggesting that they also remain where they were born (Erclik, 2017).

Conversely, it was asked whether neurons that are specified in only one region, such as the Vsx+ neurons of the cOPC, are multi-columnar in morphology. By sparsely labelling cOPC-derived neurons using the Vsx1-Gal4 driver, 13 distinct cell types were characterized that retain Vsx1 expression in the adult medulla. Strikingly, all are multi-columnar in morphology, further supporting the model that it is the multi-columnar neurons that move during pupal development (Erclik, 2017).

Finally, MARCM clones were generated in the OPC neuroepithelium and visualized using cell-type-specific Gal4 drivers in the adult medulla. Two classes of adult clone distribution were observed: clones in which neurons are tightly clustered, and clones in which neurons are dispersed. Consistent with the model, the clustered clones are those labelled with uni-columnar neuronal drivers, whereas the dispersed clones are those labelled with a multi-columnar driver (Erclik, 2017).

Taken together, these data demonstrate that neurons that do not move are uni-columnar (with cell bodies in the distal cortex), whereas most multi-columnar neurons (with cell bodies in the proximal cortex) move (Erclik, 2017).

This study shows that combinatorial inputs from the temporal and spatial axes act together to promote neural diversity in the medulla. Previous work has shown that a temporal series of transcription factors expressed in medulla neuroblasts allows for a diversification of the cell types generated by the neuroblasts as they age. This study now shows that input from the dorsal-ventral axis leads to further diversification of the neurons made by neuroblasts; at a given temporal stage, neuroblasts produce the same uni-columnar neuronal type globally as well as smaller numbers of multi-columnar cell types regionally. This situation is reminiscent of the mode of neurogenesis in the Drosophila ventral nerve cord, in which each neuroblast also expresses a (different) temporal series of transcription factors that specifies multiple neuronal types in the lineage. Spatial cues from segment polarity, dorsal-ventral and Hox genes then intersect to impart unique identities to each of the lineages. However, neuroblasts from the different segments give rise to distinct lineages to accommodate the specific function of each segment. In contrast, in the medulla, the entire OPC contributes to framing the repeating units that form the retinotopic map. It is therefore likely that each neuroblast produces a common set of neurons that connect to each pair of incoming R7 and R8 cells, or L1-L5 lamina neurons. This serves to produce 800 medulla columns with a 1:1 stoichiometry of medulla neurons to photoreceptors. The medulla neurons that are produced by neuroblasts throughout the dorsal-ventral axis of the OPC are thus uni-columnar The production of the same neuronal type along the entire OPC could be achieved by selectively 'erasing' spatial information in uni-columnar neurons, as observed in Mi1 neurons (Erclik, 2017).

Regional differences in the OPC confer further spatial identities to neuroblasts with the same temporal identity, and lead to specific differences in the lineages produced in the compartments along the dorsal-ventral axis of the medulla. These differences produce smaller numbers of multi-columnar neurons whose stoichiometry is much lower than 1:1. The majority of these neurons move during development to be uniformly distributed in the adult medulla cortex. This combination of regional and global neuronal specification in the medulla presents a powerful mechanism to produce the proper diversity and stoichiometry of neuronal types and generate the retinotopic map (Erclik, 2017).

The super elongation complex drives neural stem cell fate commitment

Asymmetric stem cell division establishes an initial difference between a stem cell and its differentiating sibling, critical for maintaining homeostasis and preventing carcinogenesis. Yet the mechanisms that consolidate and lock in such initial fate bias remain obscure. This study used Drosophila neuroblasts to demonstrate that the super elongation complex (SEC) acts as an intrinsic amplifier to drive cell fate commitment. SEC is highly expressed in neuroblasts, where it promotes self-renewal by physically associating with Notch transcription activation complex and enhancing HES (hairy and E(spl)) transcription. HES in turn upregulates SEC activity, forming an unexpected self-reinforcing feedback loop with SEC. SEC inactivation leads to neuroblast loss, whereas its forced activation results in neural progenitor dedifferentiation and tumorigenesis. These studies unveil an SEC-mediated intracellular amplifier mechanism in ensuring robustness and precision in stem cell fate commitment and provide mechanistic explanation for the highly frequent association of SEC overactivation with human cancers (Liu, 2017).

Both normal development and tissue homeostasis rely on the remarkable capacity of stem cells to divide asymmetrically, simultaneously generating one identical stem cell and one differentiating progeny. Extensive studies have unveiled how extrinsic niche signals and intrinsic cell polarity cues ensure proper orientation of mitotic spindle and, hence, asymmetric division of stem cells. However, it remains unclear whether the initial fate bias, established by unequal exposure to niche signals or differential partitioning of cell fate determinants, can be immediately and automatically consolidated and stabilized into distinct and irreversible cell fate outcomes. In fact, in vivo timelapse imaging of the developing zebrafish hindbrain using the Notch activity reporter showed that, immediately after the asymmetric division of a radial glia progenitor, Notch activity is not noticeably biased in the paired daughter cells. Instead, the differential Notch activity in the pair of daughter cells only gradually increases afterward, over a time span of 3-8 hr, indicating the existence of a progressive and tightly regulated transition phase between the initial cell fate decision and the ultimate cell fate commitment. Stem cells and progenitors, especially the fast-cycling ones, face the daunting challenges of ensuring timely, precise, and robust cell fate determination in every cell cycle and are likely to achieve so through rapid amplification of the initial small fate bias upon their asymmetric division. In electronics, a device called an amplifier magnifies a small input signal to a large output signal until it reaches a desired level. Conceivably, a similar 'amplifier' mechanism could be employed in the stem cells or progenitors to accelerate the transition phase and drive cell fate commitment. Dysregulation of such an amplifier could cause an imbalance between self-renewal and differentiation, resulting in impaired tissue homeostasis. However, the regulatory modules governing the transition phase from stem cell fate decision to fate commitment, especially the identity and control of this putative 'amplifier,' remain largely unexplored (Liu, 2017).

Drosophila type II neural stem cells (NSCs), known as neuroblasts (NBs), provide an excellent model system for studying stem cell fate commitment. Firstly, distinct from type I NB lineages, type II NB lineages contain transit-amplifying cells called intermediate neural progenitors (INPs), similar to mammalian NSC lineages in both functional and molecular criteria, yet with much simpler anatomy and lineage composition. Each type II NB undergoes stereotypic, self-renewing divisions to produce immature INPs, which, upon maturation, undergo a few rounds of asymmetric, self-renewing divisions to give rise to ganglion mother cells (GMCs) that subsequently generate post-mitotic neurons or glia. Secondly, the identity of each cell type in the NB lineages can be unambiguously determined by a combination of cell fate makers as well as by their geological positions within the lineages. Thirdly, the molecular mechanisms underlying initial NB versus INP fate decision are well understood. Unidirectional Notch signaling is both necessary and sufficient to promote type II NB self-renewal. At each division, type II NBs asymmetrically segregate differentiation-promoting determinants, such as Notch antagonist Numb, into immature INPs. As a consequence, Notch pathway effector HES (hairy and E(spl)) genes, such as E(spl)mγ, are highly expressed in NBs but not in immature INPs. HES genes, encoding basic helix-loop-helix (bHLH) transcription factors, are crucial for promoting NB self-renewal. Importantly, numb mutant immature INPs fail to complete maturation but instead revert fate back into NBs and result in tumorigenesis, indicating that the asymmetric segregation of Numb protein is critical for establishing the initial fate bias between a type II NB and its sibling INP. However, whether such initial bias is sufficient to confer differential Notch activity and achieve definitive fate commitment is currently unclear. Lastly, type II NBs undergo fast cell divisions, dividing every 2 hr, placing them under huge pressure to timely yet precisely achieve differential fate outcomes. Therefore, within type II NB lineages, a regulatory module that drives robust cell fate commitment is likely to exist, plausibly with high activity (Liu, 2017).

Overactivation of Notch signaling leads to immature INP dedifferentiation and tumorigenesis, providing a sensitized background for identifying factors pivotal for NB or INP fate commitment. In such a genetic background a genome-wide RNAi-based screen was carried out for genes whose downregulation specifically suppresses the supernumerary NB phenotype induced by Notch overactivation, and subunits of the super elongation complex (SEC) were identified. The SEC is composed of the elongation factor ELL (eleven-nineteen lysine-rich leukemia) 1/2/3, the flexible scaffolding protein AFF (AF4/FMR2 family) 1/2/3/4, the ELL-associated factor EAF1/2, eleven-nineteen leukemia (ENL)/AF9, as well as the Pol II elongation factor P-TEFb consisting of cyclin T (CycT) and cyclin-dependent kinase 9 (CDK9). The screen identified all subunits of SEC except EAF and ENL/AF9, suggesting that SEC interplays with Notch signaling in promoting NB self renewal. The SEC subunits were originally identified as frequent translocation partners of MLL (mixed-lineage leukemia) in inducing leukemogenesis, and play key roles in c-Myc-dependent carcinogenesis and HIV viral DNA transcription. Previous studies demonstrated that SEC executes its functions by inducing rapid gene transcription, mainly through phosphorylating RNA polymerase II (Pol II) C-terminal domain and releasing it from promoter-proximal pausing (Liu, 2017).

This study shows that the SEC is specifically expressed in Drosophila NBs, where it acts as an amplifier to drive type II NB fate commitment. SEC exerts its function by physically associating with Notch transcription activation complex to stimulate dHES (Drosophila HES) transcription. dHES in turn promotes SEC expression/activity. Thus, driven by a self-reinforcing feedback loop between SEC and Notch signaling, an initial small bias of Notch activity between an NB and its sibling INP is rapidly amplified and consolidated into robust and irreversible fate commitment (Liu, 2017).

Is the establishment of an initial fate bias at the end of stem cell asymmetric division truly the end, or just the beginning of the end? The current findings revealed that a progressive and tightly controlled transition phase exists between the initial fate decision and the final definitive fate commitment. The results identified the evolutionarily conserved SEC as a crucial intrinsic amplifier, accelerating this previously overlooked fate transition phase and ensuring NSC fate commitment in Drosophila type II NB lineages. Inactivation of SEC prevents the self-reinforcing feedback loop between SEC and Notch signaling from running, resulting in NBs with ambiguous stem cell identity and ultimate fate loss. Conversely, ectopic overactivation of SEC initiates and sustains this positive feedback loop within progenitors, driving dedifferentiation and tumorigenesis. It is interesting to note that, as one of the most active P-TEFb-containing complexes in controlling rapid transcriptional induction in response to dynamic developmental or environmental cues, SEC is particularly suitable for being an amplifier in driving timely cell fate commitment. Since fast-cycling stem cells are under huge pressure to achieve robust fate determination in every cell cycle, it is not surprising that they employ SEC as a regulatory component to induce immediate activation of master fate-specifying genes that in turn form a self-amplifying loop with SEC to rapidly magnify the initial fate bias and ensure prompt fate commitment (Liu, 2017).

Such an intracellular amplifier mechanism revealed by these studies might complement the well-established intercellular lateral inhibition mechanism and represent a general, cell-autonomous paradigm to ensure robustness and precision in binary cell fate commitment. Lateral inhibition is a widely used mechanism underlying cell fate diversification, whereby unidirectional Notch signaling utilizes intercellular feedback loops to amplify an initial small difference between adjacent daughter cells, and eventually confers distinct cell fates. Lateral inhibition relies on intercellular interactions between adjacent cells. This study proposes a model whereby an intracellular amplifier mechanism may also diversify cell fates (Liu, 2017).

The intracellular amplifier and intercellular lateral inhibition mechanisms, both acting through feedback loops, are not mutually exclusive. Instead, they are complementary to each other and can be used concomitantly or sequentially to achieve differential fate outcomes in a timely, precise, and robust manner. An amplifier design often employs negative feedback to prevent excessive amplification. In this study the dHES-Earmuff/Brahma-SEC double-negative regulatory mechanism that this study has revealed in NBs might also operate in neural progenitors, where the Erm/Brm complex could serve as a crucial 'brake' to prevent the Notch-SEC-Notch self-reinforcing positive feedback loop from starting (Liu, 2017).

Notch signaling plays a conserved role during vertebrate embryonic neurogenesis in maintaining the undifferentiated status of NSCs. Intriguingly, expression of HES-1, a primary target of Notch pathway in mammalian neural development, oscillates every 2 hr. It has been proposed that oscillations in HES-1 expression drive fluctuations in gene expression, resulting in differential expressions between neighboring cells, which needs be further amplified to confer distinct cell fates. How such an amplification step is triggered and modulated remains elusive. Given that SEC is highly conserved in mammals, it is interesting to speculate that a similar amplifier mechanism is employed to ensure mammalian NSC fate commitment. Whether SEC interplays with Notch signaling to drive cell fate commitment in other stem cell lineages also warrants future investigation (Liu, 2017).

Despite extensive studies elucidating how SEC regulates transcription elongation, the in vivo function of SEC in normal development and physiology remains enigmatic. The current results indicate that SEC is highly expressed in Drosophila NSCs, where it is recruited by the Notch transcription activation complex to stimulate the transcription of dHES genes and promote self-renewing fate. Interestingly, the dHES genes in fly larval brain NB lineages are non-pausing genes, raising the possibility that SEC promotes the transcriptional activation of dHES in the absence of paused Pol II. Consistent with this view, recent studies have demonstrated that the rapid transcriptional induction of some nonpausing genes, such as Cyp26a1 in human embryonic stem cells and a subset of pre-cellular genes in early Drosophila embryos, depends on SEC activity and Pol II occupancy. The current findings that SEC physically and genetically interplays with the dCSL-NICD-MAM transcription activation complex to activate dHES transcription thus provide a unique physiological context for elucidating the detailed molecular mechanisms underlying transcriptional induction of non-pausing genes by SEC (Liu, 2017).

The upstream signals and molecular mechanisms controlling SEC activity in normal development or physiology are just unfolding. It has been previously shown that the activity of SEC could be regulated by modulating the kinase activity of CDK9, the catalytic subunit of SEC. The results unveil a new and unexpected mechanism underlying the control of SEC: the Notch-HES axis spatially restricts SEC activity within NSCs by cell-autonomously promoting the protein abundance of dAFF and dELL, two regulatory subunits of SEC. Consistently, overactivation of Notch signaling led to dedifferentiation of immature INPs, in which the expression levels of dAFF/dELL and, hence, the activity of SEC evidently increase (Liu, 2017).

Dysregulation of the SEC subunits is frequently associated with various human cancers including leukemia and glioblastoma. However, in most cases, whether SEC acts as a cancer driver or passenger is unclear. Furthermore, whether SEC subunits exert their oncogenic or tumor suppressive roles as a component of SEC or independent of SEC remains poorly understood. Intriguingly, the results show that overexpression of dELL and dAFF but not either alone induces a dramatic surge of dHES expression in immature INPs and causes progenitorderived tumor. These findings strongly suggest that, in NSC lineages, SEC drives tumorigenesis as an integral complex and exerts its oncogenic function in a dose-dependent manner. Supporting this view, the kinase activity of CDK9 is essential for dELL/dAFF-induced tumorigenesis. It will be interesting to investigate whether upregulation of dELL/dAFF abundance is sufficient to induce carcinogenesis in other biological contexts. Tne findings highlighting the self-reinforcing feedback loop between SEC and Notch signaling in driving tumorigenesis further suggest that CDK9 inhibitors could be pursued as an effective therapy for Notch overactivation-induced tumors (Liu, 2017).

Notch pathway and heart development: Kuzbanian is crucial for proper heart formation in Drosophila

A collection of EMS mutagenized fly lines was screened in order to identify genes involved in cardiogenesis. The present work studied a group of alleles exhibiting a hypertrophic heart. The analysis revealed that the ADAM protein (A Disintegrin And Metalloprotease) Kuzbanian, which is the functional homologue of the vertebrate ADAM10, is crucial for proper heart formation. ADAMs are a family of transmembrane proteins that play a critical role during the proteolytic conversion (shedding) of membrane bound proteins to soluble forms. Enzymes harboring a sheddase function recently became candidates for causing several congenital diseases, like distinct forms of the Alzheimer disease. ADAMs also play a pivotal role during heart formation and vascularisation in vertebrates, therefore mutations in ADAM genes potentially could cause congenital heart defects in humans. In Drosophila, the zygotic loss of an active form of the Kuzbanian protein results in a dramatic excess of cardiomyocytes, accompanied by a loss of pericardial cells. The data presented suggest that Kuzbanian acts during lateral inhibition within the cardiac primordium. Furthermore a second function of Kuzbanian in heart cell morphogenesis is discussed (Albrecht, 2006).

Studies on the function of ADAMs in vertebrates indicate that some of the members of the gene family are involved in cardiac development. Mice, lacking functional ADAM19, exhibit severe defects in cardiac morphogenesis, e.g. ventral septal defects, abnormal formation of the aortic and pulmonic valves and abnormalities of the cardiac vasculature. Recently, it was shown that TACE (ADAM17) is essential for cardiac valvulogenesis in mice, likely by its sheddase function on EGFR. Interestingly, mice lacking a functional TACE die at birth with an enlarged fetal heart with increased myocardial trabeculation and reduced cell compaction, mimicking the pathological changes of noncompaction of ventricular myocardium. In addition, larger cardiomyocyte cell size and increased cell proliferation were reported in ventricles of the TACE knockout mouse hearts. Cardiac restricted overexpression of a non-cleavable, transmembrane TNF/tumor necrosis factor, a major substrate of TACE, in mice provokes a hypertrophic cardiac phenotype (Albrecht, 2006).

Although it is clear that some mammalian ADAMs are crucial for heart development, a role for ADAM metalloproteases during Drosophila heart formation has not reported so far. This study provides multiple lines of evidence that the metalloprotease Kuzbanian/ADAM10 is crucial for cardiogenesis in flies. Screening a collection of mutagenized Drosophila lines for displaying heart phenotypes and the subsequent analysis of selected alleles revealed that mutations in the kuzbanian genes cause a hyperplasic heart (Albrecht, 2006).

Loss of Kuzbanian function and subsequently abrogation of Notch signalling explains the heart phenotype of kuzbanian mutant embryos. Notch activity was found between 6 and 10 h of development in the dorsal mesoderm, from which the cardioblasts, the pericardial cells, and the lymph glands arise. During this time, Notch-dependant lateral inhibition is responsible for selecting cardiac precursors within the dorsal mesoderm. Elimination of Notch during the first half of this period, using a temperature-sensitive Notch mutant, results in substantially more cardioblasts, pericardial cells and lymph gland progenitors. Reducing Notch function between 8 and 10 h causes an excess of cardioblasts and a concomitant loss of pericardial cells, reflecting a crucial function of Notch for cell fate specification. In embryos carrying a deficiency for Notch [Df(1)N81K1], the number of heart cells is strongly increased, pericardial cells are missing, and cardioblasts fail to assemble into a regular tube, reflecting a combined effect on cell number and cell fate. The role of Notch for a particular subset of pericardial cells, the EPCs, has been investigated. Mutants lacking the maternal and zygotic Notch revealed initially enlarged clusters of EPC progenitors. This finding is consistent with the current observation that panmesodermal driven KuzDN causes a loss of Even-skipped expressing pericardial cells at later stages, a phenotype, which was also found in embryos mutant for mastermind, a downstream effector of Notch signalling. Hypertrophy of cardioblasts was also reported using Nts-1/Df(1)N81K1 embryos, but in this case the number of pericardial cells (Zfh1-expressing cells) was reported as being normal. These studies have shown that Notch is required in the dorsal mesoderm (stage 11 to 12) to regulate the initial commitment of cells to the cardioblasts and pericardial cell fate, but also to regulate the choice between the cardioblast and pericardial fate. In addition, a biphasic recruitment of Notch signalling in heart development has been proposed. First, Notch is needed during lateral inhibition for selecting heart precursors in a field of equally competent cells within the dorsal mesoderm, which essentially covers the function of Notch described above. Second, Notch is involved in asymmetric cell division giving rise to specific heart progenitors. The second requirement of Notch is restricted to those heart cells that arise from asymmetric cell division, in conjunction with additional components of the asymmetric cell division machinery, including Sanpodo, Numb and Inscuteable (Albrecht, 2006).

Previous reports have shown that the Notch receptor is a main substrate of Kuzbanian. Proteolytic cleavage of the membrane anchored Notch receptor is interrupted in the absence of Kuzbanian, which finally results in inactivation of Notch signalling. Thus, the observed heart phenotype of the identified EMS-induced kuzbanian alleles is caused by interruption of Notch signalling in the cardiac primordium, respectively. Therefore, a similar phenotype of kuzbanian, mastermind and Notch mutant embryos is expected. Indeed, the five kuzbanian and the three mastermind EMS alleles identified in the current screen for heart mutants as well as the previously described kuzbanian alleles kuz3 and kuz29-4 exhibit cardiac malformations resembling particular aspects of cardiac defects described for Notch mutants. The phenotypes are not completely identical, which can be explained by the maternal product that masks most of the very early recruitments of Kuzbanian. Indeed, embryos lacking any maternally derived Kuzbanian product and contain no zygotic copies of kuzbanian have a early neurogenic phenotype, which is even more severe than strong Notch phenotypes. Therefore, it is assumed that the zygotic kuzbanian mutants reflect a later requirement of Kuzbanian, manifested in the cardiac mesoderm when the maternal product is diminished in this tissue. The cardiac mesoderm develops as cardioblasts to the expense of pericardial cells (and lymph gland cells) in kuzbanian mutant embryos. It is postulated that Kuzbanian plays a dual role during cardiogenesis, first for the selection of cardiac progenitors during Notch dependent lateral inhibition and second for cell fate specification during Notch dependent asymmetric cell division. This explains the strong excess of all subtypes of heart cells and the significant loss of pericardial cells. The excess of cardioblasts firstly arises, because in the absence of Notch dependent lateral inhibition too many progenitors are selected within the dorsal mesoderm. This phenotype is further enhanced in later development when remaining pericardial cells (those that arise from the asymmetric lineage) are transformed into cardioblasts as a consequence of an abrogated Notch dependent asymmetric cell division. This explains why the hyperplasia of the heart is more prominent in segments A2–A8 than in T3 to A1, because the anterior cardioblasts arise from symmetric cell divisions only. If conversion of the pericardial cell fate into the cardioblast cell fate takes place, it occurs in the posterior heart lineage, resulting in a stronger hyperplasia in this heart region as seen in kuzbanian mutant embryos (Albrecht, 2006).

A model predicts that Kuzbanian is essential for heart development by effecting Notch signalling, which is further supported by the fact, that another key component of Notch signalling was retrieved from the screen, based on a nearly identical phenotype. Three alleles of the mastermind gene, which encodes a downstream effector of the Notch signalling pathway, were found. Mastermind has been isolated in various screens as a modifier of Notch signalling and acts on the molecular level via a direct interaction to the ankyrin repeats of the intracellular form of Notch. As in kuzbanian, mastermind mutant alleles give rise to strong excess of cardioblasts and a reduced number of pericardial cells (Albrecht, 2006).

Interestingly, mutations in another component of Notch signalling pathway reveals a slightly different phenotype. liquid facets, which encodes a Drosophila Epsin orthologue, is reported to be responsible for endocytosis and trafficking of the Notch ligand Delta in the signalling cell. Liquid facets homozygous embryos reveal a hyperplasic heart, but a normal number of pericardial cells. The excess of cardioblasts in liquid facets arises due to a preferential expansion of the tinman expressing subtype of cardioblasts (which arise from symmetric cell division), whereas the number of seven-up expressing cardioblasts (which arise from asymmetric cell division) is normal. Moreover, the additional cardioblasts presumably develop at the expense of fusion competent myoblasts from the dorsal mesoderm. In contrast, kuzbanian mutant embryos show an increased number of seven-up expressing cardioblast, although the overall excess of cardioblasts is, similar to the findings in liquid facets mutants, preferentially due to the expansion of the tinman expressing cardioblasts. One explanation is that Kuzbanian is required for the selection of both cardioblast cell types, whereas Liquid facets is not. Additionally, the maternal contribution of Kuzbanian present in kuzbanian mutant embryos might be sufficient for the selection of the correct number of the Seven-up (Svp) progenitors, but not for the symmetrically derived Tinman cardioblast progenitors. Shortly later, absence of Kuzbanian then affects the Notch dependent asymmetric cell division in the Seven-up lineage, resulting in a moderately increased number of Seven-up cardioblasts. This hypothesis is confirmed by observation of a maximum of four Svp cells per hemisegment in the kuzbanian mutant background, fully explainable by an effect on asymmetric cell division in this cell lineage. It should also be mentioned that Liquid facets is reported to be critical for endocytosis and trafficking of Delta in the signalling cells, whereas Kuzbanian is crucial not only for proteolytic processing of Notch, but presumably also for processing Delta as well. As for kuzbanian, maternal contribution of liquid facets likely masks some requirements of this molecule and hampers a direct comparison of the zygotic phenotypes (Albrecht, 2006).

To date, morphogenesis of cardiac cells is not very well understood. After specification, cardioblasts align bilaterally along the a–p axis and migrate together with the overlaying ectoderm towards the dorsal midline. During this process, all cardioblasts become flattened, polarised cells. The heart lumen is formed when the trailing edges of the cardioblasts of either side bend around and contact each other at the dorsalmost position. Recently, a growing number of genes controlling morphogenesis of cardioblasts has been identified. Among these, cell adhesion molecules play a pivotal role during the alignment of cardioblasts (faint sausage), adhesion of opposing cardioblasts and lumen formation (E-cadherin), and maintenance of the normal heart morphology at late embryonic stages (laminin A). For the alignment and migration of cardioblasts towards the dorsal midline, the Toll receptor acts as a critical cell adhesion component. The Drosophila type IV collagen-like protein Pericardin is a crucial factor for the alignment of cardioblasts and the formation of an organised heart epithelium . The polarity of cardioblasts is a prerequisite to form the organised heart tube. Recently, it was shown that the T-box transcription factor neuromancer (Nmr1/H15 and Nmr2/Midline) is required for establishing the polarity of heart cells, most likely by regulating genes that are responsible for the transition of an unpolarised cardioblast progenitor into a flattened polarised cardioblast. A number of studies have shown that Kuzbanian/ADAM10 plays a role in cell–cell communication and cell adhesion. For example, ADAM10 binds Ephrin2A, a protein with a role in neuronal repulsion, is necessary for shedding EGFR ligands and is involved in cleavage of N-cadherin and regulation of cell–cell adhesion. In Drosophila, Kuzbanian mediates the transactivation of EGF receptor as shown by in vivo studies. Kuzbanian is also important for border cell migration in the Drosophila ovary (Albrecht, 2006).

In kuzbanian mutant embryos initial heart cell differentiation takes place. This includes the specification of different subtypes of heart cell, as shown by the use of specific molecular markers, the alignment of cardioblasts on both sides of the heart primordium and the initiation of heart beating. In late embryos (stage 16/17), an abnormal morphology of cardioblasts is observed: an uncoordinated arrangement of ostia-forming cardioblasts and, instead of a single heart lumen, additional lumen-like structures. These findings point to the possibility that Kuzbanian might have a function for final heart cell morphogenesis, e.g. by processing substrates other then Notch. Evidence has been provided that Kuzbanian is involved in the repulsive guidance system in the CNS and interacts genetically with Slit. Interestingly, the Slit/Robo signalling pathway plays a pivotal role in polarity and morphogenesis control of cardioblasts as well, pointing to the possibility that one of these molecules might be a substrate for Kuzbanian. This would explain the heart cell morphogenesis phenotypes seen in kuzbanian mutant embryos. In embryos that express a dominant negative Kuzbanian form driven late in the heart cardioblast, morphogenesis is not significantly affected. This indicates that the drivers used so far are inappropriate to separate different functions of Kuzbanian. Furthermore, it cannot be excluded that the induction of morphogenetic processes requires the Kuzbanian-dependant Notch signalling pathway quite early. This would hamper significantly the separation of Kuzbanian functions using various driver lines and the dominant negative Kuzbanian form. Nevertheless, it remains to be clarified if the described effects on heart cell morphology are primarily due to the absence of Kuzbanian function or occur as a secondary consequence of hyperplasia (Albrecht, 2006).

This paper has shown that Kuzbanian (ADAM10) plays an essential role in heart development. Members of the ADAM gene family in vertebrates are also known to be critical for cardiogenesis. Interestingly, mice lacking ADAM17 exhibit a cardioblast proliferation phenotype as well, besides other defects, indicating a conserved function of ADAM proteins in heart development. It is assumed that Kuzbanian might have additional functions, e.g., as a sheddase on unknown substrates in the cardiac mesoderm, which has to be proven in future studies (Albrecht, 2006).

The convergence of Notch and MAPK signaling specifies the blood progenitor fate in the mesoderm

Blood progenitors arise from a pool of pluripotential cells ('hemangioblasts') within the Drosophila embryonic mesoderm. The fact that the cardiogenic mesoderm consists of only a small number of highly stereotypically patterned cells that can be queried individually regarding their gene expression in normal and mutant embryos is one of the significant advantages that Drosophila offers to dissect the mechanism specifying the fate of these cells. This paper shows that the expression of the Notch ligand Delta (Dl) reveals segmentally reiterated mesodermal clusters ('cardiogenic clusters') that constitute the cardiogenic mesoderm. These clusters give rise to cardioblasts, blood progenitors and nephrocytes. Cardioblasts emerging from the cardiogenic clusters accumulate high levels of Dl, which is required to prevent more cells from adopting the cardioblast fate. In embryos lacking Dl function, all cells of the cardiogenic clusters become cardioblasts, and blood progenitors are lacking. Concomitant activation of the MAPK pathway by EGFR and FGFR is required for the specification and maintenance of the cardiogenic mesoderm; in addition, the spatially restricted localization of some of the FGFR ligands may be instrumental in controlling the spatial restriction of the Dl ligand to presumptive cardioblasts (Grigorian, 2011).

In this study pursued two goals: to elucidate the precise location and cellular composition of the cardiogenic mesoderm, and to analyze the mechanism by which Notch becomes activated in the restricted subset of these cells that become blood progenitors. The findings show that the cardiogenic mesoderm is comprised of segmentally reiterated pairs of clusters (cardiogenic clusters) defined by high expression levels of Dl, L'sc and activated MAPK. The MAPK pathway, activated through both EGFR and FGFR signaling, is required for the specification (EGFR) and maintenance (EGFR and FGFR) of all cardiogenic lineages. As shown previously, the default fate of all cardiogenic cells is cardioblasts. Notch activity triggered by Dl is required for the specification of blood progenitors (thoracic cardiogenic clusters) and nephrocytes (abdominal cardiogenic clusters), respectively. One of the downstream effects of MAPK signaling is to maintain high levels of Dl in the cardiogenic clusters, and to help localize Dl expression toward a dorsal subset of cells within these clusters, which will become cardioblasts. Dl stimulates Notch activity in the surrounding cells, which triggers the blood progenitor/nephrocyte fate in these cells (Grigorian, 2011).

The cardiogenic clusters form part of a larger population of mesodermal cells defined by high expression levels of l'sc. Based on l'sc in situ hybridization, these authors mapped 19 clusters of l'sc expressing cells within the somatic mesoderm. Many of these clusters (called 'myogenic clusters' in the following) give rise to one or two cells that transiently maintain high levels of l'sc, whereas the remaining cells within the cluster lose expression of l'sc. The l'sc-positive cells segregate from the mesoderm to a more superficial position, closer to the ectoderm, undergo one final mitotic division, and differentiate as muscle founder cells. Dl/Notch mediated lateral inhibition was shown to act during the singling-out of muscle founders from the myogenic clusters. Loss of this signaling pathway caused high levels of L'sc to persist in all cells of the myogenic clusters, with the result that all cells developed as muscle founders. Interestingly, loss of l'sc had only a mild effect, consisting of a slight reduction in muscle founders. This is similar to what was find in this paper in l'sc-deficient embryos, which show only a mild reduction in cardioblasts and other cardiogenic lineages (Grigorian, 2011).

The developmental fate of most of the L'sc-positive clusters within the dorsal somatic mesoderm is different from that of the ventral and lateral myogenic clusters discussed above, even though several parallels concerning the morphogenesis, proliferation, and dependence on Dl/Notch signaling are evident. The somatic (anterior, Wg-positive) mesoderm is divided into a dorsal and ventral domain based on the expression of Tin. Initially expressed at high levels in the entire mesoderm, this gene is maintained only in the dorsal mesoderm, as a result of Dpp signaling from the dorsal ectoderm. The dorsal somatic mesoderm, which is called 'early cardiogenic mesoderm', includes four L'sc-positive clusters, C2 and C14-C16. The development of C2 has been described in detail. C2 gives rise to a progenitor that divides twice; two of the progeny become the Eve-positive pericardial cells. Meanwhile, C15 which appears later at the same position as C2, seems to behave like a 'normal' myogenic cluster. It produces a progenitor that divides once and forms the founders of the dorsal muscle DA1. As shown in this paper, the two remaining dorsal clusters, C14 and C16, give rise to the cardioblasts. It is noted that the Eve-positive progenitors, as well as the cardioblasts, resemble the muscle founders derived from the typical myogenic clusters in three aspects. First, they segregate toward a superficial position, close to the ectoderm, relative to the remainder of the cells within the clusters. Secondly, they undergo one (in case of C2: two) rounds of division right after segregation. And third, they are restricted in number by Dl/Notch signaling: in all cases, they are increased in number following Dl or Notch loss of function (Grigorian, 2011).

Cardiogenic clusters, like myogenic clusters, also depend on the MAPK signaling pathway. Past studies have shown that in the Eve-positive C2 and C15 clusters, Ras, is capable of inducing the formation of additional Eve-positive progenitors. Ras is a downstream activator of both the EGFR and FGFR tyrosine kinase pathways, both of which have been seen to be important for the formation of the Eve-positive progenitors. With a loss of the FGFR pathway, Eve-positive progenitors of both the C2 and C15 cluster are lost; by contrast, the EGFR pathway affects only C15. The balanced activity of MAPK and Notch, which in part depends on reciprocal interactions between these pathways, determines the correct number of C2/C15 derived progenitors. Ras-induced MAPK activation upregulates the expression of other MAPK signaling pathway members (autoregulatory feed-back loop), but also stimulates the antagonist Argos, as well as Dl. Dl-activated Notch, in turn, inhibits MAPK signaling (Grigorian, 2011).

Both Dl/Notch and MAPK signaling are active in the C14 and C16 clusters, which constitute the definitive cardiogenic mesoderm. MAPK activity is required for the maintenance of all lineages derived from these clusters, as shown most clearly in the EGFR LOF phenotype that entails a lack of cardioblasts, blood progenitors, and pericardial nephrocytes. Overexpression of Ras results in an increased number of all three cell types, which indicates that the C14/C16 clusters attain a larger size, possibly by an additional round of mitosis. The phenotype seen in embryos suffering from loss- or overexpression of Dl/Notch pathway members can be interpreted in the framework of a classical lateral-inhibition mechanism: Dl is upregulated in the C14/C16 derived cardioblast progenitors (analogous to the Eve-progenitors of C2/C15), from where it activates Notch in the remainder of the C14/C16 cells; these cells are thereby inhibited from forming cardioblasts, and instead become nephrocytes/blood progenitors. The level of Notch activity affects the expression of tin (low Notch) and the GATA homolog srp (high Notch), which triggers the fate of cardioblasts and blood progenitors/nephrocytes, respectively (Grigorian, 2011).

MAPK is required for the initial activation of Dl in the cardiogenic clusters (just as in the myogenic clusters). Input from the pathway is most likely also instrumental in the subsequent restriction of Dl to the cardioblast progenitors. The positive interaction between MAPK and Notch signaling could occur at several levels. A mechanism shown for the ommatidial precursors of the eye disc involves Ebi and Strawberry notch (Sno), which are thought to act downstream of EGFR signaling and lead to an upregulation of Dl through the Su(H) and SMRTER complex (Grigorian, 2011).

Generally, when one progenitor cell is seen to give rise to two different cell types it is accomplished in one of two ways. One: there is an asymmetric division, where a factor expressed by the progenitor is segregated into only one daughter cell; two: a non-uniformly expressed extrinsic signal effects one cell, but not its neighboring sibling. In the posterior (abdominal) segments of the Drosophila cardiogenic mesoderm, inhibition of Notch by Numb accounts for the asymmetric activity of Notch in a small set of cardiogenic mesoderm cells, the Svp-positive cells. If Numb function is removed, these cells, which normally produce two cardioblasts and two pericardial nephrocytes, instead give rise to four cardioblasts. However, multiple nephrocytes per segment remain in numb loss-of-function mutations; furthermore, loss of numb does not cause any defect in the blood progenitors, where asymmetrically dividing Svp-positive cells are absent. This suggests that in addition to the numb-mediated mechanism, directional activation of Notch by one of its ligands is required for the majority of nephrocytes and all of the blood progenitors. It is proposed in this study that the spatially restricted upregulation/maintenance of Dl in nascent cardioblasts acts to activate Notch in the remainder of the cells within each cardiogenic cluster, which promotes their fate as blood progenitors and nephrocytes (Grigorian, 2011).

The Notch signaling pathway is typically associated with members of two different types of bHLH transcription factors. One type act as activators, while the other act as repressors. In the context of lateral inhibition, best studied in Drosophila neurogenesis, activating bHLH transcriptions factors, including genes of the AS-C like l'sc, are expressed at an early stage in clusters of ectodermal or mesodermal cells, where they activate genetic programs that promote differentiative pathways such as neurogenesis, or myogenesis/cardiogenesis. Subsequently, Notch ligands initiate the Notch pathway in these clusters; cells with high Notch activity turn on members of the Hairy/E(spl) (HES) family of bHLH genes which act as repressors and abrogate the transcriptional programs that had been set in motion by the activating bHLH factors. This paper shows that the gene cassette consisting of the Notch signaling pathway, as well as activating and repressing bHLH factors, operates in the cardiogenic mesoderm to determine the balance between cardioblasts and blood progenitors/nephrocytes. As discussed in the following, the same cassette also appears to be centrally involved in the specification of vascular endothelial cells and hematopoietic stem cells in vertebrates, which adds to the list of profound similarities between Drosophila and vertebrate blood/vascular development (Grigorian, 2011).

Even prior to the appearance of hemangioblasts, the lateral mesoderm of vertebrates is prepatterned by sequentially activated signaling pathways and transcriptional regulators similar to those that act in flies. The Wnt/Wg pathway, for example, separates subdomains of the mesoderm in vertebrates and Drosophila, as well as more ancestral ecdysozoans. Notch signaling plays an essential role in generating boundaries between segmental, as well as intra-segmental, subdomains within the ectoderm and mesoderm. The FGF signaling pathway predates the appearance of Bilaterians and plays a highly conserved role in early mesoderm patterning. Likewise, specific sets of transcriptional regulators are the targets of these signaling pathways (e.g., twist, zfh and myostatin) and play a role during the establishments of cell fate in the mesoderm. It appears, therefore, that the bilaterian ancestor featured a mesodermal subdomain, the 'cardiogenic/lateral mesoderm, in which signals of the Wg, BMP, Notch, and FGF pathways and conserved sets of transcriptional regulators established boundaries and cell fate in the mesoderm (Grigorian, 2011).

The vertebrate gene encoding an activating bHLH factor with sequence similarity to the Drosophila AS-C genes is SCL. SCL expression in the lateral mesoderm marks the first appearance of hemangioblasts; note that SCL is also expressed widely in the developing vertebrate CNS. In Zebrafish, from their site of origin in the lateral mesoderm, SCL-positive hemangioblasts migrate dorso-medially and form the intermediate cell mass (ICM). The ICM is the site of primitive endothelial blood vessel and hematopoietic cell specification. Gain of function studies carried out in zebrafish embryos have shown SCL to be one of the genes important in specifying the hemangioblast from the posterior lateral plate mesoderm. The specification of hemangioblast here comes at the expense of other mesodermal cell fates, namely the somitic paraxial mesoderm. In mice, lack of SCL affects blood and vascular development as SCL mutants are bloodless and show angiogenesis defects in the yolk sac (Grigorian, 2011).

Vertebrate homologs of the repressive Drosophila Hairy/E(spl) family of bHLH genes are the Hes and Hey (hairy/Enhancer-of-split related with YRPW motif) genes. A well studied member of the Hey family in zebrafish is gridlock, which is required for the specification of hematopoietic progenitors from the ICM. Hey 2 mutations in mice lead to severe congenital heart defects. In addition, the Hes protein plays a role in hematopoiesis as it is a positive regulator of Hematopoietic Stem Cell (HSC) expansion (Grigorian, 2011).

Genetic studies of the Notch receptors and their ligands in vertebrates support the idea that this pathway does indeed play a crucial role in the initial determination of hematopoietic stem cells. The yolk sac and the para-aortic splanchnopleura (P-Sp)/AGM (aorta gonad mesonephros) of Notch null mouse embryos lack HSCs. A similar phenotype is observed in mutants of Jagged 1, one of the Notch ligands. Notch is thought to be the deciding factor between hematopoietic and endothelial cell fates when the two originate from a common precursor or hemangioblast. In murine mutants exhibiting lower Notch1 mRNA levels, a lack of hematopoietic precursors is seen and is accompanied by an increase in the number of cells expressing endothelial cell markers. Likewise, in Drosophila, loss of Notch is associated with an increase in cardioblast number and a loss of blood precursor cells (Grigorian, 2011).

Serial specification of diverse neuroblast identities from a neurogenic placode by Notch and Egfr signaling

The brain insulin-producing cell (IPC) lineage and its identified neuroblast (IPC NB) was used as a model to understand a novel example of serial specification of NB identities in the Drosophila dorsomedial protocerebral neuroectoderm. The IPC NB was specified from a small, molecularly identified group of cells comprising an invaginated epithelial placode. By progressive delamination of cells, the placode generated a series of NB identities, including the single IPC NB, a number of other canonical Type I NBs, and a single Type II NB that generates large lineages by transient amplification of neural progenitor cells. Loss of Notch function caused all cells of the placode to form as supernumerary IPC NBs, indicating that the placode is initially a fate equivalence group for the IPC NB fate. Loss of Egfr function caused all placodal cells to apoptose, except for the IPC NB, indicating a requirement of Egfr signaling for specification of alternative NB identities. Indeed, both derepressed Egfr activity in yan mutants and ectopic EGF activity produced supernumerary Type II NBs from the placode. Loss of both Notch and Egfr function caused all placode cells to become IPC NBs and survive, indicating that commitment to NB fate nullified the requirement of Egfr activity for placode cell survival. The surprising parallels between the serial specification of neural fates from this neurogenic placode and the fly retina are discussed (Hwang, 2011).

These observations provide a framework for understanding several key features of placodal neurogenesis in the placodes in the head midline dorsomedial procephalic NE (Pdm); the steps in placode development are summarized in Model for serial specification of NB identities from the Drosophila medial pars intercerebralis primordium (pPIm). The NE placode, comprising roughly eight cells, with its underlying gene regulatory network, appears to be highly specialized to serial specify a range of distinct neural stem cell identities, beginning from an initial state of equivalent developmental potential. In the case of the pPIm, the initial state of competence is to form IPC NBs. The first indication that the pPIm had acquired prepattern identity was the synchronized round of cell division observed before placode morphogenesis at the 4-cell pPIm stage. After the expansion to the 8-cell stage, the cells entered a cell cycle arrest and formed a neurogenic placode; the pPIm then initiated a lengthy proneural competence period as the various NB identities were produced. Mutant and temperature shift analysis of Notch signaling suggested that a window of competence for the IPC NB fate exists from the time the pPIm expanded to eight cells and acquired proneural competence until the time that IPC NB fate was normally specified (the time that it became Dac+). At roughly this point, the pPIm became dependent on Spi/Egfr activity to promote the survival of NE cells not yet specified as NBs. This Egfr-dependent specification period then extended through stage 15, while alternative NB identities were specified. Neurogenesis then ended with the specification of the single Type II NB fate (Hwang, 2011).

Among the well-studied examples of Drosophila neurogenesis, perhaps the most intriguing parallels are found between serial fate specification in the placodal pPIm and in the developing facets of the Drosophila retina, particularly between specification of the IPC NB and the R8 photoreceptor within each developing ommatidium. The R8 photoreceptor is the first of a series of photoreceptor and cone cell fates to be specified by progressive recruitment to an apically constricted cluster of twelve cells. Each R8 cell, the ommatidium founder, is specified from a proneural R8 fate equivalence group generated by the activity of the bHLH factor Atonal (Ato), and is singled out by Notch-mediated lateral inhibition. Although parallel with respect to specification from a proneural fate equivalence group, the pPIm NE requires activity of the AS-C for IPC NB specification whereas Ato is not essential. In contrast to R8 specification from a proneural group, the photoreceptor R1-7 and the cone cell fates are locally recruited within an ommatidium through inductive and serial Notch and Egfr/receptor tyrosine kinase (RTK) signaling from R8. Analogous to the pPIm and IPC NB specification, Egfr activity is not essential for proneural competence and specification of R8, but is essential for survival of all photoreceptor precursors, except for R8. Egfr-mediated cell survival in the developing retina requires that the pathway activates MAPK by phosphorylation and that pMAPK in turn phosphorylates the proapoptotic factor Head involution defective (Hid). In normal Egfr signaling, phosphorylated Hid is targeted for degradation, which permits survival of the developing ommatidium; cell survival is also promoted by the activity of Pnt1/2, which repress Hid expression. It was previously reported that hid mRNA accumulates in a pan-placodal Pdm NE pattern in stage 12-13 embryos, yet this study found that Pnt1/2 was not obviously essential for survival to stage 14. This suggests that, in contrast to the retina, most of the anti-apoptotic activity of Egfr signaling is relatively independent of Pnt1/2; hence, it might act primarily through the action of pMAPK in the turnover of Hid. This hypothesis will need to be tested more fully in future studies (Hwang, 2011).

Although there are many examples of systems in which Notch and Egfr activities are mutually antagonistic or cooperate in promoting cell fate decisions, no evidence was found of a mutual dependence between Notch and Egfr activity states; either pathway became active in the absence of the other. Admittedly, from these experiments cross-talk between the two pathways could not be definitively ruled out. By contrast, other instances of neurogenesis in Drosophila, such as in the optic lobe and notum macrochaete, depend on Egfr activity to promote neurogenesis by activating as-c genes. In these contexts, the Egfr-dependent proneural state is antagonized by Notch activity. In the pPIm proneural region, it remains unclear from these experiments whether Egfr is essential for neurogenesis subsequent to the IPC NB, since the pPIm cells are lost with the loss of Egfr activity. Indeed, there is a potential parallel with neurogenesis of the abdominal chordotonal precursors, which do form in the absence of Egfr activity but then signal back to the epithelium via Spi to activate ato and extend neurogenesis, thereby recruiting additional chordotonal precursors (Hwang, 2011).

In conclusion, the parallels between serial neural fate specification in the Pdm placode and in eye development raise the interesting possibility that some aspects of the two underlying gene regulatory networks have points of overlap. If so, it is intriguing to consider whether distinct regions of proneural epithelia that express placode genes such as sine oculis, Optix, Six4 and eya, were derived in evolution from a common ancestral neuroepithelial patterning circuit that was capable of serial specification. If this were the case, the implication would be that this mode of fate specification, in which diverse neuronal or neural stem cell identities are generated through local interactions in prepatterned cell groups, is more widely distributed than currently appreciated across animal phylogeny and vertebrate species. Hence, a deeper understanding of such a neural stem cell diversification mechanism will certainly aid efforts to control the differentiation of specific neural progenitor fates in vitro (Hwang, 2011).

FACS purification and transcriptome analysis of drosophila neural stem cells reveals a role for Klumpfuss in self-renewal

Drosophila neuroblasts (NBs) have emerged as a model for stem cell biology that is ideal for genetic analysis but is limited by the lack of cell-type-specific gene expression data. This study describes a method for isolating large numbers of pure NBs and differentiating neurons that retain both cell-cycle and lineage characteristics. Transcriptional profiles were determined by mRNA sequencing and identify predicted NB-specific transcription factors were identified that can be arranged in a network containing hubs for Notch signaling, growth control, and chromatin regulation. Overexpression and RNA interference for these factors identify Klumpfuss as a regulator of self-renewal. Loss of Klumpfuss function causes premature differentiation, and overexpression results in the formation of transplantable brain tumors. The data represent a valuable resource for investigating Drosophila developmental neurobiology, and the described method can be applied to other invertebrate stem cell lineages as well (Berger, 2012).

Unlike their differentiating sibling cells, NBs regrow to their original size after each division and maintain their identity over many cell divisions. Therefore, NBs must express a regulatory transcriptional network that is highly robust over time but can be rapidly and irreversibly modified by Numb, Pros, and Brat. Previous loss-of-function experiments revealed a surprising level of redundancy among the known TFs acting in NBs (San-Juán, 2011; Zacharioudaki, 2012). Therefore, ther transcriptome data was used to identify a complete set of predicted TFs that are highly expressed in NBs and strongly downregulated during differentiation. In total, 28 TFs matched the criteria. Assuming that functionally related TFs are more likely to be coexpressed, stage- and tissue-specific microarray data was used to infer putative regulatory interactions (Berger, 2012).

The resulting hypothetical network contains six hubs that are connected to more than five genes in the network. The first hub is HLHmγ, a direct nuclear target of the Notch signaling pathway. HLHmγ connects to Dpn, and both were shown to act redundantly in controlling NB self-renewal (Zacharioudaki, 2012). It also connects to Wor and Klu, which have been linked to Notch signaling by both coexpression and functional studies. Finally, it connects to Grh, which can regulate proliferation in NBs. Surprisingly, Grh is actually a negative regulator of HLHmγ, but it has been proposed that such paradoxical elements are frequent components of transcriptional circuits and can maintain homeostatic concentrations or contribute to robust regulation (Berger, 2012).

HLHmγ and Wor are connected to two other hubs that consist of genes involved in ribosome biogenesis and growth control. Mod is the Drosophila homolog of Nucleolin, the major nucleolar protein of growing eukaryotic cells that is thought to play a role in rRNA transcription and ribosome assembly. CG10565 is the fly ortholog of MPP11, a chaperone of the DNA-J family that is involved in ribosome assembly and has been implicated in the regulation of cell growth. In NBs, CG10565 connects to Bigmax and TFAM, two factors that may be involved in growth control because they are direct downstream targets of the major growth regulators Myc and Max. The fourth interesting hub is Ssrp, a chromatin regulator that has been identified based on its RNAi overproliferation phenotype. Surprisingly, Ssrp RNAi causes gain rather than loss of NBs, and may therefore maintain a chromatin state that allows differentiation. Finally, Ken and Coop have been implicated in Jak/STAT and wingless signaling, respectively. Both of these pathways are linked to Notch signaling, although no such functional link has been described in NBs. Thus, this hypothetical transcriptional network provides potential explanations for several aspects of NB biology. For example, it could explain the direct effect of Notch signaling on cell growth that has been described in Drosophila NBs (Berger, 2012).

Previous studies of Drosophila NBs have provided important insights into the mechanism of asymmetric cell division. Despite this progress, however, the transcriptional circuits that control self-renewal and act downstream of segregating determinants have remained elusive. Functional redundancy has already been demonstrated for some TFs acting in NBs and may have hindered the genetic identification of others. These transcriptional data lay the foundation for a more targeted search for functional interconnections between redundantly acting factors. The hypothetical transcriptional network this study has identified provides testable hypotheses for how Notch signaling might connect to the machinery for growth regulation. It suggests that Notch target genes control other TFs that regulate ribosome biogenesis and assembly in a surprisingly direct manner. With the use of this established method to purify larval NBs, and the development of new techniques for chromatin immunoprecipitation even from limited numbers of cells, it will become technically feasible to test the proposed network. The ease with which individual TFs can be removed by RNAi may ultimately result in an understanding of the architecture of this network in unprecedented detail (Berger, 2012).

The cytolinker Pigs is a direct target and a negative regulator of Notch signalling

Gas2-like proteins harbour putative binding sites for both the actin and the microtubule cytoskeleton and could thus mediate crosstalk between these cytoskeletal systems. Family members are highly conserved in all metazoans but their in vivo role is not clear. The sole Drosophila Gas2-like gene, CG3973 (pigs), was recently identified as a transcriptional target of Notch signalling and might therefore link cell fate decisions through Notch activation directly to morphogenetic changes. A null mutant was generated in CG3973 (pigs)pickled eggs’, referring to the mutant phenotype): pigs1 mutants are semi-viable but adult flies are flightless, showing indirect flight muscle degeneration, and females are sterile, showing disrupted oogenesis and severe defects in follicle cell differentiation, similar to phenotypes seen when levels of Notch/Delta signalling are perturbed in these tissues. Loss of Pigs leads to an increase in Notch signalling activity in several tissues. These results indicate that Gas2-like proteins are essential for development and suggest that Pigs acts downstream of Notch as a morphogenetic read-out, and also as part of a regulatory feedback loop to relay back information about the morphogenetic state of cells to restrict Notch activation to appropriate levels in certain target tissues (Pines, 2010).

During development, signalling pathways are repeatedly used to assign different cell fates among identical precursors. Differentiating cells then undergo regulated changes in gene expression and cell morphology appropriate for their function in a tissue. Cell shape changes are largely mediated by the cytoskeleton. Downstream effectors of developmental signalling pathways thus have to impinge on the cytoskeleton to exert the desired changes (Pines, 2010).

Spatial and temporal coordination of cytoskeletal elements can be performed by 'cytolinker' proteins that have the ability to interact with more than one cytoskeletal system at a time. Only two proteins in Drosophila contain a combination of an actin-binding Calponin homology (CH) domain and a microtubule-binding domain of the Gas2 family. One is the fly spectraplakin Short stop, and spectraplakins serve many important functions during tissue morphogenesis in both flies and mammals. The other protein is encoded by the gene CG3973, which was named pigs. There are four close paralogues of Pigs in mammals, and these are the only proteins in mammals to have Gas2 domains apart from the two spectraplakins MACF1 and BPAG1 (DST -- Human Gene Nomenclature Database). The first relative of Pigs identified in mammals was called Growth arrest specific 2 (Gas2), which also gave the domain its name. Although subsequent studies did not confirm a role for Gas2 in growth arrest induction, three further relatives were found and named Gas2-like 1 (Gas2L1), Gas2L2 and Gas2L3. All have CH and Gas2 domains and can associate with both the actin and the microtubule cytoskeleton in tissue culture (Goryunov, 2007). However, the in vivo role of Gas2 or the Gas2-like proteins is unclear, and to date, no loss-of-function analysis has been reported in any species. As these proteins share with the spectraplakins the presence of both a Gas2 domain and an actin-binding CH domain, it was suspected that they serve a similarly important, albeit non-overlapping, function in the regulation of the cytoskeleton. Thus, Drosophila, with only one Gas2-like homologue, seems an excellent system to analyse the function of this class of proteins, avoiding the possibility of redundant functions between paralogues masking phenotypes (Pines, 2010).

The pigs (CG3973) gene has recently been identified as containing sites occupied by the transcription factor Supressor of Hairless [Su(H)] upon activation of Notch signalling (Krejci, 2009), suggesting that pigs could act as a direct downstream target of Notch. The Notch receptor is activated through one of its membrane-bound ligands, such as Delta, on neighbouring cells. The intracellular domain of Notch (NICD) is then cleaved and released, free to act as a transcriptional co-activator together with Su(H). Notch signalling is required for many cell fate decisions during development, but relatively few genes directly activated by Notch signalling have been identified over the years. A recent study has identified what could be a comprehensive set of direct targets of Notch activation in Drosophila myogenic cells (Krejci, 2009). This study used genome-wide chromatin immunoprecipitation (ChIP) analysis with Su(H) antibodies to identify genomic regions occupied by Su(H). The first intron of pigs contained a peak of Su(H)-bound sites and thus might be a new target (Pines, 2010).

As most previously described targets of Notch are themselves transcription factors, it was intriging to find a potential cytolinker protein as a possible direct target, and thus effector, of Notch signalling. This study set out to analyse the phenotypes observed in the absence of Pigs function and to elucidate its potential role downstream of Notch. This study shows that, in addition to modifying the cytoskeleton and cell shape, Pigs appears to act as part of a feedback loop that negatively regulates the activity of Notch during morphogenesis in certain tissues (Pines, 2010).

The data demonstrate that not only is the Gas2-like protein Pigs essential for development in the fly and is a direct target of Notch signalling, but also that Pigs is probably part of a negative regulatory feedback loop that restricts Notch signalling to appropriate levels in certain tissues. These findings are intriguing, since the domain composition of Pigs would suggest it to function as a cytoskeletal crosslinker protein, helping to coordinate actin and microtubules and aiding tissue morphogenesis. Although neither a strong disruption of either actin nor microtubule cytoskeleton was observed in early stages of oogenesis in pigs1 mutant ovaries, at late stages during the dumping phase, actin cages appeared highly disrupted. Also, already at early stages of oogenesis in the germarium, cell shapes of FCs were very irregular. As cell shape is largely determined by the cytoskeleton, this suggests that, although not visible at the level of light microscopic analysis, cytoskeletal function is impaired in pigs1 mutant cells (Pines, 2010).

The ChIP data and reporter assays indicate that Pigs is directly regulated by Notch in at least some tissues, and in both the muscles and the ovaries, the phenotypes are compatible with Pigs being an effector of Notch. However, assays of Notch pathway activity in pigs mutants indicate that Pigs is a negative regulator of Notch activity. How can this paradox be resolved? Three alternate models for Pigs function at a molecular level can be envisaged. Pigs might facilitate cytoskeletal rearrangements induced by Notch signalling by stabilising the cytoskeleton at certain subcellular sites, and the execution of necessary changes could induce further signalling factors to terminate Notch signalling. Alternatively, Pigs could directly link Notch signalling to the cytoskeleton through sequestering Notch at a particular subcellular localisation (i.e., through linkage to the cytoskeleton), and this could bring Notch in proximity to factors that switch off the signalling appropriately. In a third scenario, the morphological changes downstream of Notch could be independent of Pigs, but Pigs could act as a molecular 'sensor' to determine if the actin and the microtubule cytoskeleton have rearranged in an appropriate fashion. In support of the second scenario, the localisation of Notch (based on detection of the intracellular domain), is changed in pigs1 mutant FCs that are attempting to interdigitate and encapsulate a germline cyst (Pines, 2010).

Although Pigs is a cytoskeleton-associated protein, the phenotypes observed in pigs1 mutant ovaries are not those generally seen in mutants for structural cytoskeletal proteins. FCs mutant for actin regulators such as CAP, Cofilin (Twinstar -- FlyBase), Profillin (Chickadee -- FlyBase), Ena or Abl show alterations in the actin cytoskeleton and, in some cases, multi-layering of the follicular epithelium, but do not lead to phenotypes resembling Notch-misregulation. This supports the notion that the function of Pigs confers more than just cytoskeletal changes in the FCs (Pines, 2010).

Pigs is likely to be only one of several downstream effectors of Notch in the tissues where it is directly regulated, and it remains to be proven that it is a target in the ovary. emc is a previously characterised effector of Notch in the ovary and has overlapping phenotypes with pigs1 (Adam, 2004), supporting the model that pigs is one of several Notch targets in the ovary. This also explains the observation that the defects in pigs1 mutant ovaries are milder than those of Notch alleles; the overall pigs loss-of-function phenotype is expected to represent just a subset of the defects caused by Notch loss-of-function. Furthermore, pigs function is needed only in a subset of tissues that depend on Notch signalling for their differentiation. For example, no sensory organ defects were observed in the pigs1 deletion, and therefore it would be concluded that Pigs does not function downstream of Notch in the differentiation of this tissue. Consistent with this, a recent genome-wide analysis of genes involved in Notch signalling that focused on phenotypes in the external sensory organs of the notum did not identify pigs as a candidate (Pines, 2010).

The data presented in this study suggest that Pigs could serve a dual function in specific Notch-dependent processes: aiding morphological changes downstream of a differentiation signal combined with a regulatory role that allows that signal to be terminated when appropriate. Thus, the Notch-Pigs interaction provides an opportunity to further dissect the link between Notch-induced differentiation, cell shape and control of the cytoskeleton (Pines, 2010).

Larval: Notch function in the eye disc

The Drosophila compound eye is specified by the concerted action of seven nuclear factors: Twin of eyeless (Toy), Eyeless (Ey), Eyes absent (Eya), Sine oculis (So), Dachshund (Dac), Eye gone (Eyg), and Optix (Opt). These factors have been called 'master control' proteins because loss-of-function mutants lack eyes and ectopic expression can direct ectopic eye development. However, inactivation of these genes does not cause the presumptive eye to change identity. Surprisingly, several of these eye specification genes are not coexpressed in the same embryonic cells -- or even in the presumptive eye. Surprisingly, the EGF Receptor and Notch signaling pathways have homeotic functions that are genetically upstream of the eye specification genes; specification occurs much later than previously thought -- not during embryonic development but in the second larval stage (Kumar, 2001).

The Egfr and Notch pathways function in the specification or determination of the eye. An ey-GAL4 driver was used to express target proteins; this element drives expression first in the eye and antenna anlagen in the embryo (by stage 11) and then in regions ahead of the furrow in just the eye imaginal disc. Egfr function was removed in this domain by expressing a dominant negative form of the receptor. Both the eye and antenna were deleted from eclosed adults indicating that both structures require Egfr signaling for their specification, determination, or survival. Under these conditions, the larval discs do not form, making analysis of later developmental phenotypes impossible. The same phenotype was obtained with dominant negative Ras indicating that this activity is Ras dependent. Wild-type and activated forms of several components of the Ras pathway were expressed in the eye anlagen using the same driver and it was found that hyperactivation of many elements leads to the homeotic transformation of the eye into a morphologically complete antenna. Homeotic transformation of the eye to antenna can also be induced by the Egfr ligand Spitz but not by two other known activators. The membrane bound version of Spitz does not induce homeotic transformations, suggesting a requirement for paracrine signaling. Wild-type and constitutively active forms of the Egfr and two other Drosophila RTKs (Breathless [Btl] and Heartless [Htl]) were expressed but only Egfr is able to induce the transformation. Expression of the constitutively active version of Egfr gives a significantly stronger phenotype than the wild-type version of Egfr, suggesting that the level of Egfr signaling is important for maintaining the balance between eye and antennal identities. The downstream elements of the pathway that can induce this transformation include Ras, Raf, and PntP1, while neither MEK, MAPK, nor PntP2 induced this effect in this assay. Aop, Tramtrack (Ttk), and BarH1/H2, each of which mediates negative feedback inhibition of Egfr signaling, delete the eye. The failure of Mek, Mapk, and PntP2 to induce this transformation reflects the existence of actual branch points in the pathway. However, it is also possible that the quantitative levels of expression of these three elements are not limiting for this signal at this time and place; indeed, their phosphorylation states may be more relevant (Kumar, 2001).

Notch and Egfr have been shown to often antagonize each other during cell fate decisions in the fly eye. Notch function was removed with a dominant negative form and results similar to the effects of Egfr signal hyperactivation were obtained. Consistent with this, when an activated form of Notch was expressed, the size of the eye was reduced and there were severe dysmorphies. Expression of dominant negative transgenes of the ligands Delta (Dl) or Serrate (Ser) also results in the eye to antenna transformation. Elevated expression levels of both Su(H) and many of the proteins of the E(spl) complex (m4, m7, m8, m8DN, malpha, mß, mgamma, and mdelta) were also expressed but no effect on either eye or antenna disc development was observed. However, homeotic eye to antenna transformations occurred when Mastermind (Mam) was expressed using a dominant negative construct. Mam is a member of the neurogenic gene group that encodes a nuclear protein of unknown function. These results suggest a Su(H) and E(spl)C independent pathway for eye and antenna disc development that involves Mam (Kumar, 2001).

Do Egfr and Notch Act upstream of the eye specification genes? A molecular epistasy study was undertaken, examining the expression of some of the eye and antennal specification genes in the transforming conditions during the third larval stage (before cell types differentiate). In eye specification gene mutants (such as ey), ommatidial development is blocked, but the eye disc remains in a reduced form. Conditions that produce eye to antenna transformations, whether through hyperactivation of Egfr or downregulation of Notch signaling, show a complete replacement of the eye disc with an antenna disc. Distal-less (Dll) and Spalt-Major are normally expressed within subdomains of the antenna disc and are required for antenna development. Dll and SalM are expressed in the correct locations in the transformed antenna disc suggesting that both endogenous and transformed antenna are also both morphologically and molecularly equivalent (Kumar, 2001).

The transcription of five of the seven known eye specification genes (toy, ey, eya, so, and eyg) was examined. In transforming conditions, transcription levels of all five of the seven genes are below the levels of detection. This is consistent with both Egfr and Notch signaling acting genetically upstream to both the eye and antennal specification genes. The downregulation of ey suggests that the ey-GAL4 driver may also be downregulated via an autoregulatory mechanism. That the transformation occurs despite this may reflect a phenocritical period for the eye-antenna transformation; once the transformation has occurred the system is refractory to the loss of Egfr signaling (Kumar, 2001).

The notch pathway signals differentially in the eye versus the antenna primordia in the second larval stage. Loss of Notch activity during the second larval stage results in the transformation of the eye into an antenna. Thus, it is predicted that Notch signaling should be elevated in the presumptive eye versus the antenna at the critical time. Cells that are actively receiving a Notch signal upregulate Notch protein expression. Thus elevated Notch antigen expression can be used as a reporter of elevated Notch signaling. Notch and ey expression were examined in imaginal discs from first, second, and third stage larvae. Both Notch and ey are expressed throughout the entire eye-antennal disc anlagen during the first larval stage. By the second larval stage, Notch is differentially upregulated within the presumptive eye. Interestingly, Notch appears especially active along the eye margins and midline, where it is thought to regulate retinal polarity. In contrast, ey appears to be exclusively within the eye field. In the third larval stage, Notch expression is upregulated in the morphogenetic furrow, where it acts to control ommatidial spacing while ey remains upregulated ahead of the furrow (Kumar, 2001).

Therefore, Egfr signaling promotes an antennal fate while Notch signaling promotes an eye fate. This role for Notch is consistent with the observation that removal of Notch signaling can partially inhibit compound eye development. Furthermore, several of the eye and antennal specification genes (ey, toy, eya, so, eyg, salM, and Dll) are downstream of the Egfr and Notch inputs. Wg and Hh pathway signaling affect this specification. The eye specification genes form a regulatory network and the direct control of any one of these genes may affect the others. Thus, which (if any) of the known eye specification genes is a direct target of Notch or Egfr signals may require direct biochemical assays (Kumar, 2001).

While activating Egfr or blocking Notch signals transforms the eye cleanly into an antenna, the reciprocal transformation is not complete, suggesting that there may be additional positive regulators of eye fate. The reciprocal transformation experiment could not be conducted (i.e., antenna to eye switch via hyperactivation of Notch or downregulation of Egfr signaling solely within the antennal anlagen). Unlike the ey-GAL4 driver, there is not an equivalent known driver that is expressed solely with the antennal anlagen. All known antennal-determining genes are also expressed in other imaginal discs. For instance, the Dll-GAL4 driver is expressed in several places within the embryonic head and leg imaginal disc. Expression of Egfr or Notch constructs with this driver results only in embryonic lethality. It may be that the antenna can be changed to an eye via alterations of Egfr or Notch signaling provided that the appropriate tools for their missexpression are available (Kumar, 2001).

Why do homozygous mutants for eye specification genes not transform the eye into an antenna? While it may be that some alleles are not nulls (e.g., ey1), a more interesting possibility is that there may be functional redundancy in some cases -- particularly that of ey and toy. Thus, only when both genetic functions are eliminated will a true null condition exist. Just such a situation confused the phenotypic analysis of two other twin homeodomain proteins, engrailed and invected. Unfortunately, mutations of the toy gene do not yet exist (Kumar, 2001).

How do the eye specification genes function? Published genetic epistasy and biochemical interaction data suggest that the seven known eye specification genes' products interact at the transcriptional and protein levels to direct cells toward eye fate. This requires that they are expressed in the same cells. Furthermore, it has been suggested that many, if not all, of these genes are 'master regulators' of eye fate -- that is, they are both necessary and sufficient for eye specification. Many very compelling experiments have been described showing the induction of ectopic eyes through the ectopic expression of these genes alone or in synergistic combinations. It is suggested that these genes come under separate regulation by different patterning signals in early development and that there are overlapping domains. Only when all of the domains coincide (during the second larval stage) do eye specification genes specify the eye. This seems to be the simplest explanation since the eye specification genes form a very tight genetic, biochemical, and transcriptional regulatory network suggesting that they are together required for eye specification. It may be that the final coexpression of the eye specification genes' products (and the exclusion of the antennal specification factors) is the last step required to allow the morphogenetic furrow to initiate in response to the next local expression of hh and for the final specification of retinal cell types and pattern (Kumar, 2001).

The hernandez and fernandez genes of Drosophila specify eye and antenna

The formation of different structures in Drosophila depends on the combined activities of selector genes and signaling pathways. For instance, the antenna requires the selector gene homothorax, which distinguishes between the leg and the antenna and can specify distal antenna if expressed ectopically. Similarly, the eye is formed by a group of 'eye-specifying' genes, among them eyeless, which can direct eye development ectopically. hernandez (distal antenna related or danr) and fernandez (distal antenna or dan) are expressed in the antennal and eye primordia of the eye-antenna imaginal disc (see Dan and Danr). Hernandez and Fernandez are the names of twin brothers in Tintin comic-books. The predicted proteins encoded by these two genes have 27% common amino acids and include a Pipsqueak domain. Reduced expression of either hernandez or fernandez mildly affects antenna and eye development, while the inactivation of both genes partially transforms distal antenna into leg. Ectopic expression of either of the two genes results in two different phenotypes: such expression can form distal antenna, activating genes like homothorax, spineless, and spalt, and can promote eye development and activates eyeless. Reciprocally, eyeless can induce hernandez and fernandez expression, and homothorax and spineless can activate both hernandez and fernandez when ectopically expressed. The formation of eye by these genes seems to require Notch signaling, since both the induction of ectopic eyes and the activation of eyeless by the hernandez gene are suppressed when the Notch function is compromised. These results show that the hernandez and fernandez genes are required for antennal and eye development and are also able to specify eye or antenna ectopically (Suzanne, 2003).

Signaling pathways can modify the activity of selector genes and are needed for proper organ formation. N signaling, for instance, is needed for eye formation and can activate ey when ectopically activated. Moreover, N has been implicated in the decision of making eye or antenna, directing eye development, and suppressing antenna formation. Therefore, whether N signaling could alter the ey and elav expression induced by the Tintin genes was examined. The coexpression of the hern gene and a dominant negative form of the Notch receptor substantially reduces ey and eliminates elav ectopic signals. Accordingly, no ectopic eyes are formed in this genetic combination. This indicates that the effect of hern on ey expression and eye formation requires N signaling (Suzanne, 2003).

Two recent models have been proposed to explain the specification of eye and antenna within the eye-antennal disc. Both models suggest that the activation of the N signaling pathway is a key element in this process. It has been suggested that N signaling activates both ey and Dll in the eye and antennal primordia; subsequently, ey represses Dll in the eye and perhaps the hth and extradenticle genes repress ey in the antenna. In this way, the exclusive expression of ey (in the eye) and Dll and hth (in the antenna) determine eye and antenna identity, respectively. It has been proposed that the N and Egfr signaling pathways (together with the hedgehog and wg genes) are instrumental in the decisions to make eye or antenna. N signaling has been proposed to promote eye development and prevents formation of the antenna, whereas Egfr signaling does the opposite. Ectopic expression of either hern or fer in the antenna induces ectopic eyes and activates ey and elav, but the coexpression of hern and an N dominant-negative protein does not result in ectopic eyes and almost eliminates ey and elav activation. This suggests that N function impinges on hern activity to form ectopic eyes. As in other cases, the combined activity of signaling pathways and selector genes determine the specification of different structures (Suzanne, 2003).

Identification of the death zone: a spatially restricted region for programmed cell death that sculpts the fly eye

Programmed cell death (PCD) sculpts many developing tissues. The final patterning step of the Drosophila retina is the elimination, through PCD, of a subset of interommatidial lattice cells during pupation. It is not understood how this process is spatially regulated to ensure that cells die in the proper positions. To address this, PCD of lattice cells in the pupal retina was observed in real time. This live-visualization method demonstrates that lattice cell apoptosis is a highly specific process. In all, 85% of lattice cells die in exclusive 'death zone' positions between adjacent ommatidia. In contrast, cells that make specific contacts with primary pigment cells are protected from death. Two signaling pathways, Drosophila epidermal growth factor receptor (Egfr) and Notch, that are thought to be central to the regulation of lattice cell survival and death, are not sufficient to establish the death zone. Thus, application of live visualization to the fly eye gives new insight into a dynamic developmental process (Monserate, 2006).

The Drosophila pupal retina was used as a model system to study spatial regulation of PCD. Apoptosis of a subset of lattice cells in the mid-pupal retina is the final patterning step in sculpting the fly eye. The static nature of ultrastructural studies made it impossible to determine whether dying lattice cells occupied specific positions between ommatidia, Live visualization of the pupal retina is advantageous because it not only allows snapshots to capture specific events but also allows one to identify an apoptotic cell, and essentially look back in time to assign the cell's position before death (Monserate, 2006).

Using time-lapse imaging of the eye of the living pupa, dying cells were identified as they lost their apical footprint, one of the earliest death events. It was determined that 50% of apoptotic lattice cells occupied a position next to the bristle group and in the horizontal face between ommatidial units. Furthermore, 35% of dying lattice cells were located next to the bristle group along an oblique face. Thus, a remarkable 85% of apoptotic lattice cells died in two very specific regions between ommatidial units and adjacent to bristle groups (Monserate, 2006).

However, bristle groups cannot be the origin of the death signal; removal of bristle groups does not block apoptosis. Instead, in the absence of bristle groups, the pattern of lattice cell death changed dramatically. Lattice cells at either the anterior or posterior portion of the horizontal face now had roughly an equal chance of dying. Together, the two horizontal regions accounted for 85% of the lattice cells undergoing PCD. It is concluded that lattice cell PCD is regulated spatially so that apoptosis removes cells occupying the anterior and posterior horizontal positions between ommatidia. This region was termed the 'death zone'. Between these two points of position-specific PCD, a single cell is protected from apoptosis. Apoptosis in the death zone ends when only this cell remains in the horizontal face between ommatidia (Monserate, 2006).

How could bristle groups so dramatically affect the pattern of PCD? One possibility is that the bristle groups attract cells to their death in the death zones. It has been shown that bristle groups secrete Spi and the data demonstrate that lattice cells respond by surrounding the source of secreted Spi. Just prior to the cell death period, there are on average 1.6 times more cells in the regions around the bristle groups than in other vertices. Additionally, in the absence of bristle groups, more cells appear to populate the horizontal regions prior to death (at the expense of the oblique regions) (Monserate, 2006).

Survival of lattice cells depends on a signal, hypothesized to be secretion of Spi, from primary pigment cells and/or cone cells. The experiments confirm that lattice cells transduce a dEgfr signal, but the interpretation is complicated by the fact that P-MAPK wss not detected in lattice cell nuclei, suggesting that none of the cells respond transcriptionally to Egfr signaling. Instead, P-MAPK was identified in the cytoplasm of all lattice cells, including those in the death zone. It was recently reported that retaining P-MAPK in the cytoplasm in cells in the larval eye disc is important for normal development. In the current study, it is unlikely that blocking of nuclear MAPK signaling by 'cytoplasmic hold' is required to induce apoptosis because ectopic transcriptional repression of activated MAPK targets by expression of the activated form of aop had no effect on apoptosis in the pupal retina. Taken together, these experiments argue that Egfr signaling in lattice cells serves solely to activate P-MAPK in the cytoplasm. Cytoplasmic P-MAPK may suppress apoptosis through phosphorylation of cytoplasmic proteins such as HID (Monserate, 2006).

In light of the fact that Notch is upstream of Egfr, a new model was put forth: primary pigment cells and/or cone cells promote survival by inhibiting Notch activation and its disruption of Egfr signaling in lattice cells. It was possible to directly test this model because the live-visualization data predicted the pattern of death signals. The data do not support this hypothesis as all lattice cells, whether living or dying, are transducing a Notch signal; surviving lattice cells show no downregulation or inhibition of Notch signaling. Instead it is proposed that Notch dampens the Spi-activated dEgfr survival signaling in all lattice cells. Thus, experimental removal of Notch (i.e., heat shock of Nts1) would cause a general increase of Egfr signaling (and survival of all cells). Conversely, loss of Egfr signaling would lead to PCD, the default pathway. This model predicts that Notch transcriptionally activates a gene whose protein product keeps dEgfr signaling poised. A second scenario is one in which the Notch protein may itself influence the life or death decision of a cell. It has been suggested that Notch acts through a noncanonical pathway. In this model, Notch might function to activate PCD only in death zone cells, acting as a third signal (Monserate, 2006).

How is the death zone molecularly defined? Cells in the horizontal region are more sensitive to a reduction of Egfr signaling, but, as demonstrated by P-MAPK levels, this is unlikely to be owing to a lower level of Egfr signaling. Thus, if the death zone is defined by Egfr signaling, this may be owing to a qualitative change in cytoplasmic P-MAPK activity. A more likely possibility is that Egfr signaling (dampened by Notch) merely poises the cell to allow a third signal to induce PCD specifically in the death zone. In support of this idea is the finding that cells in the death zone regions are more likely than cells in other regions to die in response to a brief interruption in dEgfr signaling (Monserate, 2006).

Localization of apoptosis in the lattice to death zone regions brings additional complexity to the problem of PCD regulation. Clearly, the signal that regulates this final cell fate must be localized. The most likely candidate cell to produce the regulating signal is the primary pigment cell. However, each primary pigment cell contacts numerous lattice cells; some that will survive and some that will die. Thus, a model is suggested in which the dorsal and ventral regions of each primary pigment cell localize a death-inducing factor. Evidence for such a subcellular domain has been seen in primary pigment cells. The contact regions at which the two primary pigment cells meet must either block this signal or localize a strong survival signal (Monserate, 2006).

Placed in the context of other examples in which the architecture of a tissue is shaped through selective PCD, the Drosophila retina is a unique model in that it can be manipulated genetically, microscopically and development can be visualized in the living animal. The determination that apoptosis is spatially regulated in the pupal eye directs future experiments designed to identify how the death zone is created (Monserate, 2006).

Coordinating proliferation and tissue specification to promote regional identity in the Drosophila head

The Decapentaplegic and Notch signaling pathways are thought to direct regional specification in the Drosophila eye-antennal epithelium by controlling the expression of selector genes for the eye (Eyeless/Pax6, Eyes absent) and/or antenna (Distal-less). The function of these signaling pathways in this process has been investigated. Organ primordia formation is indeed controlled at the level of Decapentaplegic expression but critical steps in regional specification occur earlier than previously proposed. Contrary to previous findings, Notch does not specify eye field identity by promoting Eyeless expression but it influences eye primordium formation through its control of proliferation. Analysis of Notch function reveals an important connection between proliferation, field size, and regional specification. It is proposed that field size modulates the interaction between the Decapentaplegic and Wingless pathways, thereby linking proliferation and patterning in eye primordium development (Kenyon, 2003).

This paper analyzes the role of Dpp and Notch in the regional specification of the eye-antennal disc. This study makes four observations: (1) domains of regional identity emerge in a complex pattern starting early in L2; (2) formation of eye and antenna primordia depend upon specific domains of dpp expression that emerge in early-L2 (eye) and mid-L2 (antenna); (3) neither Notch nor Dpp control the establishment of separate eye and antennal fields; (4) Notch can influence the establishment of an eye primordium through its control of proliferation in the eye field. Current models of regional specification have been evaluated based on these results and a new perspective on the emergence of regional identity in this tissue is presented (Kenyon, 2003).

Notch is thought to function upstream of the Ey/Pax6 pathway in promoting eye field identity. Expression of Notch antagonists in the developing discs leads to adult flies lacking eyes and sometimes displaying double antennae; this latter phenotype has been interpreted as evidence for a transformation of the eye field into a second antennal field. However, eye and antennal selector gene expression is not perturbed in loss-of-function Notch mutant backgrounds (N54/9, Nts1, and Nts2. Furthermore, expression of Ey and Dll is normally restricted to the eye field and the antennal field, respectively, in late-L2 SerDN or NDN discs. These observations indicate that Notch does not control the specification of separate eye and antennal fields in L2 (Kenyon, 2003).

The occasional appearance of double antennae in flies expressing SerDN and NDN results from a duplication of the antenna primordium rather than an eye-to-antenna transformation. Antenna duplications are frequently observed after surgical removal of eye tissue and have also been observed in mutant backgrounds that cause suppression of proliferation and/or cell death in the eye field. Thus, the formation of dual antennae in ey-Gal4 UAS-SerDN or ey-Gal4 UAS-NDN discs is likely due to the extreme suppression of proliferation observed in these genetic backgrounds (Kenyon, 2003).

The proposal that Notch controls Ey in Drosophila has been extended to the control of Pax6 in vertebrates. Although it is concluded that Notch does not regulate the Ey/Pax6 pathway in the fly, Notch does influence regional specification through its control of proliferation. Hence, the interactions uncovered in vertebrates between Notch signaling and the Ey/Pax6 pathway may reflect a nonconserved aspect of eye development or alternatively an indirect relationship between Pax6 and Notch (Kenyon, 2003).

Although not a direct inducer of regional identity, Notch function is essential for the emergence of an eye primordium. In Nts1and Nts2 discs, onset of Eya expression is delayed. In ey-Gal4 UAS-SerDN discs, Eya expression is always severely affected and most often completely absent. Yet, in these discs, dpp is still expressed; hence lack of Eya expression is not due to the absence of this inducer. The observation that stimulating cell division is sufficient to rescue eye primordium formation in ey-Gal4 UAS-SerDN discs indicates that Eya induction depends upon cell proliferation or field size rather than Notch signaling per se. Since the normal onset of Eya expression in early-L2 does not correlate with the initiation of cell proliferation at mid-late-L1 and the stimulation of proliferation by constitutive Notch signaling (NIntra) is not sufficient to directly induce Eya, the latter explanation, namely that Eya expression is influenced by the size of the morphogenetic field as opposed to cell division, is preferred (Kenyon, 2003).

In conclusion, this analysis of the effect of Notch on regional identity at the L2 stage reveals an important connection between proliferation, field size, and eye primordium formation. Through its control of proliferation in the eye field, Notch may indeed participate in generating the specific signaling environment that promotes the expression of Eya and the emergence of an eye primordium. Control of field size likely reflects a general mechanism through which cell proliferation can influence the patterning of a morphogenetic field and thereby contribute to the coordination of proliferation and patterning essential to the proper development of complex multicellular organisms (Kenyon, 2003).

The roles of cis-inactivation by Notch ligands and of neuralized during eye and bristle patterning in Drosophila

The receptor protein Notch and its ligand Delta are expressed throughout proneural regions yet non-neural precursor cells are defined by Notch activity and neural precursor cells by Notch inactivity. Not even Delta overexpression activates Notch in neural precursor cells. It is possible that future neural cells are protected by cis-inactivation, in which ligands block activation of Notch within the same cell. The Delta-ubiquitin ligase Neuralized has been proposed to antagonize cis-inactivation, favoring Notch activation. Cis-inactivation and the role of Neuralized has not yet been studied in tissues where neural precursor cells are resistant to nearby Delta, however, such as the R8 cells of the eye or the bristle precursor cells of the epidermis. Overexpressed ligands block Notch signal transduction cell-autonomously in non-neural cells of the epidermis and retina, but do not activate Notch nonautonomously in neural cells. High ligand expression levels are required for cis-inactivation, and Serrate is more effective than Delta, although Delta is the ligand normally regulating neural specification. Differences between Serrate and Delta depend on the extracellular domains of the respective proteins. Neuralized acts cell nonautonomously in signal-sending cells during eye development, inconsistent with the view that Neuralized antagonizes cis-inactivation in non-neural cells. It is concluded that Delta and Neuralized contribute cell nonautonomously to Notch signaling in neurogenesis, and the model that Neuralized antagonizes cis-inactivation to permit Notch activity and specification of non-neural cells is refuted. The molecular mechanism rendering Notch insensitive to paracrine activation in neural precursor cells remains uncertain (Li, 2004).

One difference between neural and non-neural cells may be neur, which has been proposed to relieve cis-inactivation cell autonomously by endocytosing Dl, or to promote paracrine signaling in experiments where neur appears nonautonomous. Neur might make non-neural cells less sensitive to cis-inactivation, so that only high Delta levels would be effective (Li, 2004).

Cell autonomy of neur function in the eye was investigated using FLP-mediated mitotic recombination in neur heterozygous larve to induce cell clones homozygous for neur1, a loss of function neur allele. Mitotic recombination was induced late in the third larval instar to generate small neur mutant clones. Mosaic adult eyes were sectioned and the cellular contribution of neur mutant cells recorded. In many cases presence of neur mutant cells was associated with changes in the number of photoreceptor cells. Ommatidia with too many or too few photoreceptor cells were both observed, as for other neurogenic mutations. Less often, ommatidia containing one or more neur mutant cells differentiated 8 photoreceptor cells in the normal arrangement. Forty such mosaic ommatidia were examined in more detail to identify any cells where neur function might be dispensable (Li, 2004).

Ommatidia almost never developed normally with neur mutant R8 cells. Only a single example was found. If neur activates N signaling by antagonizing cis-inactivation, then one would expect that neur would be required in cells where N is active, but dispensable where N is inactive. On this basis neur should not be required in R8 cells. By contrast the data suggested that R8 is where neur is most important. It has been found that Dl is also required in R8 cells. The possibility is excluded that either neur or Dl is required directly in the execution of the R8 differentiation pathway because many ectopic R8 cells differentiate in large neur or Dl mutant clones, or when the whole eye is mutant. Instead the data suggest that ommatidia with neur or Dl mutant R8 cells could not develop normally because neur acts in R8 to promote Dl-mediated activation of N in neighboring cells (Li, 2004).

To explore further when neur acts autonomously or nonautonomously, other aspects of retinal N signaling were also examined. During ommatidial development, Notch signaling breaks the symmetry of the R3/R4 pair. Dl from R3 activates N in R4. neur mutant cells were five times as likely to take R4 fate as R3 fate. Thus neur is important for the nonautonomous signaling activity of Dl from the R3 cell but not required autonomously for activity of N in the R4 cell. Only rarely can a neur mutant R3 cell activate N in a neighboring R4 cell, but neur R4 cells can be activated in response to wild type R3 cells (Li, 2004).

Further data from abnormally constructed ommatidia support the importance of neur in R3. These were ommatidia where the R3/R4 pair remained symmetrical. 17 symmetrical ommatidia were found with two R3 cells in place of R3/R4. In 3 such ommatidia both R3-like cells were mutant for neur. In 13 of the other 14 cases the cell in the location that should normally have become R3 was neur mutant; in a single case the cell positioned to become R4 was neur mutant. These symmetrical ommatidia indicate that when R3 cells lack neur function, the neighboring cell receives insufficient Dl signaling to take R4 fate and instead is transformed into a second R3 (Li, 2004).

N signaling is further required for R7 specification. N is activated in R7 precursors by Dl from neighboring R1 and R6 cells. R1 and R6 act redundantly but if both R1 and R6 are Dl mutant then the R7 precursor adopts R1/6-like morphology. R7 was frequently neur mutant in normally-constructed ommatidia, so neur is not essential in the R7 precursor cell. R1 and R6 were never both mutant for neur in normal ommatidia. One ommatidium was found in which both R1 and R6 were neur mutant. In this ommatidium the cell in the R7 position was wild type for neur but had R1/6-like morphology. These results indicate that neur, like Dl, is not required for N activity in the R7 cell itself. neur may be required nonautonomously in R1 and R6 for proper R7 specification (Li, 2004).

A role for Wingless and Notch in an early pupal cell death event that contributes to patterning the Drosophila eye

Programmed cell death (PCD) is utilized in a wide variety of tissues to refine structure in developing tissues and organs. However, little is understood about the mechanisms that, within a developing epithelium, combine signals to selectively remove some cells while sparing essential neighbors. One popular system for studying this question is the developing Drosophila pupal retina, where excess interommatidial support cells are removed to refine the patterned ommatidial array. Data is presented indicating that PCD occurs earlier within the pupal retina than previously demonstrated. As with later PCD, this death is dependent on Notch activity. Surprisingly, altering Drosophila Epidermal Growth Factor Receptor or Ras pathway activity has no effect on this death. Instead, a role for Wingless signaling is indicated in provoking this cell death. Together, these signals regulate an intermediate step in the selective removal of unneeded interommatidial cells that is necessary for a precise retinal pattern (Cordero, 2004).

In the course of examining hid mutant retinae, it was noticed that blocking cell death in the earliest pupal stages -- prior to known stages of cell death -- led to a clear increase in the number of interommatidial cells. With this in mind, pupal retinas was examined at earlier developmental stages from 14 to 24 h APF by using an antibody to the junction protein Armadillo; apoptotic cell death was also directly assessed with TUNEL staining. Prior to approximately 20 h APF, the retina is composed of a loosely patterned array of ommatidia consisting of photoreceptor neurons and cone cells; primary pigment cells (1°s) first emerge and enwrap the cone cells at 20 h APF, and secondary and tertiary pigment cells (2°/3°s) begin organizing at about this stage as well. Approximately one third of the interommatidial cells observed at 24 h APF (25°C) are selected to die by PCD during the following 10-12 h (Cordero, 2004).

Prior to 18 h APF, no significant amount of death was observed. At 18 h APF, a sharp band of death was observed in the anterior portion of the retina; some of this death is within the retina, and some is just outside the retinal field. Between 20 and 24 h APF, additional death is observed towards the middle of the retina in addition to the anterior death band. Levels of apoptosis are highest in anterior regions of the eye, but the center of the eye, for example, also contains significant levels of death. At 24 h APF, this early wave of death rapidly declines; the remaining interommatidial cells have reorganized end-to-end by this stage. At 26 h APF, the known, previously described burst of death commences. The increasing amount of TUNEL staining correlates with a decrease in the number of interommatidial cells. These results indicated that the pupal retina undergoes two separate surges of cell death that occur between 18-24 and 26-36 h APF; for convenience, these events are referred to as 'early-stage' and 'late-stage' cell death events in the pupal eye, respectively. During the early-stage death 1.8 cells are removed per ommatidia. The early-stage has not been described previously, and it was examined whether the pathways known to regulate late-stage death also regulate its predecessor (Cordero, 2004).

The baculovirus protein P35 interferes with apoptosis by binding to and inhibiting caspase activity; it is effective in inhibiting cell death including late-stage death in the Drosophila eye. Targeted over-expression of P35 with the eye-specific promoter GMR led to a near complete block of early-stage death: only a line of anterior cell death remained in GMR-p35 retinas. This result indicates that the early-stage cell death occurred by caspase-dependent apoptosis. In addition, it confirmed the assessment, based on TUNEL staining, that some of the anterior-most apoptotic cell death occurs in a region of future head cuticle just anterior to the retina (and is therefore outside of the expression domain of the GMR promoter (Cordero, 2004).

The head involution defective hid gene is a central regulator of cell death in Drosophila including late-stage cell death pathway in the retina. Hid induces PCD through activation of caspases. Retinas lacking functional hid activity looses all evidence of early-stage PCD. The number of cells within the GMR-p35 and hid-/- retinas at 20 and 21 h APF, respectively was in fact higher than the number of cells in a 18 h APF control retina. In these mutant genotypes the ommatidia are disorganized when compared with the control retinas due to the excess of cells. It was often found that hid-/- retinas are attached to what seems to be the antennal disc, suggesting that this early-stage death may include events required for separation of the eye-antennal discs. Together these results suggest that, similar to late-stage death, early-stage death is regulated by a caspase-mediated apoptosis pathway (Cordero, 2004).

The Egfr/Ras-1 pathway has been implicated in multiple stages of fly eye development including cell proliferation, survival and differentiation. Loss of function mutations in the Egfr leads to excessive cell death of the interommatidial cells. Activation of Egfr leads to activation of dRas1, which promotes cell survival by repressing the activity and expression of hid (Cordero, 2004).

Activated Egfr and dRas1V12 was expressed under the control of an inducible, heat shock promoter. As expected, late-stage cell death (26 h APF) is almost completely blocked by each transgene. Surprisingly, no alteration was seen in either the pattern of death or the cell number in 21 h APF retinas, suggesting that early-stage death is insensitive to the Egfr/dRas1 pathway. Consistent with these results, no effect on cell death was seen upon over-expression of the Egfr antagonist Argos. These findings are especially surprising because of the results indicating that hid is required for early-stage death: unlike larval or late-stage death, hid activity appears to be regulated by a Egfr-independent mechanism during early-stage cell death (Cordero, 2004).

Notch pathway signaling is also required to remove excess interommatidial cells during late-stage cell death. Using the temperature-sensitive allele Nts1, Notch function was reduced during early-stage cell death. A significant reduction in TUNEL positive cell was seen in Nts1 retinas when compared with controls; this correlates with the presence of excess cells in the Nts1 background. However, a role for Notch in directly regulating this cell death cannot be unambiguously assigned. In a normal 21 h APF retina, 1°s have emerged to enwrap the ommatidia and interommatidial cells have already re-organized. Reduction of Notch activity between 14 and 21 h APF leads to a block in 1° differentiation, a failure of interommatidial cells to re-organize, and improper ommatidia alignment. This is consistent with previous reports on the effects of reducing Notch in the developing retina (Cordero, 2004).

Reducing activity of the downstream wingless inhibitor has been shown to provoke cell death of photoreceptor neurons at late stages in the developing retina; its role during the stages of patterning and cell fate determination has not been assessed. The effects of inhibiting the wingless pathway on early-stage death in the pupal retina was tested by placing a temperature-sensitive allele in trans to a null. A small but significant decrease was observed in cell death in wgts/- retinas shifted to the non-permissive temperature beginning at the earliest steps in early-stage death. Similar results were observed in wg-/- clones. These data suggest that the Notch and wingless pathways provide a signal that is necessary to provoke early-stage cell death. Wingless localization primarily in the cone cells was identified by antibody staining, implicating these cells as the source of Wingless protein. Similar results were observed using an enhancer trap line. Interestingly, during late-stage cell death -- which requires different pathways such Egfr/dRas1 -- 1°s are required to regulate cell death. Early-stage cell death occurs through the period that the 1°s emerge, and one possibility is that the cone cells make use of a different signaling pathway to distinguish their influence on the interommatidial cells from the 1°s (Cordero, 2004).

These data indicate that the Notch and wingless pathways provide a signal that is required for cell death to occur during early-stage apoptosis in the Drosophila retina. Localization studies suggest that the cone cells provide Wingless to the surrounding interommatidial cells. In the wing, Notch and wingless regulate expression of each other at the DV boundary; loss of wingless activity leads to an increase in cell death in the wing, an activity opposite that of the loss of death observed in the retina. Both pathways are involved in early-stage death in the pupal retina, and further studies will be required to determine if mutual regulation occurs in the context of the retina as well. Surprisingly, no effects were found of Egfr/dRas-1 signaling during early-stage cell death. This observation leaves open the question as to which pathway opposes wingless and Notch activity during early-stage events; this opposition would ensure that a sufficient number of cells survive to undergo the second round of selection during late-stage death (Cordero, 2004).

Extracellular signals responsible for spatially regulated proliferation in the differentiating Drosophila eye

Spatially and temporally choreographed cell cycles accompany the differentiation of the Drosophila retina. The extracellular signals that control these patterns have been identified through mosaic analysis of mutations in signal transduction pathways. All cells arrest in G1 prior to the start of neurogenesis. Arrest depends on Dpp and Hh, acting redundantly. Most cells then go through a synchronous round of cell division before fate specification and terminal cell cycle exit. Cell cycle entry is induced by Notch signaling and opposed in subsets of cells by EGF receptor activity. Unusually, Cyclin E levels are not limiting for retinal cell cycles. Rbf/E2F and the Cyclin E antagonist Dacapo are important, however. All retinal cells, including the postmitotic photoreceptor neurons, continue dividing when rbf and dacapo are mutated simultaneously. These studies identify the specific extracellular signals that pattern the retinal cell cycles and show how differentiation can be uncoupled from cell cycle exit (Firth, 2005).

The EGFR holds R2-R5 cells in G1 phase and promotes G2/M progression of other cells during the second mitotic wave (SMW). Earlier regulation is now found to depend on longer-range signaling by the Hh, DPP, and N signals already known to drive the progression of the morphogenetic furrow. These studies exclude other models that show that Hh, Dpp, or N act indirectly by releasing other, cell cycle-specific signals from differentiating cells, or that patterned cell cycle withdrawal or reentry occur independent of extracellular signals, such as by synchronized growth. Instead, specific signals are necessary or sufficient for each aspect of cell cycle patterning (Firth, 2005).

G1 arrest ahead of the morphogenetic furrow depends on posterior-to-anterior spread of Hh and Dpp. Hh is secreted from differentiating cells, starting at column 0 in the morphogenetic furrow. Dpp is transcribed in ~6 ommatidial columns in the morphogenetic furrow in response to Hh. Cells accumulate in G1 about 16-17 cell diameters anterior to column 0, suggesting an effective range of ~13-17 cells for Hh and Dpp (Firth, 2005).

The contribution of Dpp to this cell cycle arrest is known already, but that of Hh was not suspected. Both Dpp and Hh signaling can promote proliferation in other developmental contexts (Firth, 2005).

S phase entry in the SMW depends on another signal, N. Expression of the N ligand Dl begins at the anterior of the morphogenetic furrow. The first S phase cells are detected 6-8 cell diameters more posteriorly, just behind column 0. The transmembrane protein Dl must act more locally or more slowly than the secreted Hh and Dpp proteins, to explain gaps between S phases (Firth, 2005).

Although N activity has been associated with growth through indirect mechanisms involving the release of other secreted growth factors and also regulates endocycles, this appears to be the first report of a specific role of N in G1/S in diploid Drosophila cells. Notably, deregulated N signaling contributes to at least two human cancers and is oncogenic in mice (Firth, 2005).

At the same time that N promotes S phase entry in the SMW, EGFR activity ensures that R2-R5 cells remain in G1. N is still required in the absence of EGFR, so N activity is a positive signal and is not required only to counteract EGFR activity. Instead, EGFR activity interferes with S phase entry in response to N (Firth, 2005).

Ligands for the EGF receptor are thought to be released from R8 precursor cells, although EGFR-dependent MAPK phosphorylation is detected one ommatidial column before the column where R8 precursor cells can be identified, which is in column 0. This means that EGFR activation begins after Dl expression but before S phase DNA synthesis starts. Later, ligands released from differentiating precluster cells activate EGFR in surrounding cells to permit SMW mitosis around columns 3-5 (Firth, 2005).

Hh and Dpp together promote expression of Dl and of EGFR ligands; in part, this occurs indirectly through Atonal and the onset of differentiation. EGF receptor activity also promotes Dl expression (Firth, 2005).

At least three genetic mechanisms arrest distinct retinal cells in G1. Arrest ahead of the morphogenetic furrow depends on Dpp and Hh. During the SMW, R2-R5 cells are held in G1 by EGFR, which counteracts the SMW-promoting N activity. In addition, R8 cells, which are defined by the proneural gene atonal, remain in G1 independent of EGFR. After the SMW, all cells remain in G1 indefinitely, independent of EGFR. Although cell cycle withdrawal roughly correlates with differentiation, many of the cells that arrest after the SMW are still unspecified (Firth, 2005).

Loss of rbf and dap together overcome all cell cycle blocks, even though cell differentiation continues. This redundancy indicates that Cyclin E/Cdk2 targets other than Rbf are needed for proliferation, consistent with many other studies. Dap may be regulated by EGFR in R2-R5 cells. If rbf regulates the normal SMW, where Cyclin E expression seems not to be limiting, then other E2F targets may be involved. Some cell cycle arrest can also be overridden by forced expression of Cyclin E, E2F/DP, dRef, and ORC1, or by mutation of the Cyclin A antagonist rux (Firth, 2005).

The results show that mechanisms that assure both short- and long-term arrest of retinal cells must operate upstream of (or parallel to) Rbf and Cyclin E activities. They might resemble the barriers to transformation and regeneration that exist in mammals (Firth, 2005).

Control of cell proliferation in the Drosophila eye by Notch signaling

Cell proliferation in animals must be precisely controlled, but the signaling mechanisms that regulate the cell cycle are not well characterized. A regulated terminal mitosis, called the second mitotic wave (SMW), occurs during Drosophila eye development, providing a model for the genetic analysis of proliferation control. This study reports a cell cycle checkpoint at the G1-S transition that initiates the SMW; Notch signaling is required for cells to overcome this checkpoint. Notch triggers the onset of proliferation by multiple pathways, including the activation of dE2F1, a member of the E2F transcription factor family. Delta to Notch signaling derepresses the inhibition of dE2F1 by RBF, and Delta expression depends on the secreted proteins Hedgehog and Dpp. Notch is also required for the expression of Cyclin A in the SMW (Baonza, 2005).

This work identifies a new cell cycle checkpoint in the second mitotic wave and describes how intercellular signaling overcomes this checkpoint. Delta signaling to Notch triggers a progression from G1 arrest in the morphogenetic furrow into the S phase of the terminal mitosis. Two effectors of this Notch requirement have been identified, dE2F1 transcriptional activity and cyclin A expression. Although the data imply that at least one other target also exists, this is unidentified. The data preclude this additional factor from being Cyclin E. Previous work has identified a later SMW checkpoint, at the G2-M transition. Together, Notch and the EGFR therefore coordinately provide spatial and temporal control of the cell cycle in the SMW (Baonza, 2005).

These results led to a proposal of the following course of events. Notch is activated by the uniform band of Delta in all cells as they emerge from the morphogenetic furrow. Cells that are uncommitted thereby enter S phase, whereas cells that are part of the precluster are blocked from responding and remain in G1. It has been shown that the G1 arrest of precluster cells is dependent on EGFR activation, although the details of the mechanism remain unclear. One of the consequences of EGFR activation in precluster cells is the upregulation of Delta expression. Cells between the preclusters would therefore end up initially receiving low-level uniform Delta, later reinforced by the upregulated Delta in the adjacent preclusters. Together, these provide a robust and modulated activation of Notch in cells that will enter the SMW (Baonza, 2005).

It is emphasized that this work uncovers a normal developmental function for Notch signaling only in the control of a specific terminal mitotic cycle. The fact that clones of Notch and Delta mutant cells can be generated implies that they are not required for the earlier, unpatterned proliferation ahead of the morphogenetic furrow. Similarly, the ability to make clones in other imaginal discs indicates that there is no requirement for Delta/Notch signaling in most cell proliferation in Drosophila. Rather, this signal requirement, and the subsequent EGFR-dependent entry into mitosis, is superimposed upon normal controls in this regulated terminal mitosis. Moreover, the ability of Notch signaling to initiate S phase is restricted to a short period. Notch has other functions later in eye development, and it has been shown that later ectopic signaling does not lead to additional proliferation. Nevertheless, Delta-expressing clones in other tissues also hyperproliferate, suggesting that ectopic Notch activity has a wider ability to trigger inappropriate proliferation (Baonza, 2005).

The EGFR and Notch signal systems play distinct roles in regulating the SMW. After completing its preliminary role in maintaining cells in G1 arrest, EGFR signaling ensures that cells only undergo mitosis if they are adjacent to developing clusters, thereby matching the number of cells born with the number that will be required to complete ommatidial differentiation. In contrast, Notch initiates the whole process by regulating whether cells emerging from the morphogenetic furrow enter the SMW or remain arrested in G1 and start to differentiate (Baonza, 2005).

The secreted protein Hedgehog has a primary role in the forward movement of the morphogenetic furrow. Hedgehog also has an important function in initiating and coordinating the onset of the SMW, specifically the initiation of S phase. Hh and Dpp together lead to the expression of Delta in the furrow. Furthermore, Hh is essential for the expression of cyclin D and cyclin E in the morphogenetic furrow, whereas Cyclin E is the main cyclin that regulates S phase onset. These data imply that Hedgehog signaling activates several independent branches of the pathway that lead to the onset of S phase in the SMW. Incidentally, the observation that Cyclin E accumulates in Notch mutant clones, which lack dE2F1 activity, indicates that, at least in this context, Cyclin E is not sufficient to inhibit RBF and thereby activate dE2F1 activity (Baonza, 2005).

Cyclin A is best characterized as a mitotic cyclin, and its destruction is a key step in the completion of mitosis. An additional function in the onset of S phase in Drosophila remains enigmatic. Mammalian Cyclin A and its associated kinase Cdk2 can drive G1 cell extracts into S phase, and anti-Cyclin A antibodies can block S phase in injected cells. But, in Drosophila, S phase can proceed normally in the absence of Cyclin A and Cyclin A does not bind Cdk2. Nevertheless, when overexpressed, Cyclin A can overcome the lack of Cyclin E and allow cells to enter S phase. Furthermore, overexpression of the Cyclin A inhibitor Roughex blocks entry into S phase in embryos, and roughex mutants show precocious S phase entry in the SMW. Ectopic BrdU incorporation is observed in the eye disc when Cyclin A is misexpressed. The data indicating that Cyclin A is one of the targets of Notch signaling further support the idea that Cyclin A is part of the machinery that controls the onset of S phase in the SMW (Baonza, 2005).

Notch signaling in mammals, as in flies, is pleiotropic and context dependent. This is highlighted in human cancer, where Notch is oncogenic in a number of cases, particularly in hematopoietic neoplasms, but in other contexts has tumor suppressor functions. Moreover, although the current work highlights a proliferative function, it has been shown that Notch inhibits proliferation in the wing disc. Notwithstanding this caveat, it is striking that Notch activity can be hyperproliferative in humans and in Drosophila, and little is known about this proliferative response. It has recently been shown in the developing Drosophila central nervous system that Notch activity can maintain cells in a proliferative state by antagonizing the p21/p27 homolog Dacapo, thereby maintaining Cyclin E expression. Similarly, the dacapo gene is downregulated in response to Notch in the mitotic-to-endocycle transition in Drosophila follicle cells. This work describes a different mechanism: Notch signaling overcomes a G1-S checkpoint via the activation of universally conserved cell cycle components, RBF1, dE2F1, and possibly Cyclin A. Although tempting to speculate that these data may provide some insight into oncogenic mechanisms, it will be important to ascertain whether the particular relationships between Notch and the core machinery that triggers S phase is indeed conserved. In fact, the data imply that Notch probably also influences the mitotic cycle at other points. If the only role of Notch were to advance cells into S phase, they would simply arrest at the next checkpoint, G2-M. The fact that Notch activity leads to overgrowth therefore implies that Notch can also, directly or indirectly, drive cells through the subsequent G2-M checkpoint (Baonza, 2005).

Transformation of eye to antenna by misexpression of a single gene

In Drosophila, the eye and antenna originate from a single epithelium termed the eye-antennal imaginal disc. Illumination of the mechanisms that subdivide this epithelium into eye and antenna would enhance understanding of the mechanisms that restrict stem cell fate. This study shows that Dorsal interacting protein 3 (Dip3), a transcription factor required for eye development, alters fate determination when misexpressed in the early eye-antennal disc, and this observation has been taken advantage of to gain new insight into the mechanisms controlling the eye-antennal switch. Dip3 misexpression yields extra antennae by two distinct mechanisms: the splitting of the antennal field into multiple antennal domains (antennal duplication), and the transformation of the eye disc to an antennal fate. Antennal duplication requires Dip3-induced under proliferation of the eye disc and concurrent over proliferation of the antennal disc. While previous studies have shown that overgrowth of the antennal disc can lead to antennal duplication, these results show that overgrowth is not sufficient for antennal duplication, which may require additional signals perhaps from the eye disc. Eye-to-antennal transformation appears to result from the combination of antennal selector gene activation, eye determination gene repression, and cell cycle perturbation in the eye disc. Both antennal duplication and eye-to-antennal transformation are suppressed by the expression of genes that drive the cell cycle providing support for tight coupling of cell fate determination and cell cycle control. The finding that this transformation occurs only in the eye disc, and not in other imaginal discs, suggests a close developmental and therefore evolutionary relationship between eyes and antennae (Duong, 2008a).

Dip3 is able to bind DNA in a sequence specific manner and activate transcription directly. Dip3 possesses an N-terminal MADF domain and a C-terminal BESS domain, an architecture that is conserved in at least 14 Drosophila proteins, including Adf-1 and Stonewall. The MADF domain directs sequence specific DNA binding to a site consisting of multiple trinucleotide repeats, while the BESS domain directs a variety of protein-protein interactions, including interactions with itself, with Dorsal, and with a TBP-associated factor (Bhaskar, 2002).

Antagonism between the N and EGFR signaling pathways influences developmental fate in the eye-antennal disc leading to a loss of eye tissue and the appearance of extra antennae. Although this phenotype was originally suspected to represent eye-to-antennal transformation, subsequent analysis suggests that it most likely represents antennal duplication. Specifically, the absence of the N signal leads to a failure in eye disc proliferation resulting in compensatory over-proliferation of the antennal disc and its subdivision into multiple antennae. Consistent with the idea that the extra antennae result from under-proliferation of the eye field, it was found that the phenotype was largely suppressed by over-expression of CycE to drive the cell cycle (Duong, 2008a).

In this study, it was found that inhibition of eye disc growth leads to antennal duplication. But in addition, it was shown that the same treatment that leads to antennal duplication can also direct the transformation of eyes to antennae. These two phenotypes are anatomically distinct. This anatomical distinction is evident in adults: antennae resulting from antennal duplication are found anterior to the antennal foramen, while the antennae resulting from eye-to-antenna transformation are found posterior to the antennal foramen. It is also apparent in larval eye-antennal imaginal discs: antennal duplication discs exhibit multiple circular dac expression domains within a single sac of epithelium (the antennal disc), while eye-to-antennal transformation discs exhibit two or more circular dac expression domains spread over both the eye and antennal discs. The two types of discs display distinct molecular signatures as well: the antennal duplication discs exhibit duplicated Dll expression domains, while the eye discs undergoing transformation to antennae lack Dll expression (Duong, 2008)a.

Perhaps the most persuasive evidence that Dip3 can direct eye-to-antennal transformation is provided by the observation of eyes that are only partially transformed to antennae since is very difficult to reconcile these partial transformations with the idea of antennal duplication. In some cases, proximal antennal segments tipped with eye tissue are observed. In accord with this phenotype, some third instar larval eye discs display a central domain of Elav-positive differentiating photoreceptors surrounded by a circular dac domain (Duong, 2008a).

These arguments support the idea that antennal duplication and eye-to-antennal transformation are mechanistically distinct phenomena, and the remainder of the discussion assumes this to be the case. However, the possibility that these two phenotypes are two manifestations of a single mechanism cannot be excluded. For example, the discs exhibiting duplicated Dll domains may represent complete transformations, while the discs lacking duplicated Dll domains, but containing Elav may represent partial transformations (Duong, 2008a).

The data show that discs undergoing antennal duplication as a result of Dip3 expression are comprised of a severely diminished eye region and an enlarged antennal region. As shown by BrdU labeling experiments, these antennal duplication discs most likely result from suppression by Dip3 of cell proliferation in the eye field leading to overproliferation of the antennal disc. This conclusion is supported by the ability of factors that drive cell proliferation (e.g., Cyclin E) to alleviate the Dip3 misexpression defect (Duong, 2008a).

Many experimental manipulations that reduce the size of the eye disc (e.g., surgical excision, induction of cell death, or suppression of cell proliferation) lead to enlargement and duplication of the antennal primordium. How might reduction of the eye field lead to antennal field over-growth? One possibility is that the eye field produces a growth inhibitory signal. Alternatively, the eye field and the antennal field may compete with each other for limited nutrients or growth factors. In support of this latter possibility, recent studies of the role of dMyc in wing development have demonstrated growth competition between groups of imaginal disc cells (Duong, 2008a).

While the results imply that antennal disc overgrowth is required for antennal duplication, overgrowth is thought not to be sufficient for duplication. This conclusion derives from experiments in which an antennal disc specific driver is used to direct over-expression of CycE or Nact. This resulted in antennal overgrowth without concurrent reduction in the eye disc. In this case, antennal duplication was not observed. Thus, in addition to antennal overgrowth, antennal duplication also appears to require reduction or elimination of the eye disc. Regulatory signals from the eye disc may act to prevent antennal duplication (Duong, 2008a).

The eye and antenna discs differ in several respects: (1) Specific expression of the organ-specification genes. The eye disc expresses the retinal determination gene network (RDGN) genes, including eyeless (ey), twin of eyeless (toy), eyes absent (eya), sine oculis (so), and dachshund (dac), while the antennal disc expresses Dll and hth. hth is also expressed in the eye disc but in a distinct pattern from that seen in the antennal disc. In the second instar eye disc, hth is expressed throughout the eye disc, and collaborates with ey and teashirt (tsh) to promote cell proliferation. The hth expression domain later retracts to only the anterior-most region of the eye disc. This pattern is different from the circular expression pattern observed in the antennal disc. (2) In the antennal disc, dpp is expressed in a dorsal anterior wedge and wg is expressed in a ventral anterior wedge. The intersection of Dpp and Wg signaling is required to specify the proximodistal axis in the leg and antenna. In the early eye disc, Wg and Dpp signaling may overlap. But as the disc grows in size, the wg and dpp expression domain are separated, so that there is probably no intersection between high levels of Wg and Dpp signaling. (3) Whereas the partial overlap of Dll and hth expression domains in the antennal disc is important for proximodistal axis specification, there is no Dll expression in the eye disc. Dll expression in the center of the antennal and leg discs is induced by the combination of high levels of Dpp and Wg signaling. Because there is no overlap of Dpp and Wg signaling in the eye disc, Dll is not induced (Duong, 2008a).

Therefore, efficient transformation of the eye disc into an antennal disc requires at least three things: (1) repression of the eye fate pathway; (2) activation the antennal fate pathway; and (3) the intersection of Dpp and Wg signaling, mimicking the situation in the antenna and leg disc that induces proximodistal axis formation. Any one of these three conditions by itself is not sufficient. (1) Loss of the RDGN genes leads only to the loss of the eye. However, if apoptosis is blocked, or cell proliferation is induced, in the ey2 mutant (ey>p35 or ey>Nact in ey2), then Dll can be induced in the eye disc and extra antenna are formed. The induction of Dll is not ubiquitous in the eye disc, suggesting that the loss of ey does not autonomously lead to the expression of Dll and the transformation to the antennal fate. (2) Simply expressing the antennal determining genes Dll or hth in the eye disc does not change the eye fate into antennal fate. It was found that uniform expression of Dll in the eye disc (ey>Dll) resulted in mild eye reduction, whereas ey>hth completely abolished eye development. E132>Dll caused the formation of small antenna in the eye in about 46% of flies, whereas ptc>Dll and C68a>Dll induced extra antenna but not within the eye field. Therefore, although Dll and hth are important determinants for antennal identity, it is their specific spatial expression patterns that determine antennal development. (3) Creating the intersection of Wg and Dpp signaling does not change the eye into antenna. Such manipulation in the leg disc turned on vg and transdetermined the leg disc into wing disc. Therefore, the specific genes induced by Dpp and Wg signaling may depend on disc-specific factors. In the eye disc, turning on Wg signaling in the dpp expressing morphogenetic furrow only blocked furrow progression (Duong, 2008a).

In this study, it was found that the ectopic expression of a single gene, Dip3, can cause eye-to-antenna transformation. Dip3 apparently satisfied all three requirements. (1) Overexpression of Dip3 repressed (non-cell-autonomously) ey and dac. The repression of ey may be due to the induction of ct. The ability of Dip3 to simultaneously repress multiple retinal determination genes is completely consistent with the many known cross-regulatory interactions between these genes. (2) ey>Dip3 turned on ct and hth. (3) By blocking cell proliferation, ey>dip3 reduced the eye field size and allowed the intersection of Dpp and Wg signaling. Furthermore, ey>Dip3 induced en, which probably created an ectopic A/P border and induced ectopic dpp/wg expression (Duong, 2008a).

Interference with cell cycle progression appears to be a common link between the two phenotypes described in this study. In the case of antennal duplication, interference with eye disc growth leads to antennal disc overgrowth, which is a prerequisite for duplication. In the case of eye-to-antenna transformation, eye disc undergrowth allows the required intersection between Dpp and Wg signaling (Duong, 2008a).

The observation that Dip3 misexpression can transform the eye field, but not other tissues, to an antennal fate suggests a close evolutionary relationship between the eye and the antenna. Previous studies have emphasized the homology between antennae and legs. The findings presented here that misexpression of a single transcription factor, namely Dip3, can transform eyes to antennae provides support for the notion that the eye and antenna may also, in some sense, be homologous to one another. Previous evidence in support of this idea comes from the observation that similar spatial arrangements of Wg and Dpp signaling along with a temporal cue provided by the ecdysone signal are required for the formation of the eye and the mechanosensory auditory organ. Small mechanosensory sensilla, such as Johnston's organ and the chordotonal organs (stretch receptors) are thought to represent the earliest evolving sense organs. Perhaps the eye resulted from a duplication and specialization of such a sensillum (Duong, 2008a).

Non-cell-autonomous inhibition of photoreceptor development by Dip3

The Drosophila MADF/BESS domain transcription factor Dip3, which is expressed in differentiating photoreceptors, regulates neuronal differentiation in the compound eye. Loss of Dip3 activity in photoreceptors leads to an extra photoreceptor in many ommatidia, while ectopic expression of Dip3 in non-neuronal cells results in photoreceptor loss (Duong, 2008a). These findings are consistent with the idea that Dip3 is required non-cell autonomously to block extra photoreceptor formation. Dip3 may mediate the spatially restricted potentiation of Notch (N) signaling since the Dip3 misexpression phenotype is suppressed by reducing N signaling and misexpression of Dip3 leads to ectopic activity of a N-responsive enhancer. Analysis of mosaic ommatidia suggests that no specific photoreceptor must be mutant to generate the mutant phenotype. Remarkably, however, mosaic pupal ommatidia with three or fewer Dip3+ photoreceptors always differentiate an extra photoreceptor, while those with four or more Dip3+ photoreceptors never differentiate an extra photoreceptor. These findings are consistent with the notion that Dip3 in photoreceptors activates a heretofore unsuspected diffusible ligand that may work in conjunction with the N pathway to prevent a subpopulation of undifferentiated cells from choosing a neuronal fate (Duong, 2009b).

This analysis suggests that expression of Dip3 in one cell population suppresses neuronal differentiation of other cells around it. Loss of Dip3 expression in photoreceptors appears to release the neuronal fate suppression in non-neuronal cells, leading to the differentiation of an extra photoreceptor in each ommatidium. Consistent with this interpretation, when Dip3 is misexpressed in undifferentiated and/or primary pigment cells, which normally do not express Dip3, it inhibits neural precursors from assuming the neuronal fate. However, the possibility cannot be formally ruled out that the loss-of-function phenotype and the gain-of-function phenotype are mechanistically distinct (Duong, 2009b).

Along with inhibiting photoreceptor development, misexpression of Dip3 also leads to ectopic non-neuronal cell specification as reflected by the appearance of extra cone and pigment cells. Whether the extra cone and pigment cells originate from the inhibited neuronal precursors or from undifferentiated cells is unclear. However, some pupal ommatidia contain 8 photoreceptors and 5 cone cells implying that at least some of the extra non-neuronal cells originate from undifferentiated cells as opposed to inhibited neuronal precursors. Furthermore, the Dip3 mutant ommatidia contain the normal number of cone and pigment cells indicating that transformed cone or pigment cells cannot be the source of the extra photoreceptors that result from Dip3 loss-of-function. Therefore, Dip3 must possess at least two independent functions: inhibition of neuronal and promotion of non-neuronal specification (Duong, 2009b).

These two properties are also seen in N signaling. In lateral inhibition, the activation of N in R8 inhibits the surrounding cells from assuming the neuronal fate. Furthermore, in the R7 equivalence group, in which EGFR is active in all cells, the level of N activity determines cell fate. Cells with low N activity differentiate into photoreceptors, while cells with high N activity differentiate into cone cells. Furthermore, restricted activation of Notch in late eye development leads to loss of photoreceptors and extra cone and pigment cells similar to over-expression of Dip3. Thus, there is likely to be an interaction between Dip3 and N signaling. Consistent with this hypothesis, reduction of N signaling suppresses the Dip3 over-expression phenotype, and ectopic expression of Dip3 leads to ectopic expression of a reporter under control of a N-responsive enhancer. Finally, misexpression of Dip3 in the wing, where Dip3 is not normally expressed, inhibits wing vein development, a phenotype similar to the N over-expression phenotype (Duong, 2009b).

While the data suggest that Dip3 potentiates N signaling, they are not consistent with the notion that Dip3 simply triggers the N signaling pathway in an indiscriminate manner. While the N signal is essential and involved in many diverse aspects of development, Dip3 is not an essential gene. Furthermore, N laterally inhibits neuronal development in all cells that surround the signal-emitting cell, but Dip3 normally only suppresses one cell from assuming the neuronal fate. Therefore, Dip3 is apparently responsible for only a subset of N functions. Consistent with this interpretation, misexpression of Dip3 in the eye results in ectopic activation of a N reporter in only a subset of photoreceptors, while misexpression of Dip3 in the wing inhibits formation of only the anterior and posterior cross veins, along with the distal segment of the L5 longitudinal vein. How Dip3 is able to have these spatially restricted effects on N signaling is still unknown. It is likely, however, that the key to this spatial restriction lies in the need for combinatorial interactions between Dip3 and other spatially restricted signaling pathways or transcription factors (Duong, 2009b).

Mosaic analysis shows that no single photoreceptor must be mutant to generate the mutant phenotype. However, the possibility cannot be competely excluded that certain subsets of photoreceptors must be mutant to generate the mutant phenotype. The cells of the R7 equivalence group arise after the second mitotic wave and so may tend to be simultaneously mutant in mosaic ommatidia more often than would be expected if there were no lineage relationships at all between R cells. Thus, one possibility is that it is sufficient for all three photoreceptors of the R7 equivalence group (R1, R6, and R7) to be mutant to generate the extra photoreceptor. However, all the mosaic ommatidia with an extra R cell (all those with nine R cells in total) contain at least six mutant R cells. Thus, it is not sufficient for all the cells of the R7 equivalence group to be mutant to generate the mutant phenotype. Alternatively, it is possible that all the precluster R cells (R2, R3, R4, R5, and R8) must be mutant to generate the mutant phenotype. However, this seems unlikely as it would imply that the five precluster R cells are always mutant together in mosaic ommatidia containing five or more mutant R cells. This would, in turn, suggest a much stronger lineage relationship between R cells than has been previously observed. Thus, it is concluded that it is likely to be the total number of Dip3+ R cells that determines whether or not an extra photoreceptor is recruited to an ommatidium (Duong, 2009b).

The finding that any four Dip3+ photoreceptors are sufficient to prevent the mutant phenotype seems most consistent with the idea of a diffusible factor that must accumulate to a minimum level to inhibit extra photoreceptor development. In theory, this diffusible factor could be a N ligand or coligand. However, given that known N ligands are membrane bound proteins, the notion is favored that a diffusible factor activates a separate pathway that works in parallel with the N pathway to prevent extra photoreceptor development, perhaps by synergistically stimulating N target genes. If it is assumed that this parallel pathway is required for some, but not all, N functions, then this mechanism would explain the specificity of the phenotype (Duong, 2009b).

If Dip3 directly activates transcription of a diffusible factor, misexpression of Dip3 might be expected to produce the same gain-of-function phenotype regardless of where within the developing ommatidium it was misexpressed. Therefore, the observation that misexpression of Dip3 in cone cells does not produce a gain-of-function phenotype, while misexpression in undifferentiated cells does suggests that Dip3 may not directly activate the ligand. Rather it may be required for the modification of a ligand that is absent from cone cells, but present in undifferentiated cells. Alternatively, the lack of a phenotype due to cone cell misexpression of Dip3 could also result if all the photoreceptors are already specified by the time Dip3 is expressed in the cone cells (Duong, 2009b).

The remarkable finding that four Dip3+ cells are always sufficient while three Dip3+ cells are always insufficient to produce a mutant ommatidium suggest that the system is exquisitely tuned to the concentration of the hypothetical diffusible ligand. A number of mechanisms (e.g., zero order ultrasensitivity) have been proposed to explain this kind of extreme sensitivity to the concentration of a ligand (Melen, 2005). Extreme concentration sensitivity would also explain the observation that the non-cell-autonomous effects apparently do not spread between ommatidia (Duong, 2009b).

The MADF-BESS transcription factors each contain a DNA binding domain (the MADF domain) and an activation domain (the BESS domain) and can function as both activators and coactivators (Bhaskar, 2002). To assess the relative importance of the MADF and BESS domains, an unbiased screen was carried out for new Dip3 loss-of-function alleles. All four missense mutations identified map to the MADF domain suggesting that DNA binding is essential for the function of Dip3 in retinal patterning. Although the screen did not identify missense mutations in the BESS domain, the conserved architecture of MADF-BESS domain factors suggests that this domain is nonetheless critical. In support of this speculation, misexpression of a Dip3 deletion lacking the BESS domain with the ey-Gal4 or GMR-Gal4 driver did not produce the lethality or the smooth eye phenotype associated with the expression of full-length Dip3 (Duong, 2009b).

The high degree of conservation of the MADF-BESS domain architecture is surprising. 36 of the 57 known BESS domain-containing proteins also contain a MADF domain, while 36 of the 141 known MADF domain-containing proteins also contain a BESS domain. Although most common in flies, this architecture is conserved across phyla. For example, two of the three recognized MADF domain-containing factors in zebrafish also contain BESS domains. This level of conservation is unusual among sequence-specific transcription factors where it is generally impossible to detect homology across phyla outside the DNA binding domain. The frequency with which the MADF and BESS domains are found together suggests that they interact with one another in the developmental control of gene expression (Duong, 2009b).

Switching cell fates in the developing Drosophila eye

The developing Drosophila ommatidium is characterized by two distinct waves of pattern formation. In the first wave, a precluster of five cells is formed by a complex cellular interaction mechanism. In the second wave, cells are systematically recruited to the cluster and directed to their fates by developmental cues presented by differentiating precluster cells. These developmental cues are mediated through the receptor tyrosine kinase (RTK) and Notch (N) signaling pathways and their combined activities are crucial in specifying cell type. The transcription factor Lozenge (Lz) is expressed exclusively in second wave cells. In this study Lz was ectopically supplied to precluster cells, and the various RTK/N codes that specify each of three second wave cell fates were concomitantly supplied. This protocol reproduced molecular markers of each of the second wave cell types in first wave precluster cells. Three inferences were drawn; (1) it was confirmed that Lz provides key intrinsic information to second wave cells, and this can now be combined with the RTK/N signaling to provide a cell fate specification code that entails both extrinsic and intrinsic information. (2) the reproduction of each second wave cell type in the precluster confirms the accuracy of the RTK/N signaling code, and (3) RTK/N signaling and Lz need only be presented to the cells for a short period of time in order to specify their fate (Mavromatakis, 2013).

This paper explored three inter-related themes bearing on the nature of the signals that specify the cell types in the Drosophila ommatidium. The ability of a transcription factor to predispose the cellular responses to developmental signals was examined, the accuracy of the signaling code that represents these developmental signals was validated, and it was inferred that both the intrinsic and extrinsic aspects are only required for a brief period of time (Mavromatakis, 2013).

Lz had long been assumed to be a key factor that distinguishes how second wave cells differ from the precluster cells in their response to developmental signals. This paper rigorously tested this concept and reproduced features typical of the three second wave cell types in the R3/4 precluster cells by supplying ectopic Lz along with the appropriate RTK/N cell fate code. It is thus inferred that the presence of Lz in R3/4 precluster cells is sufficient to endow them with the second wave cell fate response repertoire. A number of issues related to these observations and their interpretations are discussed (Mavromatakis, 2013).

Normal R3/4 precursors undergo an N-Dl interaction that results in the R4 precursor experiencing much higher levels of N activity than the R3 precursor. When Lz was supplied to R3/4 precursors (sev.lz), the cell in the R4 position frequently transformed into an R7, consistent with the requirement of high N for R7 specification. Less frequently, both R3/4 precursors adopted the R7 fate, and sometimes it was the cell in the R3 position alone that generated an ectopic R7. These results suggest that in the sev.lz flies the R3/R4 N-Dl interaction does not occur correctly. When the mδ0.5.lacZ reporter line was used as a reporter of N activity (which in wild-type larvae is robustly upregulated in R4 precursors) an erratic pattern was observed, sometimes showing the wild-type pattern, sometimes showing both R3/4 cells with high levels of lacZ expression, and sometimes showing R3 alone with high levels. Hence, by expressing Lz in the R3/4 precursors the cells were not only endowed with second wave response abilities but also they were prevented from executing their N-Dl interactions properly. Indeed, it was only when N was artificially activated to a high level with activated Notch (sev.lz; sev.N**) that the R7 fate was potently induced in both R3/4 precursors (Mavromatakis, 2013).

Native R7s critically require sev gene function; in its absence, they differentiate as cone cells. However, some ectopic R7s were able to differentiate when Lz was provided to the R3/4 precursors, even in the absence of sev (sev0; sev.lz), suggesting that normal R7 specification was not fully reiterated here. Examination of these eye discs suggested that some R3/4 precursors differentiated as R7s whereas others became cone cells. Thus, these cells appear to be on the cusp of the R7/cone cell fate choice, and some cells expressing markers for both cell types were observed. In the cells that became R7s, the presence was inferred of sufficient RTK activity, which was likely to have been supplied by endogenous DER signaling active in the precluster cells. Only when N activity was raised in these cells (sev0; sev.lz; sev.N*) did their full sev dependence for the R7 fate emerge, when all R3/4 precursors differentiated as cone cells (Mavromatakis, 2013).

The sev.N* construct is a very useful activator of the N pathway in developing eye cells. Since N activity drives sev expression, the sev.N* transgene feeds back on itself and promotes its own expression, and by subsequent iterations of this effect the cells are left with potent N activity. This level is still within the physiological range, unlike that produced using Gal4/UAS techniques, and is therefore the choice method for activating the N pathway. The transgene that was routinely use to knock down N activity [sev.Su(H)EnR] has the opposite effect; it reduces its own expression, and mildly compromises N activity. This level of reduction in N activity is usually sufficient to trigger major effects without the disadvantage of the severe downregulation that can accompany the use of Gal4/UAS technology. Since the sev.lz construct would also be downregulated by sev.Su(H)EnR, it was necessary to ectopically express Lz in the precluster using another enhancer element, and to this end the ro.Gal4 line was generated. When UAS.lz was expressed under ro control, the R3/4 precursors frequently differentiated as R7s, and crucially, when N activity was concomitantly reduced [ro.Gal4; UAS.lz; sev.Su(H)EnR] cells displaying R1/6 molecular features were now detected in the R3/4 precursors (Mavromatakis, 2013).

The cells in the R2/5 positions in ro.G4; UAS.lz developing ommatidia appear to develop normally; they express Elav and Ro, but none of the other fate markers. This suggests that R2/5 cells are insensitive to the presence of Lz, and argues that there is a major molecular difference between these cells and the R3/4 precursors. Also noteworthy is the transformation of all lz mutant second wave cells into R3/4 types characterized by the expression of Svp (a marker that is not expressed in R2/5 precursors) and Elav. Thus, it appears that ectopic Lz selectively transforms R3/4 precursors of the precluster to the second wave fate, and second wave cells lacking Lz adopt the R3/4 fate. Hence, it is suspected that Lz might not provide the intrinsic information that distinguishes the second wave cells from precluster cells per se, but rather distinguishes second wave cells from R3/4 types. Experiments to evaluate this view are currently being undertaken (Mavromatakis, 2013).

A counter-argument emerges from the fate of the majority of cells in the R3 positions in sev.lz eyes, which do not switch their fate. Only when N activity is activated or reduced in these cells is a change seen in their fates, and to be sure that the R2/5 cells are insensitive to Lz expression, it would also need to correspondingly vary N activity in them. Experiments to do this using ro.Gal4 produced severely disrupted preclusters, presumably as a result of interference with N function at earlier stages of precluster formation. Since these clusters were largely uninterpretable, the issue of whether R2/5 cells are insensitive to Lz expression remains unresolved (Mavromatakis, 2013).

For many years, the role of N in photoreceptor specification was confusing. In some contexts N appeared to oppose photoreceptor specification and in others N seemed to promote it, and this confusion prevented substantial progress in defining the fate codes that specified the different cell types. In recent work three distinct roles for N in this process were identified, and with that information the cell fate codes for R1/6, R7 and the cone cells were inferred. A major goal of this current work has to been to test this code by its reiteration in the R3/4 cells of the precluster using Lz expression to endow them with second wave cell qualities. In these experiments, each of the cell codes induced the expected cell fates, providing cogent support for the validity of the code (Mavromatakis, 2013).

DER is assumed to be ubiquitously expressed in the eye disc tissue, and its ligand, Spitz, diffusing from precluster cells, is thought to reach more distant cells with time. But the N and Sev signals are regulated in a different manner. Both their ligands are membrane bound, and their receptor activations only occur in immediate neighbors. Dl, the ligand for N, is expressed transiently by differentiating cells, and, accordingly, activates N in neighboring cells for only short periods of time (a few hours). sev is an N response gene and, in consequence, Sev is only expressed in cells for a short period of time. By contrast, Boss, the Sev ligand, is expressed for a prolonged period by the R8 precursor. Thus, both the N and Sev signaling systems are only available to the cells for restricted periods, with this restriction controlled by ligand expression in the N system and by receptor expression in the Sev system (Mavromatakis, 2013).

Although Lz is expressed in the second wave cells in a persistent manner in the eye disc, the experiments suggest that it, like the extrinsic signals, is required only for a brief developmental window. Consider the transformation of sev.lz R3/4 cells to the R7 fate. The sev enhancer is only active in these cells for a few hours and yet a complete transformation of the cells is achieved. The expression of specific cell type markers in the eye disc might erroneously indicate the transformation of a cell when only a transient effect occurs, but the presence of ectopic R7s in the adult retina argues otherwise and suggests that the transformations are potent and permanent. This view is further validated by the rescue of lz mutant second wave cells by the sev.lz transgene. This rescue is complete and is evident by the molecular markers expressed in the disc and by the morphology of the adult cells. Thus, it is inferred that Lz is only required during the same time window when the RTK/N signals are transduced, and it is further inferred that the combined activities of the RTK and N pathways, in concert with Lz, function in a short-lived manner to lock in the fate of the cells. How the presence of ephemeral extrinsic and intrinsic information is molecularly 'remembered' by the cells to allow their appropriate differentiation over a prolonged developmental period remains an intriguing question (Mavromatakis, 2013).

Larval: Notch and the segmentation of the leg

Segmentation is a developmental mechanism that subdivides a tissue into repeating functional units, which can then be further elaborated upon during development. In contrast to embryonic segmentation, Drosophila leg segmentation occurs in a tissue that is rapidly growing in size and thus segmentation must be coordinated with tissue growth. Segmentation of the Drosophila leg, as assayed by expression of the key regulators of segmentation, the Notch ligands and fringe, occurs progressively and this study defines the sequence in which the initial segmental subdivisions arise. The proximal-distal patterning genes homothorax and dachshund are positively required, while Distal-less is unexpectedly negatively required, to establish the segmental pattern of Notch ligand and fringe expression. Two Serrate enhancers that respond to regulation by dachshund are also identified. Together, these studies provide evidence that distinct combinations of the proximal-distal patterning genes independently regulate each segmental ring of Notch ligand and fringe expression and that this regulation occurs through distinct enhancers. These studies thus provide a molecular framework for understanding how segmentation during tissue growth is accomplished (Rauskolb, 2001).

In recent years the key role of the Notch signaling pathway in the segmentation and growth of the Drosophila leg has been established. Notch signaling must be localized within each leg segment to promote the formation of boundaries (joints) that separate each leg segment and to induce leg growth. This requirement for a segmentally repeated pattern of Notch activation is accomplished by restricting the expression of the regulators of Notch activation, Serrate, Delta and fringe, to one ring per segment. By examining the expression of the Notch ligands and fringe during leg development, it has been possible to determine the progressive order in which leg segmentation is established. At early third instar, a single ring of Serrate, Delta and fringe expression is present within the coxa. The next ring to arise is located within the presumptive femur. At mid third instar, expression arises within presumptive tarsal segments 2 and 5. Subsequent expression is observed in the tibia and more tarsal segments, such that ultimately, by the end of third instar, a ring of expression is present in each presumptive leg segment, adjacent to each prospective leg segment border. Thus, segmentation of the Drosophila leg occurs progressively and in a reproducible pattern (Rauskolb, 2001).

Previous studies investigating the expression of a reporter gene [E(spl)mß-CD2] regulated downstream of Notch activation led to the conclusion that the first segment boundary to form was between tarsal segments 4 and 5. Additional rings of expression were then observed in the tarsus and then eventually in all leg segments. This led to the suggestion that the first segmental boundaries to form correspond to the most distal segments. However, further examination of this reporter gene indicates that expression is observed in proximal cells prior to expression within the tarsus. Moreover, temperature shifts of a conditional Notch allele at different stages of development demonstrate that the temperature-sensitive period for Notch in proximal segmentation occurs before that in tarsal segmentation. The conclusion is reached that leg segmentation does not occur in a simple distal to proximal order, nor proximal to distal order, nor are the most proximal and distal segments established first and other segments added by intercalation. Rather, the establishment of Drosophila leg segmentation occurs in a complex sequence (Rauskolb, 2001).

Importantly, Notch signaling may actually coordinate progressive segmentation of the leg with leg growth. For example, in early leg discs there is a single ring of Serrate expression within the coxa, in Hth-expressing cells immediately adjacent to Dac-expressing cells. However, by the time the femur ring arises, the coxa ring of Serrate expression has been displaced and is no longer within cells immediately adjacent to the Dac-expressing cells; rather, there are Hth-expressing cells lying in between that do not express Serrate. Thus, it is postulated that once Serrate, Delta and fringe expression is established within the coxa, Notch is activated, which promotes local cell proliferation, thereby displacing the coxa ring. This then allows for the femur ring of expression to be established in cells that are not immediately adjacent to the coxa expression ring. This mechanism also requires that once a ring of ligand expression is established in a particular segment, this expression must be maintained such that it is not influenced by later alterations in relation to leg gap gene expression. This maintenance could be accomplished by a feedback loop between Notch activation and ligand expression, similar to what has been observed during late wing development, where Notch activation cell autonomously represses ligand expression and nonautonomously induces ligand expression in flanking cells by regulating the expression of a signaling molecule. Preliminary studies have indicated that Notch activation can influence Notch ligand expression in the developing leg (Rauskolb, 2001).

Flexible joints separate the rigid sections of the insect leg, allowing them to move. In Drosophila, the initial patterning of these joints is apparent in the larval imaginal discs from which the adult legs will develop. The later patterning and morphogenesis of the joints, which occurs after pupariation (AP), is described. In the tibial/tarsal joint, the apodeme insertion site provides a fixed marker for the boundary between proximal and distal joint territories (the P/D boundary). Cells on either side of this boundary behave differently during morphogenesis. Morphogenesis begins with the apical constriction of distal joint cells, about 24 h AP. Distal cells then become columnar, causing distal tissue nearest the P/D boundary to fold into the leg. In the last stage of joint morphogenesis, the proximal joint cells closest to the P/D boundary align and elongate to form a 'palisade' (a row of columnar cells) over the distal joint cells. The proximal and distal joint territories are characterized by the differential organization of cytoskeletal and extracellular matrix proteins, and by the differential expression of enhancer trap lines and other gene markers. These markers also define a number of more localised territories within the pupal joint (Mirth, 2002).

To identify distinct cell populations in the joints, the expression patterns of 10 joint markers were examined with respect to a posterior marker (engrailed lacZ) and a ventral marker (wingless lacZ). The leg discs of wandering larvae, and pupal legs at 24-28 and 34-38 h AP, were examined. Four of the joint markers were previously reported to be expressed in L3 and prepupal joints (Notch, disconnected lacZ, Nubbin, and odd-skipped lacZ). The rest were isolated for this study by screening Gal4 enhancer trap lines for those that drive expression of GFP in pupal leg joints (ckm78, ckm90, ckm239, ckm175, ok388, and ok483). Most of the joint markers do not change their expression domains between 24-28 and 34-38 h AP. Therefore, data is presented from wandering L3 discs only, and from legs at 34-38 h AP (Mirth, 2002).

In the L3 leg disc, joint markers fall into one of two categories, marking either the proximal joint territories (e.g., Nubbin) or the distal territories (e.g., Notch and odd-skipped lacZ). Of all the markers examined, only Nubbin (Nub), disconnected lacZ (disco lacZ), and odd-skipped lacZ (odd lacZ) are expressed in more than two joints in the L3 stage. Others mark one or two joints at this stage but are expressed in all joints during the pupal stage. Studies examining the expression of Notch and other elements of the Notch patterning cascade have also found that the joint seems to be divided into proximal and distal territories at this stage. Thus, proximal and distal joint domains have already been established by the late L3 (Mirth, 2002).

By 34-38 h AP, patterns of marker expression define three additional territories. First, a proximal-dorsal patch is high-lighted by two joint markers, ckm90 and ckm175, that drive GFP expression only in a patch above and includes the most proximal cells of the dorsal apodeme. The expression of GFP driven by ckm175 includes a greater number of cells than that driven by ckm90. The second domain identified was a mid-distal domain. Odd lacZ expression becomes largely restricted to a mid-distal group of cells in all but the tarsal joints. This corresponds to the region that does not accumulate collagen IV and marks the cells that push underneath the proximal joint cells. Odd lacZ is also expressed in the apodemes. Lastly, ok388 expresses GFP in the lateral anterior and posterior parts of the distal tibial/tarsal (but not tarsal) joint, but is excluded from the dorsal and ventral domains. This expression domain corresponds with the region of elongating cells seen in longitudinal sections of the leg (Mirth, 2002).

Two of the joint markers are expressed in both the proximal and distal portions in the developing adult joint: ckm239 and disco lacZ. Disco lacZ is expressed throughout the entire joint, and ckm239 is excluded from the ventralmost region (wingless lacZ-expressing region) (Mirth, 2002).

It seems likely that the domains of gene expression observed in the L3 leg disc correspond with those of the same genes in the developing adult joint, though this has not been verified directly. If so, proximal and distal joint domains are established before pupariation. These two joint territories separate cells that will invaginate [the cells in the odd lacZ domain, expressing the Notch target E(SPL)Mß] from those that will form the proximal palisade (the cells expressing Delta, Serrate, and Nubbin). During pupal development, the proximal and distal domains of the joint become further subdivided. Most of the enhancer trap markers identified are expressed in specific groups of cells within either the proximal or distal domain in the tibial/tarsal joint at 34-38 h AP. At the same time, the expression of some earlier markers becomes restricted to more specific territories. odd lacZ, which is expressed in some joints in the L3, is expressed most strongly in the mid-distal joint cells at 34-38 h AP. Ok388 expresses in the distalmost but not mid-distal joint cells, and is restricted to the lateral anterior and posterior sides. In the proximal joint, markers such as ckm90 and ckm175 express in only a small group of cells on the dorsal side. Thus, it seems that the tibial/tarsal joint may divided into three proximodistal domains based both on cell behavior and gene expression: proximal, mid-distal, and distalmost regions. Later during pupal development, the distalmost region subdivides into lateral anterior/posterior and dorsal/ventral domains and the proximal joint also subdivides into smaller territories. That further patterning and subdivision of the joint occurs after the prepupal stages is hardly surprising: the adult joint is too complex a structure to be derived simply from the proximodistal interactions that occur before pupal development (Mirth, 2002).

Notch signaling relieves the joint-suppressive activity of Defective proventriculus in the Drosophila leg: The joint-suppressive activity of Dve is required to repress dAP-2 expression

Segmentation plays crucial roles during morphogenesis. Drosophila legs are divided into segments along the proximal-distal axis by flexible structures called joints. Notch signaling is necessary and sufficient to promote leg growth and joint formation, and is activated in distal cells of each segment in everting prepupal leg discs. The homeobox gene defective proventriculus (dve) is expressed in regions both proximal and distal to the intersegmental folds at 4 h after puparium formation (APF). Dve-expressing region partly overlaps with the Notch-activated region, and they become a complementary pattern at 6 h APF. Interestingly, dve mutant legs resulted in extra joint formation at the center of each tarsal segment, and the forced expression of dve caused a jointless phenotype. Evidence that Dve suppresses the potential joint-forming activity, and that Notch signaling represses Dve expression to form joints (Shirai, 2007).

To achieve specific developmental programs, antagonism between Notch and EGFR signaling has been widely observed. A graded activity of EGFR signaling from the distal tip of a leg disc is crucial for patterning the distal structure, and it should be converted into the segmental activation, which is critical for suppression of inappropriate joint formation. One possible explanation is that P-D patterning genes define the segment boundary, and thereby refine the Notch signaling pathway in the distal region of each segment, where EGFR signaling should be repressed. The expanded expression of argos-lacZ in Nts mutants strongly suggests that the Notch signaling pathway antagonizes EGFR signaling. Interestingly, a similar type of regulation has been reported for Caenorhabditis elegans vulval development. Thus, the antagonistic interaction between EGFR and Notch signaling establishes the complementary activation of these pathways in neighboring cells, and is crucial for both vulval cell fate determination and leg joint formation (Shirai, 2007).

In vertebrates, the early process of body segmentation, i.e., somitogenesis, takes place sequentially from head to tail. Somites are generated from the presomitic mesoderm (PSM), the unsegmented paraxial mesoderm at the tail end of the embryo. A 'clock and wavefront' model has been proposed to explain the mechanism of sequential somite formation. Oscillated gene expression, i.e., the clock, driven by Wnt and Notch signaling in the posterior PSM is translated into the segmental units in the wavefront, which is generated in the anterior PSM in response to the decreased activities of graded Wnt and FGF signaling from the tail end. As an embryo grows caudally, the wavefront moves backwards at a constant rate. Thus, the segment boundary is set at the interface between the Notch-activated and -repressed domains in the anterior PSM (Shirai, 2007).

During vertebrate somitogenesis, it has been shown that the interface between the Notch-activated and Notch-repressed domains is generated on suppression of Notch activity through induction of the lunatic-fringe (Lfng) gene in the segment boundary. This refinement is under the control of the basic helix-loop-helix type transcription factor Mesp2, which is expressed in the rostral half of the anterior PSM, indicating that rostral-caudal polarity within a somite is important for restricted Notch activation. The results indicate that the restricted Notch activation during Drosophila leg segmentation also occurs at the segment boundary rather than the center of each segment, suggesting that a conserved mechanism in both Drosophila legs and vertebrate somites underlies the activation of Notch signaling adjacent to the segment boundary (Shirai, 2007).

A Dve-expressing region straddles the fold of the segment boundary, and the following observations indicate that Dve has joint-suppressive activity: (1) dve mutant legs resulted in extra joint formation and (2) forced expression of dve in the presumptive joint region suppressed joint formation. Thus, the mechanism of joint development can be explained as follows; Notch-mediated Dve repression on the proximal side to the intersegmental fold relieves the above joint-suppressive activity, leading to normal leg joint formation. This is reminiscent of the abdomen-suppressive activity of Hunchback, which is relieved by Nanos to induce the abdominal structure. In contrast, Dve expression on the distal side to the fold should be maintained to suppress inappropriate joint formation, because dve mutation leads to extra joint formation with reverse polarity. It appears that Dve activity is only induced to suppress joint formation and that temporally regulated Dve repression is crucial for normal leg joint formation, because dve mutations did not affect normal joint formation (Shirai, 2007).

Extra joints with reverse polarity (reverse joints) are derived from mutants deficient in the PCP or EGFR signaling pathway. Previous reports have suggested a model in which the Notch signal activation proximal to the Notch ligand-expressing domains is blocked by these signals, only allowing the Notch signal activation in a distally adjacent region, i.e., the distal region of each segment. Based on the expression pattern of the Notch ligand Ser, it is assumed that the center of a segment is highly potent for receiving Notch signaling. This idea can explain the reverse polarity of extra joints, because Ser activates the Notch signaling pathway in two different directions: from proximal to distal for normal joints, and distal to proximal for extra joints. However, it seems unlikely that ectopic activation of Notch signaling is restricted at the center of a segment. A Notch-target gene, dAP-2, is autonomously activated in response to ectopic Notch signaling, and, in pk mutants, ectopic dAP-2 expression has expanded on the distal side to the intersegmental fold, the most proximal but not the central region in a segment. Furthermore, the joint-suppressive activity of Dve is also required to repress dAP-2 expression on the distal side to the intersegmental fold. These results suggest that reverse joints are derived from the distally adjacent region to the intersegmental fold (Shirai, 2007).

Based on the results, a model is proposed in which joint-forming activity is generated from the intersegmental fold in a bidirectional manner, and that an inappropriate signal having reverse polarity is blocked by Dve activity, and the PCP and EGFR signaling pathways. In this model, Dve activity is required to suppress Notch target genes, such as dAP-2, involved in joint formation. This is very similar to the situation observed in wing discs, where the Notch target gene wg is repressed by Dve in regions adjacent to the Notch-activated D-V boundary. It is an intriguing possibility that the vertebrate somite boundary generates similar bidirectional signals, and that the inhibition of either one is closely linked to the rostral-caudal polarity within a somite. Further characterization of Drosophila leg segmentation is needed to determine whether this model is applicable to vertebrate somitogenesis or other segmentation processes (Shirai, 2007).

Influence of Notch on dorsoventral compartmentalization and actin organization in the Drosophila wing

Compartment boundaries play key roles in tissue organization by separating cell populations. Activation of the Notch receptor is required for dorsoventral (DV) compartmentalization of the Drosophila wing, but the nature of its requirement has been controversial. Additional evidence is provided in this study that a stripe of Notch activation is sufficient to establish a sharp separation between cell populations, irrespective of their dorsal or ventral identities. Cells at the DV compartment boundary are characterized by a distinct shape, a smooth interface, and an accumulation of F-actin at the adherens junction. Genetic manipulation establishes that a stripe of Notch activation is both necessary and sufficient for this DV boundary cell phenotype, and supports the existence of a non-transcriptional branch of the Notch pathway that influences F-actin. Finally, a distinct requirement has been identified for a regulator of actin polymerization, capulet, in DV compartmentalization. These observations imply that Notch effects compartmentalization through a novel mechanism, which is referred to as a fence, that does not depend on the establishment of compartment-specific cell affinities, but does depend on the organization of the actin cytoskeleton (Major, 2005).

These results have advanced the understanding of DV compartmentalization in two crucial ways. First, they demonstrate that a stripe of Notch activation is sufficient to separate cells, which, together with prior studies on the requirements and geometry of Notch activation, lends support to the idea that a separation fence rather than promotion of a compartment-specific cell affinity provides the best explanation for the role of Notch in DV compartmentalization. Second, they identify both a distinct morphology and distinct genetic requirements for F-actin organization at the DV compartment boundary. In addition to these new insights into DV compartmentalization, the results also suggest that Notch influences F-actin in the wing through an alternate, non-transcriptional pathway (Major, 2005).

The polarization of F-actin accumulation effected by Notch activation at the DV boundary cannot easily be accounted for purely by the transcriptional regulation of target genes associated with Notch activation. In the context of the normal DV boundary, or an ectopic boundary associated with altered Fng expression, a rectangular cell at the boundary has three neighbors with similar levels of Notch activation, but F-actin is elevated along only one of these cell interfaces. In the case of ectopic Delta expression, Notch activation is actually inhibited inside of Delta-expressing clones through autonomous inhibition, and so is now asymmetric with respect to the clone boundary, yet it still effects a similar modulation of F-actin. The common feature of the cellular interface at which F-actin is upregulated in all cases is that it is the cellular interface at which most Notch-ligand interaction is actually occurring. This leads to a suggestion that F-actin accumulation might be polarized through an alternate Notch pathway that impinges on the actin cytoskeleton. This inference is consistent with the observation that forms of Notch that are constituitively activated for transcriptional pathways do not result in a general, autonomous upregulation of F-actin (Major, 2005).

The possibility of links from Notch or its ligands to the actin cytoskeleton that do not involve the canonical Notch transcriptional pathway has been suggested previously, based on studies of Notch pathway components on axon guidance, neurite or filopodial extension, and keratinocyte motility. The nature of this alternate pathway or pathways is not clear. In the context of the influence of Notch on axon guidance in the Drosophila embryo, this alternate pathway is characterized by genetic interactions with Abl, and a lack of genetic interactions with the Notch pathway transcription factor Suppressor of Hairless [Su(H)]. Although Abl mutations do not noticeably affect DV compartmentalization, Abl mutants in general have relatively mild effects, presumably because Abl is partially redundant, and the elevation in phospho-tyrosine and Ena staining at the DV boundary is intriguing in light of potential links between Notch and Abl (Major, 2005).

Rather than a single mechanism for compartmentalization, studies of the DV boundary suggest that a series of distinct strategies affect what, by lineage analysis, appears to be a constant boundary. Tartan (Trn) and Capricious (Caps) are expressed specifically in dorsal cells during the second larval instar, when the boundary first forms. Ectopic expression of these proteins can cause ventral cells to sort to and associate with dorsal cells, but their contribution to compartmentalization must be transient, since their dorsal-specific expression is lost during early third instar. Elevated F-actin staining at the DV boundary could not be consistently detected during second instar, was clearly visible from early through mid-third instar, but disappeared at late third instar. Thus, the role of the F-actin-dependent fence might also be transient. At late third instar, cells near the DV boundary stop proliferating. Since the arrangement of cells in imaginal discs is largely a function of growth rather than movement, this cessation of proliferation presents a third potential mechanism for compartmentalization, which could be important at late stages (Major, 2005).

It has generally been assumed that compartmentalization is effected by the establishment of differential cell affinities, which result in cells sorting to their respective sides of a compartment boundary. Although this paradigm fits well with studies of AP compartmentalization, it is not easy to reconcile with studies of DV compartmentalization, given that Notch is activated and required on both sides of the compartment boundary, that neither mutation nor ectopic activation of Notch causes directed changes in cell location, that the requirement for Notch is non-autonomous, and that the requirement for Notch does not depend on the dorsal or ventral identity of a cell. Models that have proposed that Notch influences DV compartmentalization by affecting a compartment-specific cell affinity have required that it act in conjunction with Apterous. The crucial failing of such models is that they cannot explain how a compartmental separation of cells is achieved by the ectopic Notch activation associated with a mutation of fng, or ectopic expression of Fng, Serrate or Delta, as in all of these cases cells on both sides of the boundary are identical with respect to the presence or absence of Ap (Major, 2005).

An alternative hypothesis is that Notch activation influences a property or behavior of cells at the boundary, referred to as a fence, in a way that prevents them from intermingling. The determination that Notch signaling effects a polarized elevation of F-actin and Ena supports this hypothesis, since it demonstrates that Notch can polarize the actin cytoskeleton in conjunction with its ability to separate cells, and that this influence of Notch is independent of the dorsal or ventral identity of the cell. Additionally, the bidirectional and non-autonomous disruptions of the compartment boundary effected by capt mutant clones are consistent with the inference that compartmentalization involves an F-actin-dependent fence. When the fence is broken, cells can intermix in either direction, irrespective of their DV identity. By contrast, it is not clear how the non-autonomous affects of capt mutant clones could be reconciled with models that postulate a compartment-specific cell affinity (Major, 2005).

The possibility of a non-transcriptional influence of Notch on DV compartmentalization, as suggested above for its influence on F-actin, is appealing because it could explain the observation that Fng can influence compartmentalization even when co-expressed with N-intra. Loss-of-function studies have provided mixed results as to the requirements for Notch transcriptional pathways in DV compartmentalization. Clones of cells mutant for a hypomorphic allele of Su(H), Su(H)SF8, respect the compartment boundary, even though transcriptional targets are affected. However, this is not a null situation for Su(H), and it would be predicted that at a minimum, a Notch transcriptional pathway would be required at the DV boundary to maintain the expression of Notch ligands. Requirements for transcriptional mediator proteins confirm that transcription is required for DV compartmentalization, but a role for a transcriptional Notch pathway does not preclude a parallel role for a non-transcriptional pathway (Major, 2005).

The molecular nature of the compartmentalization fence is not yet clear, but some possibilities can be suggested. One model is based on the similarity of the F-actin stripe at the DV boundary to a prominent F-actin cable detected along the interface between leading edge cells and amnion-serosa cells during dorsal closure of the Drosophila embryo. The F-actin cable and associated proteins are thought to help keep dorsal epidermal cells in register as they move, through actin-myosin-based contraction and/or influences on the protrusive behavior of filopodia. Similar processes could maintain a smooth separation between cells at the DV compartment boundary. Intriguingly, genetic studies have suggested a potential role for Notch in dorsal closure that does not involve Su(H). The distinct requirement for a regulator of actin polymerization, capt, at the DV boundary is consistent with the hypothesis that the elevated F-actin detected at the DV boundary plays a crucial role in compartmentalization. In this view, the F-actin cable would be a physical manifestation of the Notch-dependent separation fence (Major, 2005).

An alternative possibility is suggested by the observations Notch and its ligands can, at least in cultured cell assays, act as cell adhesion molecules, and that association of Notch with its ligands can promote cleavage of both molecules. Thus, while loss- and gain-of-function studies of Notch ligands do not support the possibility that they act as compartment-specific cell adhesion molecules, it is suggested that cleavage of Notch and/or its ligands might act as a boundary-specific de-adhesion mechanism. Boundary-specific de-adhesion, rather than compartment-specific adhesion, has been suggested as a possible mechanism for Eph-Ephrin-mediated cell separation. In this model, the influence on F-actin might be a secondary consequence of the primary separation mechanism. Alternatively, because the cytoplasmic domains of Notch and its ligands have been reported to associate with proteins that can impinge on actin organization, Notch or ligand cleavage might be a direct mechanism for modulating F-actin (Major, 2005).

Notch signalling coordinates tissue growth and wing fate specification in Drosophila

During the development of a given organ, tissue growth and fate specification are simultaneously controlled by the activity of a discrete number of signalling molecules. These two processes are extraordinarily coordinated in the Drosophila wing primordium, which extensively proliferates during larval development to give rise to the dorsal thoracic body wall and the adult wing. The developmental decision between wing and body wall is defined by the opposing activities of two secreted signalling molecules, Wingless and the EGF receptor ligand Vein. Notch signalling is involved in the determination of a variety of cell fates, including growth and cell survival. Evidence is presented that growth of the wing primordium mediated by the activity of Notch is required for wing fate specification. The data indicate that tissue size modulates the activity range of the signalling molecules Wingless and Vein. These results highlight a crucial role of Notch in linking proliferation and fate specification in the developing wing primordium (Rafel, 2008).

The expression of Wg in the most ventral part of the wing disc specifies the wing field at the same time as restricting Vn expression to the most dorsal part. Vn is required to block the responsiveness of body wall cells to Wg. Thus, the relative concentration of the diffusible proteins Wg and Vn experienced by disc cells directs their wing versus body wall fate. It is interesting to note that the expression of these two molecules is established long before the wing field is induced in the presumptive wing primordium. Wg expression starts long before wing field specification takes place, as revealed by the later induction of Nub expression and the reduction in the expression of the body wall cell marker Tsh. It is therefore proposed that tissue growth modulates the cellular response to these signalling molecules and controls, in time, wing fate specification. In the early wing primordium, Vn might reach every wing cell, thereby blocking responsiveness to Wg and repressing wing fate specification. Growth induced by Notch activity might pull the sources of Wg and Vn apart and, thus, most ventral cells might not sense sufficient Vn levels, so Wg would be able to induce wing fate. Interestingly, the overexpression of Wg or overactivation of its signalling pathway is able to bypass the requirement of growth in this process, indicating that the cells sense the relative levels of Wg and Vn. Once the wing field has been specified, Wg starts to be expressed along the presumptive wing margin, where it exerts a fundamental function in the maintenance of the Notch-dependent organizing center along the DV boundary. Note that the organizing activity of Notch at the DV boundary takes place long after the early function of Notch revealed in this work, which is involved in promoting growth and facilitating wing fate specification. As revealed by the expression of the Notch target E(spl)m-β, it is not until late in the second instar that the expression of Notch is restricted to the DV boundary. During the process of wing fate specification that takes place during second instar, it is uniformly expressed in the whole wing disc. These results imply that growth also facilitates the reiterative use of signalling molecules, such as Wg and Notch, to exert different functions during the development of a multicellular organ like the wing primordium (Rafel, 2008).

At the same time that wing and body wall fate specification takes place in the wing primordium, Vn is involved in the induction of apterous expression in the dorsal region. Consistent with the model proposed above, the activity of Vn, as monitored by the expression of apterous, was modulated by tissue growth. In the absence of Notch activity, even though Vn expression is not affected, Vn appears to reach every wing cell, as apterous expression was expanded ventrally. Increased levels of Wg expression or growth promoted by CycE appear to re-establish the dorsally restricted range of activity of Vn, as apterous expansion was blocked under these circumstances (Rafel, 2008).

Growth promoted by Notch has also been shown to be directly involved in the specification of the eye within the Drosophila eye-antenna primordium, a process that also depends upon the opposing activities of two secreted signalling molecules, in this case Dpp and Wg. Thus, Notch coordinates in a very elegant manner both eye and wing primordia tissue growth and eye/wing specification, by modulating the response of the cells to the activities of signalling molecules. These results indicate that the same mechanism might be commonly used in animal development to coordinate tissue growth and fate specification (Rafel, 2008).

The evolution of wings was crucial in the process of adaptation, allowing insects to escape predators or colonize new niches. The loss and recovery of wings has occurred during the course of evolution. This would suggest that wing developmental pathways are conserved in wingless insects and are being re-used. According to the current results, it is speculated that adaptive changes in animal size could modulate the cellular response to signalling molecules such as Wg, thereby helping to drive some of these extraordinary reversible transitions (Rafel, 2008).

Robustness and stability of the gene regulatory network involved in DV boundary formation in the Drosophila wing

Gene regulatory networks have been conserved during evolution. The Drosophila wing and the vertebrate hindbrain share the gene network involved in the establishment of the boundary between dorsal and ventral compartments in the wing and adjacent rhombomeres in the hindbrain. A positive feedback-loop between boundary and non-boundary cells and mediated by the activities of Notch and Wingless/Wnt-1 leads to the establishment of a Notch dependent organizer at the boundary. By means of a Systems Biology approach that combines mathematical modeling and both in silico and in vivo experiments in the Drosophila wing primordium, this regulatory network was modeled and tested; evidence is presented that a novel property, namely refractoriness to the Wingless signaling molecule, is required in boundary cells for the formation of a stable dorsal-ventral boundary. This new property has been validated in vivo, promotes mutually exclusive domains of Notch and Wingless activities and confers stability to the dorsal-ventral boundary. A robustness analysis of the regulatory network complements the results and ensures its biological plausibility (Buceta, 2007).

In silico evidence is presented that refractoriness to the Wg signal in boundary cells provides stability to the gene regulatory network. Boundary cells are characterized by high levels of Notch activity, thus suggesting Notch is responsible for making boundary cells refractory to the Wg signal. The role of Notch in this process was analyzed in the developing wing primordium. Ectopic activation of Notch in non-boundary cells represses Wg target gene expression. Note that Notch, in this case, causes ectopic Wg expression in non-boundary cells, which induces target gene expression only in Wg non-expressing cells. By contrast, ectopic expression of Wg alone induces the expression of target genes in both Wg-expressing and non-expressing cells. When boundary cells lack Notch activity, either by mutation or by expression of a dominant negative form of Delta known to titrate out the Notch receptor, these cells start to express target genes of Wg. It can then be concluded that either Notch activity itself, or one or several of its target genes inhibits the expression of Wg target genes in boundary cells (Buceta, 2007).

High levels of Notch activity induce expression of the homeobox gene cut in boundary cells and Cut has been previously shown to be required to repress Delta and Serrate expression in these cells. Then, whether Cut mediates the activity of Notch in inhibiting the expression of other Wg target genes was examined. In the absence of Cut activity, either in a homozygous mutant background or in clones of mutant cells, boundary cells start expressing genes regulated by the Wg signal, and ectopic Notch activation in non-boundary cells is now unable to repress Wg target gene expression. Note that Notch, in this case, causes ectopic expression of Wg, which induces target gene expression in both Wg-expressing and non-expressing cells. Finally, forced expression of Cut in non-boundary cells represses the expression of Wg target genes. Taken together, these results indicate that Cut is not only required but also sufficient to inhibit Wg target gene expression in boundary cells downstream of Notch (Buceta, 2007).

Cut might exert its function either by blocking the Wg signaling pathway or, alternatively, by inhibiting the expression of every Wg target gene. The Wg signaling pathway is activated by controlling the levels and subcellular localization of the transcriptional co-activator Armadillo (Arm, known as β-catenin in vertebrates). In the absence of Wg signal, Arm levels are kept low through degradation. This degradation depends on the phosphorylation of Arm by the kinase Shaggy/Zeste white-3/Glycogen synthase kinase-3β (GSK-3β). Phosphorylated Arm is recognized rapidly by the proteasome and destroyed. Following Wg ligand binding, this degradation is inhibited, which enables Arm to accumulate, enter the nucleus and activate a transcriptional response. In the Drosophila wing, Arm protein levels are severely reduced in boundary cells, when compared with adjacent cells, even though extracellular Wg protein is available in both types of cells. This observation indicates that the activity of the Wg signaling pathway is repressed in these cells at the level or upstream of Arm. Consistent with this observation, a dominantly activated form of Arm (ArmS10), which lacks the GSK-3ß phosphorylation sites and escapes degradation, induces expression of Wg targets in boundary cells. Overexpression of any other limiting factor of the Wg pathway that acts upstream of Arm is unable to induce Wg target gene expression in these cells (Buceta, 2007).

Cut appears to mediate this type of repression of the Wg signaling pathway. In the absence of Cut activity, Arm protein levels are not reduced in boundary cells, and ectopic expression of Cut in non-boundary cells reduces Arm protein levels and represses the expression of Wg target genes. Moreover, ArmS10 can bypass the effects of ectopic Cut expression and restores Wg target gene expression in non-boundary cells. Co-expression of limiting factors of the Wg pathway acting upstream of Arm does not cause this effect. Taken together, these results indicate that Cut blocks the Wg signaling pathway at the level or upstream of Arm. Cut might exert its function through transcriptional regulation of a gene product involved in regulating the degradation of Arm (Buceta, 2007).

So far in vivo evidence has been provided that Cut is required in boundary cells to repress the Wg signaling pathway and also, by means of in silico experiments, it has been shown that such repression leads to a stable DV boundary formation. In silico implementation of the refractoriness to the Wg signal via Cut leads to stable DV boundary formation. The stationary pattern of gene expression and activity observed in this case is in agreement with in vivo results (Buceta, 2007).

The conclusions can be extended further with regard to the role played by Cut in DV boundary formation. In the absence of refractoriness to the Wg signal (provided by the activity of Cut in boundary cells) an initial increase in Notch activity and Wg expression takes place. This result suggests that Cut is dispensable for the onset of the DV boundary. This and the evolution predicted by modeling are in agreement with the in vivo results. In cut mutant discs, the early activation of Notch at the DV boundary, as shown by the expression of Wg, is comparable to wild-type discs. However, in mature third instar discs Notch activity and Wg expression are not maintained in the mutant background. Taken together, these results indicate that refractoriness of boundary cells to the Wg signal provided by the activity of Cut is required to shape a stationary and stable DV boundary in the developing wing primordium (Buceta, 2007).

This study analyzed the properties of the regulatory network for the establishment and maintenance of the DV organizer in the Drosophila wing imaginal disc. Evidence is provided that that a mathematical model can convert the initial DV asymmetric expression pattern of Notch ligands into the DV symmetric and mutually exclusive domains of active receptor and Notch ligands in boundary and non-boundary cells, respectively. To model the network 'circuitry', and test and verify the proposal, advantage was taken of a combination between in vivo and in silico experiments that has allowed checking of the analytical and predictive capacity of the modeling (Buceta, 2007).

The most striking finding of this research is that a novel property is required in the regulatory network for a robust and stable maintenance of the DV organizer: namely boundary cells must be refractory to the Wg signal. This property is conferred by the activity of Notch through its target gene cut. The role of Cut in repressing the Wg signaling pathway in boundary cells, and Wg in repressing Notch in non-boundary cells, generates two mutually exclusive domains of Notch and Wg activities, corresponding to boundary and non-boundary cells, respectively. Consequently, Notch ligands and receptors are expressed in two distinct non-overlapping cell populations. This helps to restrict the width of the boundary population to few (two-three) cells and contributes to polarizing ligand-receptor signaling towards the boundary and not against it, i.e., flanking ligands signal Notch towards the boundary but not against it since down-regulation of the Notch pathway in non-boundary cells inhibits the receptors' activity in those cells. In addition, light has been shed on several dynamical properties of the network, such as the refinement of Notch activity (Buceta, 2007).

At the time the role of Cut in the repression of Delta and Serrate expression was described, Cut and the concomitant restriction of ligand expression to non-boundary cells were postulated to be essential for the stability of the DV boundary. However, the other negative input of Wg into the Notch pathway through the activity of Dishevelled was not taken into account. In silico results have predicted that a general repression of the Wg pathway is required for stable activity of Notch at the DV boundary. In vivo results indicate that this repression takes place at the level or upstream of Armadillo. In order to be refractory to the inhibitory effect of Dishevelled on Notch, this repression should be taking place close to Dishevelled if not further upstream in the Wg signaling cascade (Buceta, 2007).

Finally, the conclusions are placed into a broader context. Boundary formation between adjacent rhombomeres in vertebrates relies on the same Wnt/Notch-dependent regulatory network. Therefore, it is speculated that boundary cells also need to be refractory to the Wnt signal to generate stable boundaries. To close, it is concluded that the robustness and stability of this network, in which the interconnectivity of the elements is crucial and even more important than the value of the parameters used, might explain its use in boundary formation in other multicellular organisms (Buceta, 2007).

A novel interaction between hedgehog and Notch promotes proliferation at the anterior-posterior organizer of the Drosophila wing

Notch has multiple roles in the development of the Drosophila melanogaster wing imaginal disc. It helps specify the dorsal-ventral compartment border, and it is needed for the wing margin, veins, and sensory organs. Evidence is presented for a new role: stimulating growth in response to Hedgehog. This study shows that Notch signaling is activated in the cells of the anterior-posterior organizer that produce the region between wing veins 3 and 4, and strong genetic interactions are described between the gene that encodes the Hedgehog pathway activator Smoothened and the Notch pathway genes Notch, presenilin, and Suppressor of Hairless and the Enhancer of split complex. This work thus reveals a novel collaboration by the Hedgehog and Notch pathways that regulates proliferation in the 3-4 intervein region independently of Decapentaplegic (Casso, 2011).

This article shows activation of N signaling at the wing AP organizer by defining with cellular resolution the expression patterns of N protein and N pathway reporters in relation to the AP organizer, and dependence on Hh signaling is shown. Strong interactions are also shown between hh- and N-signaling pathways, and it is confirmed that the activation of N signaling is necessary for the normal growth of the AP organizer. This work uncovers a previously unknown activity of the Hh pathway in mitogenesis at the AP organizer: the activation of N signaling. These results are surprising in that they show that the roles of N signaling in the growth of the wing are not limited to the function of the DV organizer and a general growth-promoting function in the wing: N signaling also induces growth downstream of hh at the AP organizer (Casso, 2011).

N is essential for the cells that give rise to the DV margin, veins, and sensory organs of the wing, and its expression is elevated in the progenitors that produce these structures. The DV margin progenitors, which transect the wing disc in a band that is orthogonal to the Hh-dependent AP organizer, express wg in response to N. These wg-expressing cells function as a DV organizer, and several lines of evidence suggest that the AP and DV organizers function independently: Hh signaling along the AP axis is not N-dependent, N signaling along the DV axis is not hh-dependent, and targets regulated by the AP and DV organizers are not the same. The findings reported in this study show that, separately from its roles elsewhere in the wing disc, N signaling has an essential mitogenic role in the cells of the AP organizer region (Casso, 2011).

While N can stimulate growth by inducing expression of wg (as it does in the DV organizer), hyper-activation of N signaling near the AP border of the wing pouch causes overgrowth that is independent of wg. wg is not normally expressed along the AP axis, but this study found that N signaling is activated at the AP compartment border in late third instar discs, pupal discs, and pupal wings. Through vn expression, Hh signaling at the AP compartment border increases expression of Dl flanking the organizer, and Hh signaling activates N in the 3-4 intervein region. While a role for Ser at the AP organizer has not been directly investigated, Ser expression in the wing disc is very similar to that of Dl, with high levels of Ser in the vein 3 and 4 primordia as well as along the DV border. The results show that growth of the 3-4 intervein region, long known to be dependent on Hh, is also dependent on Hh-induced activation of N (Casso, 2011).

Expression of N pathway reporters and components and genetic interactions support this model of regulation of the intervein region. The reporters Su(H)lacZ and E(spl)m-α-GFP express at the AP border in a Hh-dependent manner. Elevated levels of N protein expression on the anterior side of the AP border require Vn signaling. This N region is flanked by Dl expression in the vein 3 and vein 4 primordia; Dl expression is known to be dependent upon expression of the Hh target vn. Genetic interactions between smo RNAi and N and between smo RNAi and N pathway components [e.g., the Psn intramembrane protease, which activates N; the Su(H) transcriptional co-activator; the Su(dx) E3 ubiquitin ligase, which monitors levels of N protein; and the E(spl) complex of N transcriptional targets] also indicate a functional link between the Hh and N systems (Casso, 2011).

The model for the role of N in the 3–4 intervein region is consistent with previous reports of expression patterns of the E(spl) genes E(spl)m8, M-β, and M-α. Ectopic expression of HLH-mδ and m8 rescues smo RNAi. Although HLH-mδ does not appear to be expressed in the AP organizer in a wild-type wing because the E(spl) genes are thought to have partially overlapping functions, the fact that mδ phenocopies the rescue by m8 reinforces the conclusion that the function of the E(spl) genes is critical to inducing growth at the AP organizer. Importantly, these findings show that the cells that activate N are the anterior cells of the AP organizer and are not associated with development of veins in pupal wings. Vein 4 develops within the posterior compartment and in many cases has posterior cells between it and the AP border. Since activation of these reporters was never observed extending into posterior territory, their expression correlates better with the position of the AP organizer than with vein/intervein territories at the stages that were examined. It should be noted that no single readout currently available marks all tissues in which N is activated. The E(spl) genes, for example, express in a variety of spatial and temporal patterns in response to N, and these patterns are only partially overlapping. The possibility cannot be excluded that N signaling is also activated along the stripe of Dl expression in the vein 3 primordium or that signaling could be occurring in the entire broad stripe of elevated N expression in the AP organizer. No changes were seen in proliferation using a direct readout such as phosphohistone staining of mitotic cells to visualize increases or decreases in growth at the AP organizer. These proliferation assays mark cell cycle progression at a single time point in fixed tissues, and the changes that were seen in the adult wing could be due to one or two fewer cell division cycles occurring over the course of days of development (Casso, 2011).

The findings indicate a link between the Hh and N pathways and suggest a model in which the domain of N activation at the AP border [manifested by Su(H)lacZ expression] is a consequence both of flanking cells that express high levels of Dl and of Hh signaling. The proposed role for Hh signaling is multifaceted: Hh is required for vn expression, which is itself required for high levels of Dl expression in the vein 3 stripe and the vein 4 stripe and for N expression at the AP organizer. Although whether Dl expression in veins 3 and 4 activates N signaling has not been directly tested, vn function is necessary for N activation, and the reciprocal relationship between cells expressing high levels of Dl and neighboring cells expressing high levels of N is well established (Casso, 2011).

Interactions between the Sonic hedgehog (SHH) and N signaling pathways have been identified previously in vertebrates. Particularly noteworthy for their relevance to the interactions that were found in the Drosophila wing disc are the increased expression of the Serrate-related N ligand, Jagged 1, in the mouse Gli3Xt mutant; reduced expression of Jagged1 and Notch2 in the cerebella of mice with reduced SHH signaling; regulation of the Delta-related ligand, DNER, by SHH in Purkinje neurons and fetal prostate; activation of N signaling in neuroblastomas in Ptch+/– mice with elevated SHH signaling; and Notch2 overexpression in mice carrying an activated allele of smo. These studies establish a positive effect of SHH signaling on the N pathway, consistent with the current data (Casso, 2011).

In Drosophila, there have been several reports of interactions between the N and Hh pathways. In the wing pouch, for example, expression levels of the Hh targets ptc, ci, col, and en are markedly lower at the intersection of the AP and DV borders than elsewhere in the AP organizer. This repression is mediated by wg. In addition, N and col function together to determine the position of wing veins 3 and 4. However, loss of function of either col or vn did not show interactions with smo RNAi (Casso, 2011).

N functions in two types of settings. One is associated with binary fate choices; it involves adjacent cells that adopt either of two fates on the basis of the activation of N signaling in one cell and inactivation in the other. In these settings, activation of N not only induces differentiation in a designated cell, but also blocks activation of N in the neighbors. The second type of setting does not induce a binary fate choice, but instead activates the pathway at the junction of two distinct cell types. N pathway activation at the DV border in the wing is one example; in this setting, N is activated in a band that straddles the DV border and the N ligands Dl and Ser signal from adjacent domains from either the dorsal (i.e., Dl) or the ventral (i.e., Ser) side. Activation of N in the 3-4 intervein region at the AP border appears to be of this second type: it occurs adjacent to regions of elevated Dl expression at the apposition of anterior and posterior cell types. There is no apparent binary fate choice in this region of the wing (Casso, 2011).

In ways that are not understood well, development of the 3-4 intervein region is controlled differently from other regions of the wing pouch. Whereas Hh induces expression of Dpp, and Dpp orchestrates proliferation and patterning of wing pouch cells generally, Dpp does not have the same role in the 3-4 intervein cells. For these cells, Hh appears to control proliferation and patterning directly. For example, the lateral regions of wings that develop from discs with compromised Dpp function are reduced, but their central regions, between veins 3 and 4, are essentially normal. Downregulation of Dpp activity and repression of expression of the Dpp receptor appears to be the basis for this insensitivity. In contrast, partial impairment of Hh signal transduction that is insufficient to reduce Dpp function (such as in fu mutants or in the smo RNAi genotypes that were characterized) results in wings that are normal in size and pattern except for a small or absent 3-4 intervein region. Since the 3-4 intervein cells divide one to two times in the early pupa during disc eversion and wing formation, the direct role of Hh in regulating these cells may be specific to this post-larval period. N signaling has a well-described mitogenic function in the wing. Ectopic signaling causes hyper-proliferation, while clones that impair the activation of the pathway reduce growth. The current findings indicate that Hh regulates proliferation of cells in the 3-4 intervein region at least in part by activating N signal transduction (Casso, 2011).

The idea that this model promotes is that Hh-dependent activation of N at the AP organizer is stage- and position-specific. This model is consistent with the complex pattern of N expression and activation in the wing, since different pathways may regulate N in different locations. It is also consistent with the proposed role of N regulating the width and position of veins 3 and 4, since the processes that establish the veins and control proliferation of the intervein cells need not be the same, even if they are interdependent. The temporal specificity that this study describes represents an example of how complex patterns are generated with a limited number of signaling pathways -- in this case by using N signaling for different outcomes at different times and in different places. Throughout larval development, Dpp regulates proliferation and patterning in the wing disc. In the pupal wing, Dpp takes on a new instructive vein-positioning function. There is no evidence that Hh regulates Dpp in the pupal wing, and moreover, the cells that had produced Dpp at the AP organizer no longer do so and no longer function as a AP organizers. These data show that N also takes on a new role during late larval and pupal stages: functioning at the AP organizer to regulate growth in response to Hh signaling (Casso, 2011).

Sequential Notch signalling at the boundary of fringe expressing and non-expressing cells

Wing development in Drosophila requires the activation of Wingless (Wg) in a small stripe along the boundary of Fringe (Fng) expressing and non-expressing cells (FB), which coincides with the dorso-ventral (D/V) boundary of the wing imaginal disc. The expression of Wg is induced by interactions between dorsal and ventral cells mediated by the Notch signalling pathway. It appears that mutual signalling from dorsal to ventral and ventral to dorsal cells by the Notch ligands Serrate (Ser) and Delta (Dl) respectively establishes a symmetric domain of Wg that straddles the D/V boundary. The directional signalling of these ligands requires the modification of Notch in dorsal cells by the glycosyltransferase Fng and is based on the restricted expression of the ligands with Ser expression to the dorsal and that of Dl to the ventral side of the wing anlage. In order to further investigate the mechanism of Notch signalling at the FB, the function of Fng, Ser and Dl was analyzed during wing development at an ectopic FB and at the D/V boundary. Notch signalling was found to be initiated in an asymmetric fashion on only one side of the FB. During this initial asymmetric phase, only one ligand is required, with Ser initiating Notch-signalling at the D/V and Dl at the ectopic FB. Furthermore, the analysis suggests that Fng has also a positive effect on Ser signalling. Because of these additional properties, differential expression of the ligands, which has been a prerequisite to restrict Notch activation to the FB in the current model, is not required to restrict Notch signalling to the FB (Troost, 2012).

During wing development, the activity of the Notch pathway is required to establish a stripe-like domain of expression of several genes along the D/V boundary that control wing growth and patterning, chief among them are wg and vg. The D/V boundary is a FB, which provides an interface that is crucial for the activation of Notch and establishment of this organising centre. The current understanding is that Fng promotes Dl signalling and prevents Ser signalling through the modification of Notch. As a result, Dl signals strongly from ventral to dorsal and Ser from dorsal to ventral cells boundary cells. The simultaneous signalling of the ligands in opposite direction establishes the expression of Notch target genes at both sides of the FB. It is essential for this model to work at the D/V boundary that induction of expression of Ser is restricted to dorsal and that of Dl to ventral cells. If e.g. Dl expression could also be induced in dorsal cells by the Notch pathway, the activity of Notch would immediately spread throughout the dorsal half of the wing anlage. In agreement with this requirement, it has been observed that expression of Ser is restricted to dorsal cells upon expression of activated Notch in dorsal and ventral cells. The combination of spatially restricted expression of the ligands and the Dl/Ser loop restricts the activation of Notch to the D/V boundary during early stages of wing development. At the middle of the third larval instar stage the Dl/Ser/Wg loop takes over to maintain the activity of Notch. Thus, a critical step is the establishment of expression of Wg in boundary cells. Once this is achieved the second feedback-loop assures expression of Wg and Notch signalling throughout wing development (Troost, 2012).

While this model can explain the events at the D/V boundary, it cannot explain the events at an ectopic FB, since differential expression of the ligands is unlikely to occur there. Nevertheless, the expression of Wg is also restricted at the ectopic FB. The presented work provides further evidence for the current model of Fng action, but adds new details that enable it to explain also the events at an ectopic FB. One addition is the initial sequential establishment of the expression domain of Wg through asymmetric Notch signalling during early stages of wing development. Asymmetric expression of Wg was observed only in ventral boundary cells (VBCs) at the D/V boundary, indicating that these cells achieve sufficiently high activation of Notch to initiate expression of Wg. The existence of the early asymmetric phase of Notch activation was surprising given the fact that activation of Notch results in the activation of the expression of Dl and Ser. Consequently, the activation of Notch in ventral cells should immediately lead to up-regulation of expression of Dl in ventral cells and back-signalling to dorsal cells. It was therefore expected that if an asymmetric phase exists, it would be too short in time to be detected. Importantly, the initial asymmetric phase appears to be a general property for Notch signalling at a FB, since it was observed on both analysed FBs. The existence of the asymmetric phase also indicates that a FB can be used to generate activity domains of the Notch pathway where the feedback-loop that regulates the expression of the ligands through Notch activation does not occur. So far the loop has been found only in the wing pouch. In the absence of the loop the asymmetric state would remain and thus, a defined stripe of high Notch activation would be generated in a field of cells that uniformly express Dl even in the absence of Ser (Troost, 2012).

At the ectopic FB, it was found that eliminating the activity of the Notch pathway in PBCs does not result in the loss of expression of Wg in ABCs. It only prevents the late symmetric phase. This indicates that establishment of a Dl/Ser feedback loop in A/P boundary cells is not essential for reaching sufficient high levels of Notch signalling to induce Wg expression during the asymmetric phase. The same was observed for the D/V boundary: Depletion of Su(H) function or over-expression of H causes a restriction of expression of Wg to VBCs, but not its abolishment. Thus, the Dl/Ser loop is probably mainly required for the later occurring patterning of the future wing margin, but not for the establishment of the wing primordium (Troost, 2012).

A further addition is that the basic expression of Dl is independent of Ser signalling. This is indicated by the observation that 1) Dl is expressed throughout the wing anlage in early discs and 2) Dl signals to DBCs at the D/V boundary in absence of Ser function. This holds true also for the ectopic FB: here Dl signals to the anterior boundary cells (ABCs) in the absence of Ser. However, in both cases Dl signalling is not strong enough to initiate Wg expression, which is a crucial event for wing development (Troost, 2012).

The results also reveal an unanticipated requirement of Ser in ABCs for the expression of Wg. This requirement could be explained through Ser signalling from the ABCs to PBCs to up-regulate Dl there, which in turn signals back to ABCs (Ser/Dl loop). This explanation would imply that the levels of Notch activation required for the induction of Dl expression are lower than that for Wg. Otherwise, the expression of Wg would not stay asymmetric as it is observed. However, it was found that Notch signalling is not required in PBCs during the early asymmetric phase. This excludes the mentioned explanation and suggests that Ser must activate Notch in the Fng expressing ABCs. This assumedly weak activation contributes to the total activity of Notch in these cells and guarantees levels of Notch signalling above the threshold required for expression of Wg. In the absence of Ser the activation by Dl from PBCs appears to fail to reach the threshold level in a fraction of discs. In agreement with this notion it has been shown that expression of Ser is broadly induced upon ectopic expression of Fng (Troost, 2012).

It was further observed that concomitant loss of Ser and Dl function in ABCs always abolishes the expression of Wg at the ectopic FB. Hence, Dl must also signal in Fng expressing ABCs and contribute to Notch signalling in these cells. The results suggest that the total amount of Notch activity in ABCs is the sum of signalling by Dl and Ser in the Fng domain and Dl signalling from PBCs to ABCs. Whereby the signal from posteriors to anterior is the more important one, since its loss always abolishes expression of Wg. A similar requirement at on both sides of the boundary for Dl had been described for the D/V boundary. Interestingly, the data indicate that Dl plays a similar role there as Ser at the ectopic FB (Troost, 2012).

Another addition to the current model is that Fng has two antagonistic effects on each ligand. The modification of Notch by Fringe is known to polarise signalling at the D/V-boundary by enhancing Dl- and suppressing Ser-signalling. Loss of function of fng during early stages of wing development abolishes expression of Wg at a time where it is solely dependent on Ser signalling. This observation indicates that Fng has a positive effect on Ser signalling in addition to its known negative one: it enhances Ser-signalling from Fng expressing dorsal to non-expressing ventral cells. This enhancement is required to activate expression of Wg in cells across the boundary. This finding is in good agreement with previous work that reports that Fng can enhance the ability of Ser to induce ectopic wing margins upon their co-overexpression. It is a possibility that this positive influence on Ser is indirect: The modification of Notch mediated by Fng results in a decrease of binding Ser to Notch. As a consequence high levels of free Ser are available in Fng expressing boundary cells at the FB that can bind in trans to the unmodified Notch on the adjacent Fng non-expressing boundary cells. At the analysed ectopic FB, it was found that Dl is required in Fng non-expressing PBCs to raise the activity of Notch signalling to a level that is sufficient for expression of Wg in Fng expressing anterior boundary cells, although Dl is expressed ubiquitously in early discs and thus, present in both cell populations at the same levels. This finding indicates that although Dl can induce activity of Notch in the Fng domain, this activity is insufficient to initiate the expression of Wg. The activity rises beyond the threshold only if the cells receive an additional Fng-enhanced Dl signal from non-expressing. This suggests that Fng has a suppressing effect on Dl signalling within its domain. The opposing effects on the activity of each ligand have important implications for restricted Notch-signalling at the FB. Restricted expression of the ligands on opposite sides of the FB is not required to restrict Notch activation to the FB. Only Dl outside the Fng domain is sufficiently active to induce Wg expression in Fng expressing cells. In turn only Ser in Fng expressing cells is sufficiently active to induce Wg expression in Fng non-expressing cells. These conditions are only met at the FB. These properties assure that expression of Wg is restricted to a FB even in a tissue where Dl or Ser are initially expressed uniformly, as it is the case for the ectopic FB (Troost, 2012).

A difference between the ectopic FB and the D/V boundary is that the roles of the ligands are reversed: at the ectopic FB, Dl signalling is required for the establishment of the initial asymmetric phase and Ser to establish the symmetric phase and to maintain expression of Notch activity at a high level. In contrast, Ser signalling is required for the initial asymmetric phase at the D/V boundary and Dl for the establishment of the later symmetric phase and probably maintenance during later stages. Thus, activation of Notch-signalling at a FB can be initiated by both ligands. Another difference is the location of the asymmetric stripe of wg expression, which is located in Fng expressing cells at the ectopic FB, but in non-expressing cells at the DV boundary. It is believed that the events observed at the ectopic FB represents the more general mode of interactions, since the interactions occur solely in the ventral pouch where the cells differ mainly with respect to the expression of Fng. In contrast, at the D/V boundary dorsal cells differ from ventral cells by expression the selector Ap, which might modify the outcome of the interactions (Troost, 2012).

On the basis of these results, the events at the ectopic boundary can be summarized. During early stages of wing development, Dl is ubiquitously expressed throughout the wing anlage. Ectopic expression of fng with ptcGal4, creates a band-like Fng domain in the ventral pouch. During early stages Dl and Ser (probably induced by Dl) activate Notch signalling throughout the Fng domain at low levels that are not sufficient for activation of Wg. At the sharp posterior FB, Dl signalling from PBCs to ABCs, enhanced by Fng, raises the levels of Notch in ABCs beyond the threshold required for expression of Wg. The asymmetric phase is established. Dl signalling also up-regulates expression of Ser in ABCs. Over time Ser signalling from ABCs to PBCs (enhanced by Fng), induces Wg expression in PBCs. After the solid induction of symmetric expression of Wg, the Dl/Ser/Wg loop takes over and maintains Notch signalling at the D/V boundary. It was observed that the expression of Gbe+Su(H)-lacZ is broader upon ectopic expression of Fng in Ser mutant early third instar discs. Moreover, Wg is ectopically expressed throughout the ventral ptc domain upon ectopic expression of Fng by with ptcGal4 in Ser H, but not in H mutant discs. Thus, Ser probably contributes to keeping the Notch activity in the Fng domain at low level. It is known that the expression of Ser can contribute to the suppression of Notch activity in a cell-autonomous manner through cis-inhibition. Cis-inhibition has been discovered during analysis of wing development and appears to be involved in regulation and directional Notch signalling in several processes. This mechanism causes strong Ser signalling only from Ser expressing to non-expressing cells. Since Ser expression is induced in the Fng domain, strong signalling occurs from ABCs to PBCs. Thus, it is likely that cis-inhibition contributes to the directional signalling of from ABCs to PBCs to induce the later symmetric phase (Troost, 2012).

At the D/V boundary signalling is initiated differently. This study has shown that loss of the activity of the Notch pathway in dorsal cells does not prevent the establishment of expression of Wg along the D/V boundary, but restricts it to VBCs and allows wing development to proceed. Thus, the dorsal to ventral signal, which is mediated by Ser is most important. This notion is also in agreement with the null phenotype of Ser. Ser and Fng are initially expressed in all dorsal cells. Fng enhances Ser signalling to VBCs, but suppresses signalling among dorsal cells. This strong polarised signalling results in the activation of Wg expression and up-regulation of Dl expression in VBCs. It is likely, that the cis-inhibitory effect of Ser contributes to the suppression of the activation of Notch in dorsal cells through the initial phase of wing development, since the expansion of the expression of Gbe+Su(H)–lacZ over the whole dorsal wing anlage in early Ser mutant discs. This is probably induced by the weak ubiquitous expression of Dl that was observed in early wing discs. The analysis of the Ser null and Ser H double mutants indicates that Dl can activate Notch signalling in absence of Ser function, but not strong enough to induce Wg expression, despite the presence of the FB. It appears that at the D/V boundary, Ser has to up-regulate the expression of Dl in VBCs over time beyond the threshold that is required to induce Wg expression in DBCs. Fng contributes to induction of Wg expression by enhancing Dl signalling from Fng non-expressing VBCs to expressing DBCs. The requirement for accumulation of Dl could contribute to the observed delay of the establishment of the symmetric phase of expression. After the initial asymmetric phase, the Ser/Dl/Wg loop is established to maintain Notch signalling and symmetric expression of Wg. These results indicate that the initial Fng enhanced Ser signal is sufficiently strong to induce Wg in VBCs in a manner that enables also the establishment of the Ser/Dl/Wg loop. This is indicated by the observation that suppression of Notch activity in all dorsal cells throughout wing development does not prevent maintenance of expression of Wg in late stages of the third instar and even allows the development of adult wings with only minor patterning problems. Thus, the later signal from VBCs to DBCs is mainly required for positioning and patterning of the wing margin (Troost, 2012).

Asymmetric signalling through the Hh pathway has been shown to establish the organising centre for the A/P axis at the anterior side of the boundary. It has been assumed that one difference in the establishment of the D/V organising centre is its symmetric placement on the D/V boundary. The current results suggest that at least during initial phases, the signalling at the D/V boundary is also asymmetric and that the established organising centre can also work if it is displaced ventrally. Thus, it appears that the signalling events at both boundaries are more similar than previously anticipated (Troost, 2012).

Delta, acting throught the Notch receptor, and Hairy establish a periodic prepattern that positions sensory bristles in Drosophila legs

In vertebrates and invertebrates, spatially defined proneural gene expression is an early and essential event in neuronal patterning. This study investigates the mechanisms involved in establishing proneural gene expression in the primordia of a group of small mechanosensory bristles (microchaetae), which on the legs of the Drosophila adult are arranged in a series of longitudinal rows along the leg circumference. In prepupal legs, the proneural gene achaete (ac) is expressed in longitudinal stripes, which comprise the leg microchaete primordia. It has been shown that periodic ac expression is partially established by the prepattern gene, hairy, which represses ac expression in four of eight interstripe domains. This study identifies Delta (Dl), which encodes a Notch (N) ligand, as a second leg prepattern gene. Hairy and Dl function concertedly and nonredundantly to define periodic ac expression. The regulation of periodic hairy expression was explored. In prior studies, it was found that expression of two hairy stripes along the D/V axis is induced in response to the Hedgehog (Hh), Decapentaplegic (Dpp) and Wingless (Wg) morphogens. This study shows that expression of two other hairy stripes along the orthogonal A/P axis is established through a distinct mechanism which involves uniform activation combined with repressive influences from Dpp and Wg. These findings allow for the formulation of a general model for generation of periodic pattern in the adult leg. This process involves broad and late activation of ac expression combined with refinement in response to a prepattern of repression, established by Hairy and Dl, which unfolds progressively during larval and early prepupal stages (Joshi, 2006).

Patterning of the leg imaginal disc along its circumference axis is controlled by the Hh, Dpp and Wg morphogens. In this and previous studies, attempts have been made to elucidate the molecular mechanisms through which these signals give rise to specific morphological features of the leg, the mechanosensory microchaetae. Patterning of leg mechanosensory microchaetae requires spatially defined expression of the proneural gene ac and its repressor Hairy. Expression of hairy in two pairs of longitudinal stripes, the D/V-hairy and A/P-hairy stripes, is directed by separate enhancers that are Hh-, Dpp- and Wg-responsive. In this study, it is reported that the D/V-hairy and A/P-hairy stripes are differentially regulated by Dpp and Wg and that distinct mechanisms are utilized to control hairy expression along the A/P and D/V axes. D/V-hairy expression is locally induced near the A/P compartment boundary by Hh signaling. In addition, Dpp and Wg positively influence expression of the dorsal and ventral components of the D/V-hairy stripes, respectively, by acting together with Hh to define the register of these stripes relative to the compartment boundary. In contrast, the A/P-hairy stripes, which are expressed orthogonal to the D/V-hairy stripes and A/P compartment boundary, are not activated via local induction. Rather, it appears that they are broadly activated along the leg circumference and repressed by Dpp dorsally and Wg ventrally to define their dorsal and ventral boundaries. This model for A/P-hairy regulation is supported by the observations that hairy is ectopically expressed in dorsal, but not ventral, clones lacking tkv or Mad function and that A/P-hairy expression is compromised by elevation of Dpp signaling. Furthermore, ventral, but not dorsal, clones lacking dsh function also ectopically express hairy and high-level Wg signaling results in loss of A/P-hairy expression (Joshi, 2006).

A potential caveat to this model for regulation of A/P-hairy expression is that conclusions were drawn from analysis of endogenous hairy expression rather than by examining expression directed by isolated A/P-hairy enhancer(s). Hence, it is possible that the ectopic hairy expression seen in tkv, Mad and dsh mutant clones is a result of expansion of D/V-hairy rather than A/P-hairy expression. However, several lines of evidence argue against this interpretation. First, through genetic and molecular analyses of D/V-hairy enhancer function, it has been demonstrated that Dpp and Wg positively regulate D/V-hairy expression, an observation that is inconsistent with the suggestion that D/V-hairy is ectopically expressed in clones unable to respond to Dpp or Wg signaling. Furthermore, in 3rd instar and early prepupal leg discs, stages at which the A/P-hairy stripes are not expressed, ectopic hairy expression is not observed in tkv mutant clones. Second, it has been found that the D/V-hairy stripes can only be expressed in anterior compartment cells near the A/P boundary, which are the cells that receive and respond to Hh signal. Thus, it is unlikely that ectopic hairy expression observed in clones at distance from the compartment boundary, which receive little or no Hh signal, and in the posterior compartment, in which cells do not respond to Hh signal, corresponds to D/V-hairy expression. Finally, it has been found that elevation of Dpp or Wg signaling specifically disrupts A/P-hairy but not D/V-hairy expression. Taken together, these findings are consistent with the conclusion that A/P-hairy rather than D/V-hairy is expressed in clones compromised in their response to Dpp and Wg signaling (Joshi, 2006).

The expression of the A/P-hairy stripes at a distance from the dorsal and ventral organizers implies that A/P-hairy expression is repressed even at low threshold levels of Dpp and Wg signaling. This raises questions regarding the mechanisms through which Dpp and Wg define the sharp boundaries of the A/P-hairy stripes. A mechanism for Dpp-mediated repression in imaginal discs has been described, in which a complex of activated Mad with the Schnurri transcription factor acts directly through a repression element in the brinker (brk) gene. However, Dpp does not establish sharp boundaries of brk expression. Rather, brk expression drops off in a graded fashion toward the source of Dpp. Since the dorsal boundary of A/P-hairy expression is sharp and at distance from the Dpp source, this would imply that A/P-hairy expression is very sensitive to Dpp-mediated repression. Hence, it will be of interest to further investigate this process. Also of interest are the mechanisms of Wg-mediated repression, which are poorly understood (Joshi, 2006).

In this study, Dl was identified as a second prepattern gene that functions together with hairy to establish ac expression in the leg microchaete proneural fields. Several lines of evidence that support this conclusion. (1) It was found that, beginning at 4 h APF, Dl expression is up-regulated in domains overlapping the microchaete proneural fields. This distribution of Dl is similar to that, in the notum, where Dl has been shown to regulate proneural ac expression. (2) It was shown that ac expression is expanded in legs with reduced Dl function. (3) It was found that elevated N signaling throughout the tarsus results in severely reduced ac expression. (4) Activation of N signaling was observed within the hairy-OFF interstripes, in agreement with the genetic requirement for Dl/N signaling in these domains. Based on these results, it is proposed that ac expression is activated broadly during mid-prepupal leg development but is confined to the microchaete proneural fields by a previously generated prepattern of repression, established by Hairy and Dl/N signaling. This hypothesis is supported by analysis of cis-regulatory elements that direct ac expression in the leg microchaete proneural fields. By generating rescue and reporter constructs, an enhancer was identified that specifically controls expression of ac in the microchaete proneural fields. Unlike the hairy leg enhancers, no modular organization is observed of the cis-regulatory elements that control expression of ac stripes in different regions of the leg. Rather, preliminary analyses suggest that there is one enhancer consisting of an activation element that directs broad expression of ac along the leg circumference and two repression elements, which are N- or Hairy-responsive. This finding is consistent with genetic studies and the model for regulation of ac expression in the leg microchaete proneural fields (Joshi, 2006).

hairy and Dl function to repress ac expression in complementary domains. hairy encodes a transcriptional repressor which has been shown to directly repress ac expression in the wing by binding a specific site in the ac promoter. It is likely that Hairy acts through a similar site to repress ac expression in the leg. Dl represses ac expression via a different mechanism: presumably, cells of the microchaete proneural fields, which express high levels of Dl, signal to adjacent cells to activate N. This suggestion is supported by the observation that expression of two N-responsive reporters is specifically activated in cells corresponding to the hairy-OFF interstripes. One of the reporters used in this study, E(spl)mβ-CD2, and other similar reporters recapitulate endogenous E(spl)mβ-CD2 expression in wing and leg imaginal discs. E(spl)mβ is one of seven genes in the E(spl)-C that encode bHLH repressors related to Hairy. Hence, it appears that ac expression in the leg microchaete proneural fields may be established by a prepattern of periodically expressed bHLH repressors (Joshi, 2006).

N signaling is not activated within ac-expressing cells, even though these cells express high levels of Dl. This could be explained by a dominant-negative effect of Notch ligands on N signaling, which has been observed in the wing. In the wing, it has been shown that N signaling is not activated within cells expressing high levels of Dl and Ser but, rather, that these cells signal to adjacent cells to activate N signaling within the wing margin. Consistent with the hypothesis of a potential dominant-negative function for Dl in the leg microchaete proneural fields is the observation that over-expression of Dl along the leg circumference results in expansion of ac expression into the hairy-OFF interstripes, which would be expected if N signaling was disabled. Over-expression of N ligand expression has been shown to exert a similar effect in other tissues (Joshi, 2006).

A curious observation of this study is that, as suggested by genetic evidence and the expression of two N-responsive reporters, N signaling, with one exception, is not activated within the hairy-ON interstripes, even though each Hairy stripe is straddled on either side by a Dl stripe. This suggests either that Dl signals asymmetrically or that there is an asymmetric response to N signaling and raises questions regarding the underlying mechanism of asymmetric activation of N-target gene expression. A potential mechanism for asymmetric signaling by Dl is suggested by studies in the notum, in which it has been shown that the N receptor is distributed in a pattern complementary to Dl. If N levels were higher within the hairy-OFF vs. the hairy-ON interstripes in the leg, this could allow for preferential signaling within these domains. However, N expression was assayed in prepupal legs and it was found that N appears to be uniformly distributed along the leg circumference. Hence, either there is an asymmetric response to N or alternative mechanisms are responsible for establishing the directionality of Dl signaling in the leg, such as post-translational modification N signaling pathway components. For example, glycosylation of N by the Fringe glycosyltransferase influences its interactions with its ligands (Joshi, 2006).

Another intriguing finding is the overlap of N signaling with the V-Hairy stripe. This result was surprising because it would suggest redundancy between hairy and Dl/N signaling in this region. However, an absolute requirement for hairy function was observed in the ventral leg. An explanation for this puzzling finding is suggested by the specific loss of the V-Gbe+Su(H)m8-lacZ stripe in hairy mutant legs, which indicates that Dl/N signaling or responsiveness in the ventral leg is dependent on hairy function. The specific loss of N signaling in the ventral leg could be a result of the expansion of Dl expression in hairy mutant legs, which as explained earlier might have a dominant-negative effect on N signaling. This proposal is corroborated by the expansion of ac expression along the circumference of legs ectopically expressing Dl throughout the tarsus. The overlap of hairy and Dl/N signaling in the ventral leg raises questions regarding the function of Dl/N signaling in this domain. It was observed that V-hairy and Gbe+Su(H)m8-lacZ expression overlap only partially, suggesting that combined function of Dl and Hairy in the ventral leg could serve to establish a broader domain of repression in this region in comparison to other interstripe domains. This idea is supported by the morphology of the adult leg tarsus in which the spacing of bristles is most pronounced along the ventral midline. However, the function of N in the ventral leg is not as yet clear. It is plausible that there is a role for Dl/N signaling in the ventral leg that is unrelated to regulation of ac expression (Joshi, 2006).

The potential function of Dl as a regulator of proneural ac expression in the leg was suggested by studies in the notum, on which mechanosensory microchaetae are also organized in longitudinal rows. In the notum, Dl/Notch signaling, rather than Hairy, regulates periodic ac expression. The current studies suggest a distinct mechanism for leg microchaete patterning in which Hairy and Dl act together and nonredundantly to define periodic ac expression. In both the leg and notum, Dl signals to adjacent cells to repress ac expression. However, whereas in the notum Dl activates N signaling in cells on either side of each Dl/Ac stripe, in the leg, N signaling is activated (with one exception) only within the hairy-OFF interstripes. Although the pattern of mechanosensory bristles on the leg and notum is overtly similar, the bristle rows are more precisely aligned in the leg. The more organized pattern on the leg may be a consequence of the combined function of Hairy and Dl which might more precisely define the domains of proneural gene expression (Joshi, 2006).

Dl function is essential for proper patterning of ac expression, and it is suggested that accurate positioning of the Dl stripes is necessary for activation of Notch signaling within appropriate domains. Hence, regulation of Dl expression is an important aspect of leg microchaete patterning. In legs lacking hairy function, Dl expression expands into four broad domains and ectopic hairy expression greatly reduces Dl expression, indicating that periodic expression of Dl is regulated in part by hairy. Concomitant with the expansion of Dl expression, there is loss of N signaling in the ventral leg, suggesting that hairy functions to create an apposition of cells expressing high levels of Dl to cells expressing low levels of Dl, which allows for activation of N signaling in the ventral leg. Regulation of Dl expression in proneural fields is not understood. A plausible hypothesis is that, like hairy, Dl expression is established in response to the morphogens that control pattern formation during leg development (Joshi, 2006).

This and previous studies suggest an outline of a general genetic pathway for the regulation of ac expression in the leg microchaete proneural fields. This process involves broad and late activation, by an unknown factor, of ac expression along the leg circumference combined with refinement in response to a prepattern of repressors, which is established during larval and early prepupal stages. Hairy and Dl have been identified as the primary prepattern factors that regulate ac expression along the leg circumference. Position-specific expression of both hairy and Dl in longitudinal stripes is essential for proper ac expression. It has been determined that the longitudinal stripes of hairy are established in direct response to the Hh, Dpp and Wg signals, which globally pattern the leg, indicating that hairy acts as an interface between ac and these morphogens. Dl expression is regulated by Hairy, but its regulation is otherwise poorly understood. In addition to elucidating a pathway for establishment of periodic ac expression during leg development, these studies also provide insight into the mechanisms through which morphogens function to generate leg morphology (Joshi, 2006).

Periodic ac expression is established progressively. The first evidence of periodicity is expression of the longitudinal stripes of hairy expression. The D/V-hairy stripes are expressed first in the early 3rd instar leg disc followed by the A/P-stripes between 3 and 4 h APF. Between 4 and 6 h APF, Dl expression within the mechanosensory microchaete primordia is established. Then, ac expression is activated uniformly along the leg circumference. By the time that ac expression is activated, the interstripe domains have been defined by the four Hairy stripes and Dl/N signaling (Joshi, 2006).

The delay of ac expression in the microchaete proneural fields until mid-prepupal stages is likely due to the requirement of ac function for formation of all leg sensory organs. Leg sensory bristles can be grouped into two broad categories based on their time of specification: one group includes the early-specified mechanosensory macrochaetae (large bristles) and chemosensory microchaetae, and the second group includes the more numerous late-specified mechanosensory microchaetae. During the 3rd instar and early prepupal stages, ac is expressed in small clusters of cells that define the primordia of early-specified bristles, while expression of ac in the mechanosensory microchaete primordia is activated later in the mid-prepupal stage. This late expression of ac is activated broadly along the leg circumference and is presumably delayed to allow for expression of the hairy and Dl stripes during earlier stages. Premature expression of this normally late ac expression would likely lead to disturbances in sensory organ patterning, suggesting that temporal control of ac expression is an important aspect of its regulation (Joshi, 2006).

Role of Notch signaling in establishing the hemilineages of secondary neurons in Drosophila melanogaster

The secondary neurons generated in the thoracic central nervous system of Drosophila arise from a hemisegmental set of 25 neuronal stem cells, the neuroblasts (NBs). Each NB undergoes repeated asymmetric divisions to produce a series of smaller ganglion mother cells (GMCs), which typically divide once to form two daughter neurons. The two daughters of the GMC consistently have distinct fates. Using both loss-of-function and gain-of-function approaches, this study investigated the role of Notch signaling in establishing neuronal fates within all of the thoracic secondary lineages. In all cases, the 'A' (NotchON) sibling assumes one fate and the 'B' (NotchOFF) sibling assumes another, and this relationship holds throughout the neurogenic period, resulting in two major neuronal classes: the A and B hemilineages. Apparent monotypic lineages typically result from the death of one sibling throughout the lineage, resulting in a single, surviving hemilineage. Projection neurons are predominantly from the B hemilineages, whereas local interneurons are typically from A hemilineages. Although sibling fate is dependent on Notch signaling, it is not necessarily dependent on numb, a gene classically involved in biasing Notch activation. When Numb was removed at the start of larval neurogenesis, both A and B hemilineages were still generated, but by the start of the third larval instar, the removal of Numb resulted in all neurons assuming the A fate. The need for Numb to direct Notch signaling correlated with a decrease in NB cell cycle time and may be a means for coping with multiple sibling pairs simultaneously undergoing fate decisions (Truman, 2010).

The great diversity of cell types within the nervous system has been appreciated since the studies by Cajal. Understanding the rules that are used for generating such diversity remains one of the major goals of neurodevelopment, and the insect CNS has contributed significantly to understanding this problem. With the possible exception of the optic lobes, the generation of central neurons is strictly a lineage-related process, with no regulation of cell fates seen between lineages. In the embryo, the neurons arising from the division of the GMC typically differ from each other, but for mushroom body neurons born during larval life, the two siblings are indistinguishable. The mushroom body pattern, however, is quite different from that inferred from studies in the ventral nervous system of Manduca, which indicate that the two siblings assume different fates. The data in this paper and the study on the antennal lineages by Lin (2010) indicate that the mushroom body pattern is atypical. The pattern across the 25 thoracic lineages is for the GMC to produce two different daughters. In lineages that are monotypic, a situation seemingly similar to that seen in the mushroom body, one of the siblings is consistently removed by programmed cell death soon after its birth (Truman, 2010).

Previous studies showed that Notch was not needed for the maintenance and division of NBs during the larval neurogenic period. These data are completely consistent with that finding. Notch also seems dispensable for the early differentiation and survival of the young neurons. With Notch loss-of-function, sometimes an NB was seen with a cluster of immature cells but no neurites from maturing neurons were seen exiting from the cluster, but this was only seen in lineages in which the B (NotchOFF) sibling normally dies. Similarly, expressing NotchCA also resulted in 'disembodied' NBs and cell clusters, but only in the lineages in which the A (NotchON) sibling is fated for death. These disembodied clusters were especially impressive in lineages, such as lineage 16, that also showed supernumerary NBs due to constitutive Notch activation. Overall, though, these data and those from the loss of the initiator caspase Dronc, show that the abnormal death seen with Notch manipulation is a result of the role of Notch in determining cell fate, rather than a requirement for Notch for survival or early differentiation (Truman, 2010).

An interesting question is whether there are global rules for fate determination that apply across the lineages. In monotypic or almost monotypic lineages, there is no consistent relationship of the dominant sibling to the state of Notch signaling. In seven of the monotypic thoracic lineages the 'A' sibling is the dominant surviving cell type, whereas in nine lineages, the 'B' sibling is the dominant cell type. Axonal projection, however, does correlate strongly with whether or not the Notch pathway is activated. Only four bundles (6ci, 7c, 18c and 19c) project into the longitudinal tracts, and these are the B siblings of their respective lineages. Five lineages produce motoneurons (from NBs 15, 20, 21, 22 and 24) and they also represent the 'B' fate. In addition, six more lineages [0, 4, 8, 12, 13 and 19] have one sibling that has a local primary target, whereas the other sibling projects to the periphery or across a commissure to the contralateral side of the CNS. Lineage 4 is the only one of this group in which the B sibling stays within its hemineuropil, whereas the A sibling projects across a commissure. The remaining ten lineages are uninformative because they are situations like that in lineage 3, in which both siblings stay local, or lineage 1, in which both siblings are projection cells. These last examples notwithstanding, there is a strong bias for the A (NotchON) sibling to stay local and for the B (NotchOFF) sibling to project to distant targets. Notch signaling works through the Suppressor of hairless [Su(h)] transcription factor, so one might suspect that targets downstream of Su(h) might promote features of local interneurons and suppress projection neuron characteristics (Truman, 2010).

Although the role of Notch in establishing sibling identity is consistent across the lineages and through time, that of numb is not. During embryonic neurogenesis, the loss of numb function results in constitutive activation of Notch and the production of only A siblings. Early in the postembryonic period, however, it was found that the GMCs produce daughters of both fates despite the loss of numb function, but by the start of the third instar numb becomes essential for directing Notch activity. Therefore, early in the secondary phase of neurogenesis, Notch signaling is not dependent on numb, and other factors must be at play to allow Notch to establish the differences in sibling identity. The nature of these factors is not known (Truman, 2010).

It may be important that the lack of a requirement for numb is correlated with rate of division of the NB. In the embryo and in the third larval instar the cell cycle of the NB is less than an hour. In the latter case, two or more neurons in a given cluster showing nuclear-localized Notch are seen suggesting that siblings from successive GMCs undergo fate decisions in an overlapping manner. Using Numb protein at this time to bias sibling identity would ensure that a given sibling would not be given ambiguous signals from a cousin. When the NBs first reactivate in the second instar, however, they are dividing much more slowly. Assuming that the dynamics of GMC lifespan are similar to those seen later, it is suspected that at these earlier times only one sibling pair in a cluster may be undergoing Notch-dependent decisions at a time. This would permit the types of cell-cell interactions, such as seen in some peripheral sensory precursor cells or postulated for grasshopper lineages, to come into play. The biasing of cells by Numb may be an adaptation for rapid cell cycles when multiple neuron pairs are sensitive at the same time (Truman, 2010).

Recently, it has been shown that some NBs, the posterior asense-negative (PAN) NBs, differ from the classic scheme in that their GMCs undergo additional divisions before making postmitotic neurons (Bello, 2008; Boone, 2008; Bowman, 2008). PAN neuroblasts respond dramatically to enhanced Notch signaling; clones that either express NotchCA or are numb negative show a dramatic expansion in the number of NBs in a given cluster, as some of the GMCs transform into NBs (Bowman, 2008). In contrast to the PAN NBs, 'classic' brain NBs are unaffected by enhanced Notch activity (Truman, 2010).

A dichotomy was found in how the NBs in the ventral CNS respond to the loss of Numb or expression of NotchCA. Most NBs and GMCs characteristically maintain their normal pattern of division despite constitutive Notch activation. A few lineages (NBs 8, 9, 13, 16, 17, 19 and the 20s), however, resemble the PAN lineages of the brain in that they respond to constitutive Notch signaling by generating multiple NBs. Their responses are more muted than the brain NBs, however, with only a few extra NBs being generated in a given cluster (Truman, 2010).

As also seen for an antennal lobe lineage by Lin (2010), the sensitivity to constitutive Notch signaling is greatest early in the postembryonic life of the NB. This transient sensitivity to Notch activation suggests that some thoracic NBs may have PAN neuroblast characteristics early after their reactivation but later establish a traditional mode of behavior for the remainder of their lineage. However, no other GMC clones were found with more than two siblings, which would be expected if some thoracic lineages made a few 'transiently amplifying' progeny. Clones were not systematically induced through the early period of larval neurogenesis, however, so the possibility that a few transiently amplifying GMCs are produced among the earlier GMCs in these lineages cannot be excluded (Truman, 2010).

The production of supernumerary NBs has also been seen in caterpillars of Manduca sexta, after treatment with hydroxyurea, a drug that blocks nucleotide reductase. Although the drug treatment was devised to kill cycling NBs, it was found that there was a brief window at the start of postembryonic neurogenesis when drug treatment caused some NBs to duplicate, rather than die, and resulted in twice as many neurons of the appropriate phenotype. It may be that some NBs in moths may also have PAN neuroblast characteristics early in larval life and that hydroxyurea causes a transiently amplifying precursor to cross a line that changes it into a fully fledged NB (Truman, 2010).

It is interesting that the lineages that are the most sensitive (i.e. show extra NBs in over 50% of the clones) to either Numb loss or Notch activation are ones that supply local interneurons to the leg neuropil (lineages 8, 9, 13, 14, 16, 19, 20, 21 and 22). Providing PAN neuroblast characteristics to these NBs may be a strategy to enhance the cell number or cellular diversity in this highly integrative region of the CNS (Truman, 2010).

This paper has presented a comprehensive analysis of the role of Notch signaling in generating neuronal phenotypes within the secondary lineages of the thoracic ventral CNS. The universal pattern is for a GMC to produce two neurons of different phenotypes, A and B, with cell death involved in making some lineages monotypic. A clear division of labor between these A and B cell types suggest that the components of circuitry of the thoracic nervous system are generated in developmental units termed 'hemilineages'. It is believed that viewing the construction of this part of the nervous system through the lens of the hemilineage will lead to insights into the way by which the genome generates units of connectivity and how these are constructed into circuits underlying behavior (Truman, 2010).

Lineage-specific effects of Notch/Numb signaling in post-embryonic development of the Drosophila brain

Numb can antagonize Notch signaling to diversify the fates of sister cells. Paired sister cells acquire different fates in all three Drosophila neuronal lineages that make diverse types of antennal lobe projection neurons (PNs). Only one in each pair of postmitotic neurons survives into the adult stage in both anterodorsal (ad) and ventral (v) PN lineages. Notably, Notch signaling specifies the PN fate in the vPN lineage but promotes programmed cell death in the missing siblings in the adPN lineage. In addition, Notch/Numb-mediated binary sibling fates underlie the production of PNs and local interneurons from common precursors in the lAL lineage. Furthermore, Numb is needed in the lateral but not adPN or vPN lineages to prevent the appearance of ectopic neuroblasts and to ensure proper self-renewal of neural progenitors. These lineage-specific outputs of Notch/Numb signaling show that a universal mechanism of binary fate decision can be utilized to govern diverse neural sibling differentiations (Lin, 2010).

In contrast to MB lineages, in which GMCs divide to make two indistinguishable neurons, the three AL neuronal lineages examined produce GMCs that consistently undergo asymmetric cell division and yield daughter cells with distinct fates. This mechanism allows doubling of neuron types, as in the lAL lineage. However, in the adPN and vPN lineages, only one from each pair of daughter cells persists into the adult stage. They are both present as hemilineages. Notably, about 50% of central brain lineages exist as hemilineages, as revealed by clonal analysis with twin-spot MARCM using a pan-neuronal driver. Recovery of the missing hemilineages in the Drosophila VNC has implicated the Notch/Numb-mediated asymmetric cell division as a mechanism for divergent configuration of distinct insect brains. In sum, asymmetric cell division is broadly utilized; depending on the lineages, a GMC may divide to make two identical neurons, two distinct neurons, or only one mature neuron (Lin, 2010).

Notch and Numb underlie asymmetric cell division in diverse contexts, including the asymmetric cell divisions of diverse AL PN precursors. Notably, the output of Notch signaling is grossly opposite in the adPN versus vPN lineage. Each GMC in both lineages makes one PN and one mysterious sibling. Interestingly, Notch-on specifies the PN fate in the vPN lineage but antagonizes the PN fate in the adPN lineage. The cell-fate determinants for PNs of different lineages could be more distinct than their gross phenotypes suggest. In addition, the mysterious siblings of adPNs versus vPNs, upon rescued, might acquire very different fates. These lineage-dependent outputs of Notch signaling support the argument for its involvement in modulating cell differentiation, rather than specifying any de novo cell fate. It appears that two, possibly mutually exclusive, cell fates pre-exist in each precursor, and that Notch signaling, which occurs only in Numb-negative daughter cells, triggers cell differentiation along one rather than the other pre-programmed path (Lin, 2010).

Notch/Numb-dependent asymmetric cell division underlies the derivation of two complex lAL hemilineages that both persist into the adult stage. Distinct PN types are made along the Notch-off hemilineage, whereas diverse types of non-PNs, including various AL LNs and most Acj6-positive progeny, differentiate from Numb-negative daughter cells. As in other neuronal lineages, specific neuron types of the lAL lineage are made at specific times of development. However, it remains uncertain whether specific PN types consistently pair with specific non-PN types through the production of the sister hemilineages. Superficially, there exist many more non-PN types than the recognizable PN types in the lAL lineage, raising the possibility that neuronal temporal identity is altered in distinct paces between the two lAL hemilineages. Determining individual lAL GMCs and their derivatives is essential for resolving the detail and further elucidating how two parallel sets of temporal cell fates can be generated by a common progenitor through repeated self-renewal (Lin, 2010).

Besides governing neuronal cell fates following asymmetric cell division of GMCs, Numb, together with other basal complex proteins, including Brat and Prospero, is selectively segregated into GMCs during self-renewal of Nbs. However, in contrast with its essential role for preventing the transit-amplifying precursors from undergoing tumor-like overproliferation in PAN lineages, the function of Numb in restraining the basally situated Nb offspring from adopting Nb fate varies among non-PAN lineages and depends on the stage of development. Notably, Numb is required in certain non-PAN neuronal lineages, including the lAL lineage, for preventing production of ectopic Nbs. Although Notch is dispensable for maintaining the stem cell fate in lAL Nbs, it remains likely that loss of Numb leads to ectopic Notch signaling, which in turn promotes stem cell fate in otherwise GMCs. The differential requirement of Numb for proper specification of GMCs of different origins could be due to lineage- and/or stage-dependent variations in the abundance of Notch signaling components. Interestingly, the ectopic Nbs apparently maintain proper temporal identity and could make diverse neuron types as the endogenous progenitor. These raise the possibility that dynamic Notch signaling might be utilized in vivo to promote self-renewal versus amplification of Nbs (Lin, 2010).

Taken together, most neuron types in the Drosophila central brain are specified not only according to their lineage origin as well as birth order, but also depending on whether Numb exists to suppress Notch signaling in newly derived postmitotic neurons. It appears that postmitotic neurons are born with two opposing cell fates that were pre-determined in their immediate precursors based on their lineage and temporal origin. Notch signaling then suppresses the otherwise dominating fate. In addition, in certain neuronal lineages, Numb plays a subtle role in ensuring production of GMCs while Nbs undergo self-renewal. A conserved Notch/Numb-dependent mechanism probably governs diverse neural developmental processes through evolution (Lin, 2010).

dFezf/Earmuff maintains the restricted developmental potential of intermediate neural progenitors in Drosophila; Erm restricts the potential of intermediate neural progenitors by activating Prospero to limit proliferation and by antagonizing Notch signaling to prevent dedifferentiation

To ensure normal development and maintenance of homeostasis, the extensive developmental potential of stem cells must be functionally distinguished from the limited developmental potential of transit amplifying cells. Yet the mechanisms that restrict the developmental potential of transit amplifying cells are poorly understood. This study shows that the evolutionarily conserved transcription factor dFezf/Earmuff (Erm) functions cell-autonomously to maintain the restricted developmental potential of the intermediate neural progenitors generated by type II neuroblasts in Drosophila larval brains. Although erm mutant intermediate neural progenitors are correctly specified and show normal apical-basal cortical polarity, they can dedifferentiate back into a neuroblast state, functionally indistinguishable from normal type II neuroblasts. Erm restricts the potential of intermediate neural progenitors by activating Prospero to limit proliferation and by antagonizing Notch signaling to prevent dedifferentiation. It is concluded that Erm dependence functionally distinguishes intermediate neural progenitors from neuroblasts in the Drosophila larval brain, balancing neurogenesis with stem cell maintenance (Weng, 2010).

Tissue development and homeostasis often require stem cells to transiently expand the progenitor pool by producing transit amplifying cells. Yet the developmental potential of transit amplifying cells must be tightly restricted to ensure generation of differentiated progeny and to prevent unrestrained proliferation that might lead to tumorigenesis. Transit amplifying cells are defined by their limited developmental capacity, a feature specified during fate determination. It is unknown whether an active mechanism is required to maintain restricted developmental potential in transit amplifying cells after specification. This study used intermediate neural progenitors (INPs) in developing Drosophila larval brains as a genetic model to investigate how restricted developmental potential is regulated in transit amplifying cells (Weng, 2010).

A fly larval brain hemisphere contains eight type II neuroblasts that undergo repeated asymmetric divisions to self-renew and to generate immature INPs. Immature INPs are unstable in nature and are mitotically inactive, and they lack the expression of Deadpan (Dpn) and Asense (Ase). Immature INPs commit to the INP fate through maturation, a differentiation process necessary for specification of the INP identity. INPs express Dpn and Ase, and undergo 8-10 rounds of asymmetric divisions to self-renew and to produce ganglion mother cells (GMCs) that typically generate two neurons. While 5-6 immature INPs and 1-2 young INPs are always in direct contact with their parental neuroblasts, the older INPs become progressively displaced from their parental neuroblasts over time (Weng, 2010).

During asymmetric divisions of type II neuroblasts, the basal proteins Brain tumor and Numb are exclusively segregated into immature INPs, and function cooperatively, but nonredundantly, to ensure that immature INPs undergo maturation and commit to the INP fate. brain tumor or numb mutant type II neuroblasts generate immature INPs that fail to mature and do not commit to the INP fate. Instead, brain tumor or numb mutant immature INPs adopt their parental neuroblast fate, leading to supernumerary type II neuroblasts. Thus, brain tumor and numb specify the INP fate, and the ectopic expansion of type II neuroblasts in these mutant genetic backgrounds occurs due to failure to properly specify the INP fate. Although Brain tumor is also asymmetrically segregated into GMCs during asymmetric divisions of INPs, the mosaic clones in brain tumor mutant INPs contain only differentiated neurons. This result indicates that Brain tumor is dispensable for maintaining the restricted developmental potential of INPs. How restricted developmental potential is maintained in INPs is currently unknown (Weng, 2010).

To identify genes that regulate self-renewal of neuroblasts, a genetic screen was conducted for mutants exhibiting ectopic larval brain neuroblasts. One mutation, l(2)5138, specifically resulted in massive expansion of neuroblasts in the brain but did not affect neuroblasts on the ventral nerve cord. The l(2)5138 mutation mapped to the 22B4-7 chromosomal interval that contains the earmuff (erm) gene (Pfeiffer, 2008). The erm transcripts are first detected at embryonic stage 4-6 in the specific domain preceding formation of the embryonic brain and remain highly expressed in the brain throughout development. Tbis study reports that Erm functions to restrict the developmental potential of INPs by promoting Prospero-dependent termination of proliferation and suppressing Notch-mediated dedifferentiation. By restricting their developmental potential, Erm ensures that INPs generate only differentiated neurons during Drosophila neurogenesis (Weng, 2010).

All neuroblasts in l(2)5138 homozygous mutant brains were proliferative, expressed all known neuroblast markers, and lacked neuronal and glial markers. The l(2)5138 mutation mapped to the erm gene, which encodes a homolog of the vertebrate Forebrain embryonic zinc-finger family (Fezf) transcription factors. The l(2)5138 mutants contained a single A/T nucleotide change in the erm coding region, leading to the substitution of a leucine for a conserved histidine in the third C2H2 zinc-finger domain. Consistent with its predicted molecular function, ectopic expression of Erm transgenic proteins tagged with a HA epitope at the amino- or carboxyl-terminus driven by neuroblast-specific Wor-Gal4 was detected in the nuclei of neuroblasts. However, the expression of the HA-tagged Erm transgenic protein bearing the identical leucine-to-histidine substitution as in the l(2)5138 mutant was undetectable, suggesting that the mutant Erm protein is unstable. It is concluded that l(2)5138 is a mutant allele of erm (Weng, 2010).

To determine whether erm mutant brains have ectopic type I and/or type II neuroblasts, the expression pattern was examined of Ase and Prospero (Pros), which are only expressed in type I neuroblasts. It was found that erm mutant brains contained over 20-fold more type II neuroblasts (Dpn+Ase-) than wild-type brains, with no significant change in the number of type I neuroblasts (Dpn+Ase+). Next, the localization of Prospero was examined in mitotic neuroblasts in larval brains expressing GFP induced by Ase-Gal4 (Ase > GFP), which mimicked the expression pattern of the endogenous Ase protein. In erm mutant larval brains, all mitotic type I neuroblasts (GFP+) showed formation of basal Prospero crescents, but none of the mitotic type II neuroblasts (GFP-) showed the expression of Prospero. Furthermore, GFP-marked erm mutant type II neuroblast clones consistently contained multiple type II neuroblasts, whereas erm mutant type I neuroblast clones always contained single type I neuroblasts and neurons. It is concluded that erm mutant brains exhibit an abnormal expansion of type II neuroblasts (Weng, 2010).

To determine the cellular origin of ectopic type II neuroblasts in erm mutant brains, the identity of cells in the GFP-marked clones derived from wild-type or erm mutant type II neuroblasts was examined using specific cell fate markers. At 30 hr after clone induction, wild-type and erm mutant neuroblast clones appeared indistinguishable, containing single parental neuroblasts (Dpn+Ase-; R10 mm) in direct contact with 5-6 immature INPs (Dpn-Ase-), while most of the INPs (Dpn+Ase+; R6 mm) were 1 cell or more away from the parental neuroblasts. At 48 hr after clone induction, the overall size of both wild-type and erm mutant neuroblast clones increased significantly due to an increase in cell number, reflecting continuous asymmetric divisions of the parental neuroblasts. In both wildtype and erm mutant clones, the parental neuroblasts remained surrounded by 5-6 immature INPs, while INPs and differentiated neurons (Dpn-Ase-Pros+) were found several cells away from the parental neuroblasts. However, erm mutant clones contained fewer INPs than the wild-type clones. Importantly, erm mutant clones consistently contained 4-6 smaller ectopic type II neuroblasts (Dpn+Ase-; 6-8 mm in diameter). Thus, Erm is dispensable for both the generation and maturation of immature INPs (Weng, 2010).

Ectopic type II neuroblasts in 48 hr erm mutant clones were always several cells away from the parental neuroblasts. This result strongly suggests that ectopic type II neuroblasts in erm mutant clones likely originate from INPs and Erm likely functions in INPs. However, it was not possible to assess the spatial expression pattern of the endogenous Erm protein in larval brains due to lack of a specific antibody and low signals by fluorescent RNA in situ. Alternatively, the expression of the R9D series of Gal4 transgenes was analyzed, in which Gal4 is expressed under the control of overlapping erm promoter fragments (Pfeiffer, 2008). The expression of R9D11-Gal4 was clearly detected in INPs, but was undetectable in type II neuroblasts and immature INPs even when two copies of the UAS-mCD8-GFP transgenes were driven by two copies of R9D11-Gal4 at 32°C for 72 hr after larval hatching. Consistently, the expression of Erm-Gal4 was virtually undetectable in brain tumor mutant brains that contain thousands of type II neuroblasts and immature INPs. While the expression of UAS-erm induced by the neuroblast-specific Wor-Gal4 driver led to premature loss of type II neuroblasts, expression of UAS-erm driven by Erm-Gal4 failed to exert any effect on type II neuroblasts. Importantly, targeted expression of the fly Erm or mouse Fezf1 or Fezf2 transgenic protein driven by R9D11-Gal4 restored the function of Erm and efficiently rescued the ectopic neuroblast phenotype in erm mutant brains. Therefore, R9D11-Gal4 (Erm-Gal4) contains the enhancer element sufficient to restore the Erm function in INPs leading to suppression of ectopic type II neuroblasts in erm mutant brains (Weng, 2010).

Mutant clonal analyses and overexpression studies strongly suggest that Erm functions to suppress reversion of INPs back into a neuroblast state. This study directly tested whether INPs in erm mutant brains can dedifferentiate back into type II neuroblasts. βgal-marked lineage clones originating exclusively from INPs were induced via FRT-mediated recombination. A short pulse of flipase (FLP) expression was targeted in INPs by heat-shocking larvae carrying a UAS-flp transgene under the control of Erm- Gal4 and tub-Gal80ts at 30°C for 1 hr. At 72 hr after heat shock, INP clones in wildtype brains contained only differentiated neurons (Dpn-Ase-). In contrast, INP clones in erm mutant brains contained one or more type II neuroblasts as well as immature INPs, INPs, GMCs, and neurons. This result indicates that while INPs in wild-type larval brains can give rise to only neurons, INPs in erm mutant brains can dedifferentiate into type II neuroblasts that can give rise to all cell types found in a normal type II neuroblast lineage. It is concluded that Erm functions to maintain the restricted developmental potential of INPs and prevents them from dedifferentiating back into a neuroblast state (Weng, 2010).

To determine how Erm maintains the restricted developmental potential of INPs, microarray analyses was performed, and prospero mRNA was found to be drastically reduced in erm mutant brains compared to the control brains. It was confirmed that the relative level of prospero mRNA was indeed reduced by 60%-70% in erm mutant brain extracts by using real-time PCR. These data supported that Erm is necessary for proper transcription of prospero, and prompted a test to see if overexpression of Erm might be sufficient to induce ectopic Prospero expression. A short pulse of Erm expression in brain neuroblasts was induced by shifting larvae carrying a UAS-erm transgene under the control of Wor- Gal4 and tub-Gal80ts to from 25°C to 30°C. A 3.5 hr pulse of Erm expression was sufficient to induce nuclear localization of Prospero in larval brain neuroblasts. Consistent with nuclear Prospero promoting termination of neuroblast proliferation, ectopic expression of Erm induced by Wor-Gal4 resulted in decreased neuroblasts compared to wild-type brains (Figure 5B). Thus, it is concluded that overexpression of Erm can restrict neuroblast proliferation by triggering nuclear localization of Pros (Weng, 2010).

The data suggest that Erm might restrict the developmental potential of INPs in part by limiting their proliferation by activating Prospero-dependent cell cycle exit. If so, it was predicted that overexpression of Erm should induce ectopic nuclear Prospero in INPs and overexpression of Prospero should suppress ectopic neuroblasts in erm mutant brains. In wild-type brains, 9.6% of INPs (32/325) showed nuclear localization of Prospero. However, overexpression of Erm driven by Erm-Gal4 led to nuclear localization of Prospero in 41.5% of INPs (105/253), likely restricting their proliferation potential and resulting in some parental type II neuroblasts surrounded only by differentiated neurons. Importantly, ectopic expression of Prospero induced by Erm-Gal4 efficiently suppressed ectopic neuroblasts and restored neuronal differentiation in erm mutant brains. Thus, Erm likely restricts the proliferation of INPs by promoting nuclear localization of Prospero. To confirm that Prospero indeed functions downstream of Erm to restrict the proliferation of INPs, genetic epistatic analyses were performed. Consistent with previously published results, prospero mutant type I neuroblast clones contained ectopic type I neuroblasts. In contrast, prospero mutant type II neuroblast clones exhibited accumulation of ectopic INPs while maintaining single parental neuroblasts. Furthermore, overexpression of Erm failed to suppress ectopic INPs in prospero mutant type II neuroblast clones, consistent with Prospero functioning downstream of Erm. These results indicate that blocking differentiation is not sufficient to trigger the dedifferentiation of INPs back into type II neuroblasts. Thus, Erm’s restriction on the proliferation of INPs is dependent on Prospero function, but its suppression of the dedifferentiation of INPs is independent of Prospero (Weng, 2010).

Previous studies showed that overexpression of constitutively active Notch (Notchintra) in both type I and II neuroblasts is sufficient to trigger ectopic neuroblasts. This study tested whether Erm suppresses the dedifferentiation of INPs by inhibiting Notch signaling. Indeed, knockdown of Notch function by RNAi in erm mutant brains led to a dramatic reduction in ectopic type II neuroblasts compared to erm mutant brains alone. Complementarily, ectopic expression of constitutively active Notch (Notchintra) induced by Erm-Gal4 transforms INPs into ectopic type II neuroblasts. Thus, reduced Notch function suppresses the dedifferentiation of INPs in erm mutant brains whereas ectopic activation of Notch induces the dedifferentiation of INPs. Next, whether Erm suppresses the dedifferentiation of INPs by antagonizing a Notch-activated mechanism was tested. Coexpression of Erm under the control of Erm-Gal4 is sufficient to suppress ectopic neuroblasts induced by the expression of Notchintra. Thus, it is concluded that Erm can suppress the dedifferentiation of INPs by negatively regulating a Notch-activated signaling mechanism (Weng, 2010).

This study has reported a mechanism that actively maintains the restricted developmental potential of transit amplifying cells after specification of their identity. The evolutionarily conserved transcription factor Erm/Fezf functions to maintain the restricted developmental potential of INPs by limiting their proliferation potential and suppressing their dedifferentiation capacity. Combining proper specification of the transit amplifying cell identity and active maintenance of their restricted developmental potential ensures the generation of differentiated progeny and prevents aberrant expansion of stem cells (Weng, 2010).

The lineage clones derived from single INPs in erm1/erm2 mutant brains contain dedifferentiated neuroblasts, immature INPs, INPs, GMCs, and neurons. Several mechanisms could lead to the diversity of cells within the clones. First, INPs in erm mutant brains might generate GMCs and neurons initially due to the presence of maternally deposited Erm. However, erm transcripts are undetectable in both adult male and female germlines by microarray analyses and in stage 1-3 embryos by RNA in situ. Furthermore, the erm1/erm2 allelic combination resulted in little to no zygotic Erm in the brain because the erm1 mutation likely leads to the production of an unstable Erm protein, whereas the erm2 mutation deletes the entire erm open reading frame. Additionally, the ectopic neuroblast phenotype in erm1/erm2 mutant brains can be observed as early as 36-48 hr after larval hatching. Thus, generation of GMCs and differentiated neurons by INPs in erm1/erm2 mutant brains is unlikely due to the maternal effect. Alternatively, erm may promote GMC differentiation in the type II neuroblast lineage, and in erm mutant brains, GMCs might dedifferentiate back into neuroblasts. If so, an ectopic accumulation of INPs would be predicted in similarly staged mosaic clones derived from erm mutant type II neuroblasts as compared to wild-type clones. However, 48 hr erm mutant single neuroblast clones consistently contained fewer INPs when compared to the wild-type clones. In addition, blocking GMC differentiation by removing Prospero function resulted in ectopic accumulation of INPs but did not lead to ectopic neuroblast formation. Therefore, the diversity of cells within erm mutant clones is also unlikely due to blocking GMC differentiation. The interpretation is favored that erm mutant INPs dedifferentiate into apparently normal neuroblasts that can give rise to all cell types found in a type II neuroblast lineage. Consistently, the dedifferentiated neuroblasts in erm mutant brains exhibited normal cortical polarity and proliferation potential. Furthermore, the dedifferentiated neuroblasts in erm mutant brains also lost the expression of Pros-Gal4 and Erm-Gal4 and established ectopic type II neuroblast lineages encapsulated by the cortex glial membrane.Thus, it is concluded that Erm likely restricts the developmental potential of INPs by limiting proliferation and suppressing dedifferentiation (Weng, 2010).

Although mutations in erm, brain tumor, and numb genes all lead to ectopic type II neuroblasts, the proteins appear to regulate INPs at distinct steps in the type II neuroblast lineage. Numb and Brain tumor function cooperatively, but nonredundantly, to ensure that immature INPs undergo maturation and commit to the INP fate (Boone, 2008; Bowman, 2008). While ectopic expression of Numb induces premature differentiation of type II neuroblasts and immature INPs, overexpression of Numb is not sufficient to suppress ectopic neuroblasts in brain tumor mutant brains. Thus, Numb likely promotes differentiation of immature INPs whereas Brain tumor likely prevents immature INPs, which are unstable in nature, from adopting their parental neuroblast fate. More studies will be necessary to discern whether ectopic neuroblasts in brain tumor mutant brains arise from dedifferentiation of partially differentiated immature INPs or failure of immature INPs to initiate differentiation. In contrast, immature INPs in erm mutant brains mature into functional INPs that exhibit normal cortical polarity and proliferation potential and can generate GMCs and neurons. Additionally, overexpression of Brain tumor or Numb in INPs was not sufficient to suppress ectopic neuroblasts in erm mutant brains. Finally, lineage clones derived from single INPs in erm mutant brains always contain ectopic type II neuroblasts, multiple immature INPs, INPs, GMCs, and neurons. These results indicate that Erm is dispensable for maturation of immature INPs and is not within the genetic hierarchy specifying the INP identity. Instead, Erm maintains the restricted developmental potential of INPs after specification of their identity (Weng, 2010).

Prospero encodes a homeodomain transcription factor, and nuclear Prospero has been shown to trigger cell cycle exit and GMC differentiation. In the wild-type brain, 9.6% of INPs showed nuclear Prospero and were likely undergoing differentiation. prospero mutant type II neuroblast clones showed ectopic accumulation of INPs but contained single neuroblasts, indicating that blocking differentiation is not sufficient to trigger the dedifferentiation of INPs. Thus, Prospero restricts the proliferation potential of INPs but does not suppress dedifferentiation of INPs (Weng, 2010).

While ectopic expression of Prospero in INPs can restore neuronal differentiation in erm mutant brains, targeted expression of Erm in neuroblasts or INPs was sufficient to induce rapid nuclear localization of Prospero in these cells and terminate their proliferation. In wild-type brains, Prospero is sequestered in a basal crescent by the adaptor protein Miranda in mitotic neural progenitors. Interestingly, mitotic neural progenitors including neuroblasts and INPs transiently overexpressing Erm also showed basal localization and segregation of Miranda and Prospero. As such, Erm likely restricts the proliferation potential of INPs by indirectly promoting nuclear localization of Prospero. Therefore, Prospero does not localize in the nuclei of mitotically active INPs, which express Miranda, but does localize in the nuclei of GMCs that do not express Miranda (Weng, 2010).

How does Erm suppress the dedifferentiation of INPs? The results show that reduced Notch function can efficiently suppress ectopic neuroblasts in erm mutant brains while constitutive activation of Notch signaling induced the dedifferentiation of INPs. Importantly, coexpression of Erm is sufficient to suppress the dedifferentiation of INPs triggered by expression of constitutively active Notchintra. Together, these results strongly suggest that Erm prevents the dedifferentiation of INPs by antagonizing a Notch-activated mechanism through interfering with the assembly of the Notch transcriptional activator complex or inhibiting the expression of Notch targets. Intriguingly, the amino terminus of all Fezf proteins contains an engrailed homology 1 domain. This domain can mediate direct interaction with the conserved transcriptional corepressor Groucho that can function as a corepressor of Notch signaling. Additional experiments will be needed to discern how Erm antagonizes Notch-activated dedifferentiation of INPs (Weng, 2010).

Downregulation of Notch mediates the seamless transition of individual Drosophila neuroepithelial progenitors into optic medullar neuroblasts during prolonged G1

The first step in the development of the Drosophila optic medullar primordia is the expansion of symmetrically dividing neuroepithelial cells (NEs); this step is then followed by the appearance of asymmetrically dividing neuroblasts (NBs). However, the mechanisms responsible for the change from NEs to NBs remain unclear. In this study a detailed analyses was performed demonstrating that individual NEs are converted into NBs. This transition occurs during an elongated G1 phase. During this G1 phase, the morphological features and gene expressions of each columnar NE changed dynamically. Once the NE-to-NB transition was completed, the former NE changed its cell-cycling behavior, commencing asymmetric division. It was also found that Notch signaling pathway was activated just before the transition and was rapidly downregulated. Furthermore, the clonal loss of the Notch wild copy in the NE region near the medial edge caused the ectopic accumulation of Delta, leading to the precocious onset of transition. Taken together, these findings indicate that the activation of Notch signaling during a finite window coordinates the proper timing of the NE-to-NB transition (Orihara-Ono, 2011).

This study provides a detailed description of the differentiation of optic medullar progenitors in Drosophila larval brains. Cells with features that were intermediate between NEs and NBs were identified and whose locations were also intermediate, forming a band between the NE and NB regions. Considering the fact that NBs are derived from NEs and that all NEs divide in an exclusively symmetric manner, only the transition of existing individual NEs into NBs without mitosis can explain the observed division patterns and increases in cell populations accounting for the expansion of the medullar primordia. In fact, it was demonstrated that the intermediate cells between the NEs and the NBs were in a transitional state and were suspended in a prolonged G1 phase (Orihara-Ono, 2011).

Genetic cell lineage analyses have shown that outer optic anlagen/outer proliferation center (OOA/OPC) NBs are derived from NEs. Consistent with this observation, a distinct population of cells was identified intermediate between NEs and NBs that did not incorporate BrdU during a labeling period of around 8 h, indicating that the BrdU-negative cells never passed through S phase during the labeling period. The BrdU-negative clusters present at the medial edge of the NE region were probably identical to a previously reported [3H]-thymidine-free band of cells, which was also reported recently (Reddy, 2010). The cell cycle of the BrdU-negative cells was investigated using Mcm2 antibody, which is specific for the pre-replication complex that begins to form during the late M phase and increases until it reaches a peak during S phase. The BrdU-negative NEs were found to be in G1 phase, since they expressed Mcm2. Furthermore, a BrdU-negative cell population was retained even after 16 h of labeling, indicating that the G1 phase of BrdU-negative NEs intermediate between the BrdU-positive NEs and NBs is extremely prolonged. Taken together, these results indicate that the NEs at the medial edge are converted into NBs during a prolonged G1 phase (Orihara-Ono, 2011).

Cell cycle progression patterns differ from cell to cell. For example, syncytial blastodermal cells lack the G1 and G2 phases entirely. The G1 phase is also reportedly absent in some neural lineages of the Drosophila peripheral nervous system. The so-called label-retaining cells in the germinal centers of the adult brain also have an extremely long cell-cycle period. Therefore, the current study may have revealed a common mechanism by which stem cells can retain their capability to reenter the cell cycle despite long periods of quiescence (Orihara-Ono, 2011).

Analogous with the optic medullar anlagen, the formation of the morphogenetic furrow of the Drosophila larval eye disc requires several processes, including the conversion of the cytoskeletal scaffold to a fluid, mobile state and the side-by-side synchrony of the cell-cycle progression. As a result, the G1 cells in the morphogenetic furrow adopt a drop-like shape with a narrow apical 'footprint' when viewed from the surface, whereas the cells in the S-G2-M phases are spherical and exhibit a larger apical surface area. Other evidence supporting this analogy was recently reported in a study examining the proneural wave, in which EGF signaling is involved. The similarities between the maturation of the optic medullar progenitors and the cells in the morphogenetic furrow strongly suggest the existence of a common mechanism regulated by Notch. Notch has also been reported to act in combination with Wg to arrest posterior compartment wing margin cells in the G1 phase and anterior compartment side cells in the G2 phase, supporting the idea that Notch regulates the cell cycle under a number of different circumstances. These mechanisms would explain our current results quite well (Orihara-Ono, 2011).

Notch signaling has been shown to mediate a wide array of cell fate decisions during development. In a recent study of fly brain homozygous for the Notch ts allele, the fate specification was perturbed and the precocious determination may have involved the malformation of the larval optic lobes (Ngo, 2010). Similar evidence has been provided by an overexpression study using a dominant-negative form of the Notch-Delta transduction cascade and mitotic mosaic production of the Notch null allele. These results are consistent with the present findings showing that Notch downregulation forced the precocious transition of NEs. Moreover, this study provides evidence that this effect occurred as a result of a cell non-autonomous process. Since Delta was used as a sensitive effector of the reception of Notch-Delta signaling, attempts were made to identify the Delta pattern as precisely as possible, especially regarding the morphological changes. Previous work demonstrated that the downregulation of Notch signaling by the ectopic expression of Numb in NEs caused a reduction in the progenies derived from a single NE, demonstrating that Notch signaling is required for the proliferation of neural cells in the OOA/OPC (Orihara-Ono, 2011).

Focus was placed on the role of Notch signaling in the NE-to-NB transition at the mid-third instar larval OOA/OPC, which was early enough to examine the acute effects of Notch-depletion. Notch signaling was found to be activated just before the NE-to-NB transition at the medial edge, although Notch was expressed throughout the entire NE population. Both the activation and downregulation of Notch signaling occurred during a finite window for the proper timing of the NE-to-NB transition. However, if Notch signaling was depleted from the NEs at a much earlier stage and NEs devoid of Notch signaling were allowed to develop for a much longer period, the number of NBs that transitioned from the NEs did not change noticeably, compared with the wild-type situation. One plausible explanation is that NEs at the medial edge region cannot be sustained without Notch signaling. Thus, NEs at the medial edge were converted into NBs via the downregulation of Notch. Even if the Notch-deficient clonal cells at the medial edge region are converted into NBs earlier than the surrounding wild-type cells, the final number of NBs generated from the clonal region should not change, since the NEs in the medial edge are supposedly not yet able to divide. In addition, if the fates of the NBs are further specified depending on the timing of their determination from the NEs, precocious transition may cause changes in the fates of the NBs. Further analyses of NB subtypes in Notch-mutant clones might provide intriguing findings (Orihara-Ono, 2011).

Notch's involvement in neural fate decisions has been described in almost all the animals in which this topic has been studied. In a study examining the embryonic Drosophila optic primordium, it has been implied that the loss of Notch causes a precocious epithelial-to-neuroblast transition typically seen in wild-type development during late embryonic stages. The findings of the present paper further explored this precocious transition during the larval stage. Furthermore, evidence is provided that additional factors, such as Delta and Su(H)-dependent downstream genes, might be involved in this process. The downstream effects of Notch are broad and include the regulation of G1/S progression via myc and translational control via Hes proteins, resulting in cell differentiation. In addition, cross-talk between the EGF and the Wnt/βcatenin pathways can alter the morphological features, which may in turn alter the signal strengths of various pathways, including Notch (Orihara-Ono, 2011).

Notch-signal transducing components include numerous ligands, modifiers, and mediators, and each step in Notch-mediated fate changes appears to be controlled by the trafficking and localization of these components. The specific localizations of these components contribute to the efficiency of downstream signaling. This study has presented evidence that a reduction in Notch, which activates the expression and trafficking of Delta, forces a cell-fate change (Orihara-Ono, 2011).

During the course of the NE-to-NB transition of optic medullar progenitors, two peaks in Notch expression were typically observed: the first peak, which occurred early during the NE-to-NB transition, was followed by a period of reduced expression during the cell-type transition, after which Notch expression once again increased as the new NBs resumed division. After several divisions, Notch was downregulated and ultimately became undetectable. Interestingly, similar changes have been reported in the vertebrate spinal cord primordium, suggesting that this pattern of Notch expression may be conserved during vertebrate neurogenesis (Orihara-Ono, 2011).

The frequent apical constriction of single-layered epithelial cells and the subsequent downregulation of E-cad are often coupled to dynamic morphogenetic movements involving the invagination of bottle-shaped cells during gastrulation, which is one of the most important examples of the epithelial mesenchymal transition (EMT). The polarity of cells is sometimes maintained throughout development, but sometimes the polarity is progressively lost and the cells' morphology is remodeled. Typically, the cell cycle is slowed (mostly by a prolonged G1 phase) during this remodeling phase. In other words, a morphogenetically active zone often corresponds to a mitotically inactive one, in which the cells are arrested in the G1 phase. Examples include bottle cells in gastrulating Xenopus embryos, the Drosophila eye morphogenetic furrow, and the laminar furrow of the optic lobes. In most cases, these events are associated with JAK signaling pathways as well as Wnt signaling and TGF-β signaling pathways. In addition, in view of the similarity between this phenomenon and the EMT, cross talk between the Notch and Delta signaling pathways must be considered. Confirming this notion, the upregulation of both Snail and Worniu, Drosophila homologs of the EMT promoting factor Snail1, has been reported in embryonic NBs. Although the relative contribution of various factors to the cell-fate decision remains unclear, it has been reported that the Jak/Stat signal is strong during the neuroepithelial phase but weakens after the NEs commit to a progenitor fate, suggesting that the Jak/Stat signal maintains the cell in its undifferentiated state. Developmental stage-dependent signaling from the environment might change the signal balance and push the cell toward differentiation (Orihara-Ono, 2011).

This paper has focused on the Notch-mediated NE-to-NB transition within a specific, transitional phase of the cell cycle. The work highlights the usefulness of this model system for studying early neurogenesis in the neural plate stage, and these findings may be applicable to general neural stem cell biology (Orihara-Ono, 2011).


Delta and Notch are required for partitioning of vein and intervein cell fates within the provein during Drosophila metamorphosis. Partitioning of these fates is dependent on Delta-mediated signaling from 22 to 30 hours after puparium formation at 25 degrees C. Within the provein, Delta is expressed more highly in central provein cells (presumptive vein cells) and Notch is expressed more highly in lateral provein cells (presumptive intervein cells). Accumulation of Notch in presumptive intervein cells is dependent on Delta signaling activity in presumptive vein cells; constitutive Notch receptor activity represses Delta accumulation in presumptive vein cells. When Delta protein expression is elevated ectopically in presumptive intervein cells, complementary Delta and Notch expression patterns in provein cells are reversed, and vein loss occurs because central provein cells are unable to stably adopt the vein cell fate. These findings imply that Delta-Notch signaling exerts feedback regulation on Delta and Notch expression during metamorphic wing vein development, and that the resultant asymmetries in Delta and Notch expression underlie the proper specification of vein and intervein cell fates within the provein (Huppert, 1997).

Peripodial cells regulate proliferation and patterning of Drosophila imaginal discs

Molecular-genetic analyses of pattern formation have generally treated imaginal discs as single epithelial sheets. Anatomically, however, discs comprise a columnar cell monolayer covered by a squamous epithelium known as the peripodial membrane. During development, peripodial cells signal to disc columnar cells via microtubule-based apical extensions. Ablation and targeted gene misexpression experiments demonstrate that peripodial cell signaling contributes to growth control and pattern formation in the eye and wing primordia. These findings challenge the traditional view of discs as monolayers and provide foundational evidence for peripodial cell function in Drosophila appendage development (Gibson, 2000).

Imaginal discs are a favored system for understanding how fields of cells can autonomously regulate growth and pattern formation. Originating as invaginations in the embryonic epidermis, discs grow into flattened sacs comprising two distinct cell layers: a columnar epithelium and a peripodial membrane. Little is known about peripodial cells, but they are thought to regulate metamorphic events. As epithelial invaginations, discs must evert during the pupal stages such that the appendages lie on the external surface of the adult fly. Eye peripodial cells have been shown to give rise to parts of the adult head and contraction of the peripodial membrane during metamorphosis is required for eye disc eversion. A similar contraction-eversion function has been reported for wing disc peripodial epithelia in the Lepidopteran Manduca sexta. Subsets of peripodial 'edge' cells employ the JNK signaling cascade to regulate the process of metamorphic interdisc fusion in Drosophila (Gibson, 2000 and references therein).

Previous studies have not addressed possible function of peripodial membranes during early growth and patterning. The adult appendages are almost entirely derived from columnar cells and consequently appendage development has been considered a two-dimensional problem with intercellular signaling restricted to idealized columnar cell monolayers. The discs-as-monolayers concept likely originated from two-dimensional fate maps produced 30 years ago. Peripodial Hedgehog signaling has been shown to induces engrailed expression in leg disc columnar cells during fragmentation-induced regeneration. This observation was an initial indication that peripodial and columnar cells might also interact during normal development. This study now shows that wing and eye disc peripodial cells form microtubule-based 'translumenal' extensions that traverse acellular space and terminate on the surface of developing disc columnar epithelia. These structures are plainly suggestive of communication between peripodial and columnar cells. Consistent with this, ablation experiments have shown that peripodial cells are required for pattern formation in the disc columnar epithelia (Gibson, 2000 and references therein).

The lipophilic membrane marker DiI has been used to describe the morphology of live wing peripodial cells. Optical sectioning obtained by confocal microscopy reveals 'translumenal extensions' that traverse a cellular space (the disc lumen) and terminate on the surface of the columnar epithelium. These structures initiated on the apical surface of each peripodial cell, range from 5–30 µm in length, and often appear to be highly vesiculated. In general, only one extension has been observed per cell. Similar structures have been observed in eye, leg, and haltere discs, suggesting a generalized morphological basis for translumenal signaling. The lumenal cavity of imaginal discs does not form until the early third instar, so it is unlikely that these structures are present during earlier stages and indeed, they were not detected. Further, not all peripodial cells produced extensions. In the wing, extensions are numerous in the presumptive dorsal hinge and notum regions but absent from the presumptive wing blade where peripodial and columnar epithelia are in direct contact (Gibson, 2000).

The subcellular morphology of peripodial cells further supports a translumenal signaling function. In wing discs, peripodial cell nuclear membranes are associated with a membranous organelle that tapers into a funnel-shaped sac within each cellular extension. It is speculated that this unusual internal membrane represents a mechanism for targeting specific RNAs or proteins to the translumenal extensions. Mitochondria labeled with Rhodamine 123 localize to the extensions and are suggestive of locally enhanced ATP consumption. To observe peripodial microtubule networks, a peripodial Gal4 driver (c311-Gal4) was identified and the Gal4/UAS system was used to direct expression of a fluorescently tagged microtubule binding protein (UAS-tau-GFP). In live analysis, translumenal extensions are packed with microtubules, demonstrating that they are not cytonemes, threadlike processes observed in disc columnar cells. Based on these observations, it is concluded that peripodial cells possess structurally specialized translumenal extensions. Evidence is provided that these structures mediate peripodial-columnar signaling during development (Gibson, 2000).

In the eye disc, peripodial translumenal extensions correlate with the position of the morphogenetic furrow, a wave of cell division and photoreceptor cluster formation that sweeps across the columnar epithelium during the latter third instar and early pupal development. To test peripodial function in furrow progression, the peripodial membrane was surgically removed during the late third instar and 'naked' eye columnar epithelia were cultured in vivo overnight. This treatment abolishes the mitotic waves normally associated with the furrow and causes a marked reduction in photoreceptor cluster formation relative to intact cultured controls. These results are consistent with the notion that furrow progression requires peripodial signaling (Gibson, 2000).

A parallel experiment was performed in situ by genetic ablation of the peripodial epithelium. The c311-Gal4 driver is peripodial membrane specific in the eye disc and c311-Gal4 and the flp-out technique could be combined to ablate eye peripodial cells with a toxic UAS <w+<RicinA transgene. Peripodial expression of RicinA was induced in late third instar animals and then eye cuticle was recovered from pupae with uneverted eyes. In all cases, peripodial ablation results in significant eye size reduction and severe pattern defects including square ommatidia. The observed size reductions are consistent with a failure in furrow progression (Gibson, 2000).

Further ablation experiments have revealed that peripodial function is not unique to the eye. Surgical ablation of wing peripodial membranes results in specific loss of the specialized bristles and hairs along the wing margin. Translumenal extensions are not observed in the presumptive wing margin region where there is no detectable lumenal cavity between the opposing peripodial and columnar epithelia. Therefore, it is inferred that direct cellular contact mediates peripodial-columnar signaling in the absence of visible translumenal processes (Gibson, 2000).

Together, these ablation experiments reveal a novel requirement for intact peripodial epithelia in patterning both eye and wing primordia. However, it is important to note that the phenotypes observed following ablation could be due to myriad effects other than disruption of signaling through translumenal extensions. For example, ablation of the peripodial epithelium could result in direct exposure of columnar cells to hemolymph or dilution of critical extracellular factors normally concentrated in the enclosed disc lumen. To circumvent the many caveats associated with cell ablation, the Gal4/UAS system was employed to inactivate functional aspects of peripodial cells while maintaining the integrity of the peripodial membrane (Gibson, 2000).

While it is anticipated that microtubule-associated motor function is required to transport signals within the extensions, the observed phenotypes might also result from an indirect structural defect or failure to form extensions at all. Glued mutant flies have reduced, rough eyes and ubiquitously expressed dominant-negative Glued causes columnar-cell mitotic delays (resulting in an increase in mitotic figures), as well as rough and reduced eyes. These phenotypes were not observed in Gal4 driven dominant negative Glued flies and it is therefore likely that the distinctive slow/arrested furrow phenotype reported here is peripodial cell specific (Gibson, 2000).

Several complex genetic networks are known to govern Drosophila eye development. If indeed the peripodial membrane signals to the disc columnar epithelium, then some described eye mutant phenotypes would be expected to derive from failed peripodial function. The action of Fringe (Fng), a Golgi-localized glycosyltransferase known to affect eye development on several levels has been explored. A ventrally restricted Fng expression boundary is thought to regulate eye disc growth; abolishing this boundary by ubiquitous overexpression of UAS-fng in the second instar severely reduces eye size. Surprisingly, clonal analysis demonstrates that while fng is required for growth of the ventral eye, this requirement lies somewhere outside the eye columnar epithelium (Papayannopoulos, 1998). A test was performed to see whether disc growth is sensitive to peripodial fng activity (Gibson, 2000).

fng-LacZ is expressed in a subset of eye peripodial cells in the first and second larval instars, growth stages where peripodial and columnar epithelia appear to be in direct contact. This is consistent with the notion that fng functions in peripodial cells. Further, ectopic expression of UAS-fng throughout the peripodial membrane (using c311-Gal4) results in dramatically reduced eye size and predominantly square-shaped ommatidia similar to those observed following genetic ablation of the peripodial membrane. The eye size reduction is consistent with the idea that proliferative growth of the eye disc is regulated by a Fng expression boundary in the peripodial membrane. An alternative interpretation is that overexpression of Fng in the peripodial membrane interfers with furrow progression, resulting in a smaller eye. However, it has been noted that ectopic fng causes size reduction of the eye disc prior to furrow movement (Papayannopoulos, 1998); thus, the former interpretation is favored. The square ommatidia phenotype is more difficult to explain, but may reflect variant cell numbers within each ommatidia or some other failure in retinal morphogenesis. It is noted that an identical phenotype is obtained by overexpression of a dominant-negative form of the Notch ligand Serrate in a broad domain of the eye. In both cases, uniformly square ommatidia are arranged in a highly regular array (Gibson, 2000 and references therein).

It was questioned whether the small eyes seen in c311-Gal4;UAS-fng flies are due to a peripodial-specific effect since the same phenotype is obtained when UAS-fng is expressed under the control of eyeless-Gal4. How could eyeless- and c311-Gal4 elicit the same fng-dependent phenotype? Intriguingly, eyeless-Gal4 drives UAS-GFP expression in eye columnar and peripodial cells throughout larval development consistent with the idea that peripodial fng activity regulates disc growth (Gibson, 2000).

During normal development Fng acts as a glycosyltransferase that modifies the ligand preference of the extracellular receptor Notch. Accordingly, the results of the previous section indicate that reception of signals via Notch could be a key feature of peripodial cell function in retinal patterning. Consistent with this idea, Notch is expressed in peripodial cells. The involvement of the Notch pathway in peripodial cell signaling was further explored by examining the distribution of its ligands, Delta (Dl) and Serrate (Ser). Dl is expressed extensively in the eye disc columnar epithelium and in some limited regions of the peripodial membrane. In contrast, high levels of Ser are observed in subcellular vesicles throughout the eye peripodial epithelium while only minimal levels are detected in the columnar cell layer. Since Ser is required for eye development, it is proposed that Ser is a strong candidate for a peripodial-to-columnar signal. Consistent with this, peripodial-specific expression of a secreted, dominant-negative form of Serrate [UAS-Ser(s)] results in flies with reduced eyes and highly irregular ommatidial patterning (Gibson, 2000).

While highly suggestive, it is noted that the c311 Gal4;UAS-Ser(s) phenotype does not indicate whether the defective Ser signal travels through translumenal extensions or is released into the lumenal cavity. Further, these results do not allow the authors to state where the dominant-negative ligand is acting. The findings do, however, clearly demonstrate that peripodial expression of a defective N ligand is sufficient to disrupt development of the eye columnar epithelium. Combining this observation with the results of fng overexpression, the peripodial localization of vesicular Ser in wild-type discs, and the requirement for Ser in eye development, it is proposed that components of the Notch signaling pathway participate in translumenal signaling. Presently the data do not directly implicate Ser in this process. However, a peripodial function of Ser could explain an outstanding paradox regarding its role in eye development: Ser mutants have rough and reduced eyes, but Ser- clones in the columnar epithelium have no apparent phenotype (Gibson, 2000 and references therein).

The identification of peripodial translumenal extensions is new evidence that specialized cellular processes effect long-range signaling during development. Interestingly, many cell signaling pathways have been genetically linked to components of the cytoarchitecture. These genetic connections take on new significance in light of evidence for long-range filopodia with specialized cytoskeletal organization. The characterization of a novel cellular extension in Drosophila will permit detailed analysis of the formation and function of long-range extensions on a molecular and genetic level. Peripodial function in disc development is further revealed by the results show that several secreted signaling molecules (including wingless, decapentaplegic, and hedgehog) are expressed in eye peripodial cells where they function to regulate the early development of the eye columnar epithelium (Cho, 2000). Together, these studies necessitate a reconsideration of the two-dimensional models for disc morphogenesis and suggest a new parallel between appendage development in Drosophila and vertebrates. It now appears that limb and retinal development require functional interactions between opposing epithelial sheets in both systems (Gibson, 2000).

Dorsal-ventral signaling in the Drosophila eye

Notch activation at the midline plays an essential role both in promoting the growth of the eye primordia and in regulating eye patterning. Specialized cells are established along the dorsal-ventral midline of the developing eye by Notch-mediated signaling between dorsal and ventral cells. D-V signaling in the eye shares many similarites with D-V signaling in the wing. In both cases an initial asymmetry is set up by Wingless expression. Both Eye and wing cells then go through a distinct intermediate step: in the wing, Wingless represses the expression of Apterous, a positive regulator of fringe (fng) expression; in the eye, Wingless promotes the expression of mirror (mrr), which encodes a negative regulator of fringe (unpublished observations of McNeill, Chasen, Papayannopoulos, Irvine, and Simon, cited by Papayannopoulos, 1998). Both wing and eye cells share a Fng-Ser-Dl-Notch signaling cassette to effect signaling between dorsal and ventral cells and establish Notch activation along the D-V midline. Local activation of Notch leads to production of diffusible, long-range signals that direct growth and patterning, which in the wing include Wingless, but in the eye remain unknown. At least one downstream target of D-V midline signaling, four jointed (fj), is also conserved. four jointed is also expressed in the wing and its expression there is indirectly influenced by Notch (Papayannopoulos, 1998 and references).

During early eye development, fringe is expressed by ventral cells. This expression appears to be complementary to that of the dorsally expressed gene mrr. During early to mid-third instar, additional expression of fng appears in the posterior of the eye disc. This line of posterior fng expression is just in front of the morphogenetic furrow and moves across the eye ahead of the furrow. In the wing disc, Dl and Ser induce each other's expression, and become up-regulated along the D-V border where they can productively signal. Dl and Ser are also preferentially expressed along the D-V midline during eye development. Ser expression, like fng expression, is complementary to that of mrr, whereas Dl expression partially overlaps that of mrr. The spatial relations among fng, Ser, and Dl expression in the eye are thus similar to those in the wing, although in the wing, their expressions are inverted with respect to the D-V axis (Papayannopoulos, 1998).

The four-jointed gene is expressed in a gradient during early eye development, with a peak of expression along the D-V midline. Together with Ser and Dl, Fj serves as a molecular marker of midline fate. Ubiquitous expression of Fng during early eye development, generated by placing fng under the control of an eyeless enhancer, eliminates detectable expression of Ser and Dl along the midline. Conversely, misexpression of Fng in clones of cells, can result in ectopic expression of Ser and fj that is centered along novel borders of Fng expression in the dorsal eye. Ectopic Ser and fj expression can also be detected along the borders of fng mutant clones in the ventral eye. These observations show that Fng expression borders play an essential and instructive role in establishing a distinct group of cells along the D-V midline of the developing eye. Animals with reduced fng activity have small eyes. Moreover, ubiquitous fng expression also results in a dramatic loss of tissue. Tissue loss is detectable in the developing imaginal disc, before the morphogenetic furrow moves across the eye. Moreover, eye loss is observed when fng is ectopically expressed during early development, but not when fng is ectopically expressed behind the furrow. These observations indictate that a Fng expression border is required for eye growth, specifically during early eye development (Papayannopoulos, 1998).

Fng differentially modulates the action of Notch ligands in the eye just as it does in the wing. Clones of cells ectopically expressing Dl can induce Ser expression in ventral, Fng-expressing cells, but not in dorsal cells. Fng alone can induce Ser expression in dorsal cells, but only near the D-V midline. When Fng and Dl are co-misexpressed, Ser expression can be induced in dorsal cells even when the clones are far from the D-V midline. Clones of cells ectopically expressing Ser are able to induce increased expression of Dl in dorsal cells but not in ventral, Fng expressing cells. However, if Ser is ectopicallly expressed in fng mutant animals, it can induce Dl expression in ventral cells (Papayannopoulos, 1998).

Notch function is also necessary for normal D-V midline cell fate. The ability of Ser and Dl to induce one another's expression indicates that the expression of either one is a marker for Notch activation in the eye. Analysis of loss-of-function mutants of Notch and its ligands, as well as ectopic expression studies, indicate that Notch activation also regulates eye growth. Several observations indicate that the D-V midline is the focus of Notch activation required for growth. Moreover, the midline corresponds to a fng expression border, which is essential for growth and modulates Notch signaling during early eye development. Because local activation of Notch has long-range effects on growth and four-jointed expression, it is inferred that Notch induces the expression of a diffusible growth factor at the midline. Notch activation influences ommatidial chirality. fng mutant clone borders within the ventral eye can be associated with reversals of ommatidial chirality, whereas mutant clones that cross the D-V midline disrupt the normal equator. The equatorial bias in the influence of ectopic Notch activation implies that the equator is the normal source of a Notch-dependent, chirality-determining signal (Papayannopoulos, 1998).

The scabrous protein can act as an extracellular antagonist of notch signaling in the Drosophila wing.

Notch is a receptor for signals that inhibit neural precursor specification. Since N and its ligand Delta are expressed homogeneously, other molecules may be differentially expressed or active to permit neural precursor cells to arise intermingled with nonneural cells. During Drosophila wing development, the glycosyltransferase encoded by the gene fringe promotes N signaling in response to DI, but inhibits N signaling in response to Serrate, which encodes a ligand that is structurally similar to DI. Dorsal expression of Fng protein localizes N signaling to the dorsoventral (DV) wing margin. The secreted protein Scabrous is a candidate for modulation of N in neural cells. Mutations at the scabrous locus alter the locations where precursor cells form in the peripheral nervous system. Unlike fringe, sca mutations act cell non-autonomously. Targeted misexpression of Sca during wing development inhibits N signaling, blocking expression of all N target genes. Sca reduces N activation in response to DI more than in response to Ser. Ligand-independent signaling by overexpression of N protein, or by expression of activated truncated N molecules, is not inhibited by Sca. These results indicate that Sca can act on N to reduce its availability for paracrine and autocrine interactions with DI and Ser, and can act as an antagonist of N signaling (Lee, 2000).

scabrous modifies epithelial cell adhesion and extends the range of lateral signaling during development of the spaced bristle pattern in Drosophila

The role of scabrous in the evenly spaced bristle pattern of Drosophila has been explored. Loss-of-function of sca results in development of an excess of bristles. Segregation of alternately spaced bristle precursors and epidermal cells from a group of equipotential cells relies on lateral inhibition mediated by Notch and Delta (Dl). In this process, presumptive bristle precursors inhibit the neural fate of neighboring cells, causing them to adopt the epidermal fate. Dl, a membrane-bound ligand for Notch, can inhibit adjacent cells, in direct contact with the precursor, in the absence of Sca. In contrast, inhibition of cells not adjacent to the precursor requires, in addition, Sca, a secreted molecule with a fibrinogen-related domain. Over-expression of Sca in a wild-type background, leads to increased spacing between bristles, suggesting that the range of signaling has been increased. scabrous acts nonautonomously, and evidence is presented that, during bristle precursor segregation, Sca is required to maintain the normal adhesive properties of epithelial cells. The possible effects of such changes on the range of signaling are discussed. It is also shown that the sensory organ precursors extend numerous fine cytoplasmic extensions bearing Dl molecules, and these structures may play an active role during signaling (Renaud, 2001).

sca functions in the inhibition of the neural fate, since in its absence an excess of neural precursors form. The sca mutant phenotype is very similar to that of hypomorphic N and Dl mutants and indeed Notch and Dl are known to be the main components of the signaling pathway regulating the spaced pattern of bristles. Thus, Sca is likely to positively modulate N signaling. During bristle precursor selection, like Dl, sca acts nonautonomously and is not required for reception of the signal that places its activity upstream of N. What are the respective roles of Dl and sca in the segregation of bristle precursors? Both genes are expressed in proneural domains and then at high levels in the bristle precursors, and their products act nonautonomously on neighboring presumptive epidermal cells. Both proteins associate with N, but so far only Dl has been shown to be an activating ligand. Scabrous is a secreted molecule, whereas data accumulated to date suggest that active Dl is membrane-bound. In the complete absence of Dl, all cells adopt the neural fate and thus bristle precursors arise adjacent to one another. In the complete absence of Sca, there is an excess of bristle precursors but they are never adjacent and are always separated by at least one epidermal cell. This indicates that sca is not needed for the bristles to be spaced apart by a short distance, and that Dl, which is expressed in sca mutants, is able to inhibit cells immediately adjacent to the precursors without any help from Sca. In contrast, in the absence of Sca, Dl is unable to inhibit those cells not in direct contact with the precursor. This suggests a role for Sca in the inhibition of cells not adjacent to the precursor. These two, possibly separable events, are referred to as 'short' and 'long' range signaling (Renaud, 2001).

Formally, there are three possible mechanisms for 'long' range signaling. The first would be that sca is part of a relay mechanism whereby cells immediately adjacent to the precursor are inhibited by Dl and then relay the signal farther out by means of Sca. This hypothesis is very unlikely, however, because one would expect sca to be expressed in cells adjacent to the precursor following activation of N by Dl. In fact, sca protein is only detectable in the precursor itself, although sca is earlier expressed at low levels in the proneural domain. Furthermore, sca is expressed in the absence of Dl and therefore does not require a prior signaling event mediated by Dl (Renaud, 2001).

A second possible mechanism is that there may be two independent signals, one acting at a 'short' range (Dl) and another at a 'long' range (Sca). Two observations suggest that this, too, is unlikely: (1) in the absence of Dl, all cells adopt the neural fate and so the process of bristle spacing is completely abolished. scabrous is expressed in cells mutant for Dl so this result indicates that Sca alone is unable to repress the neural fate; (2) the results indicate that sca is not involved in the process of lateral inhibition whereby a pattern of alternating neural and epidermal cells is generated. Along a border between wild-type cells and cells mutant for Dl, the precursors are nearly always selected from the pool of wild-type cells, rather than from the mutant cells. This is thought to be because the mutant cells produce little or no signal and are inhibited by the Dl-producing wild-type cells. Along sca mosaic borders, the mutant cells can adopt the neural fate, indicating that they are not defective in the production of the activating ligand Dl. Furthermore, since wild-type precursors also form along the mosaic border, neural precursors can be chosen from cells of either genotype. Thus, cells choose the neural or epidermal fate regardless of whether or not they express sca (Renaud, 2001).

This would explain the fact that precursors are never adjacent in sca mutants and is not inconsistent with the observed sca phenotype of excess bristles. During segregation of both the normal component of precursors, as well as the additional precursors, adjacent cells have to choose between epidermal and neural fates. Any failure of this process would result in the presence of adjacent bristle precursors, a phenotype characteristic of N and Dl mutants, but not sca mutants. Segregation of single neural precursors surrounded by epidermal neighbors is thus probably mediated by Dl alone, which would explain why bristle spacing is completely abolished in the absence of Dl, even though Sca is present (Renaud, 2001).

The third possibility is that 'long' range signaling requires both Dl and Sca. Under this hypothesis, Dl would be the activating ligand in 'long' range signaling, but would require Sca in order to inhibit cells not directly in contact with the precursor. This could be the case regardless of whether the signal originates exclusively in the precursor or additionally in proneural cells. One observation in favour of this hypothesis is afforded by examination of flies mosaic for Dl. Along the edges of Dl mutant clones, mutant cells are able to differentiate as epidermis under the influence of an inhibitory signal from neighboring wild-type cells. This 'rescue,' due to expression of Dl in the wild-type neighbours, can extend up to four cell diameters. If there is no relay mechanism and no other signal, then Dl must somehow be able to activate N several cell diameters away from the cell in which it is produced. Scabrous is of course present in both the Dl+ and the Dl- cells, but is unable to effect any rescue of cells mutant for Dl that are situated more than about four cells away from the wild-type Dl-expressing cells. Examination of Dl mutant clones in a mutant sca background would indicate the range of Dl signaling in the absence of Sca (Renaud, 2001). Clones doubly mutant for sca and Dl, however, fail to differentiate the cuticular components of the bristles (for all allelic combinations tested) and so are uninformative (Renaud, 2001).

It is suggested that bristle spacing may be the result of two signaling events. The first step of lateral signaling involves Dl alone and allows a group of cells to choose a single neural precursor that will inhibit adjacent cells. This step proceeds normally in the absence of Sca. The second step would act to inhibit cells that are not adjacent to the precursor and would require the activity of both Dl and sca. This step is impaired in both sca and Dl mutants. In Dl mutants, in the absence of the inhibitory signal, lateral inhibition fails, leading to adjacent precursors and a loss of epidermal cells. In sca mutants, an excess of precursors form, but they segregate singly and are spaced by at least one epidermal cell through the activity of Dl. Examination of the nascent precursors with neu-lacZ, fail, to reveal two temporally separate waves of precursor formation in sca mutants. Staining of wild-type pupal nota with DE-cadherin, however, may provide a visual correlate of the two groups of target cells. A rosette-like ring of wedge-shaped cells surrounds the bristle precursor; it is reminiscent of the cell preclusters that precede segregation of the R8 photoreceptor during development of ommatidia. These supra-cellular structures may allow more cells to enter into direct contact with the precursor during the first step of inhibitory signaling (Renaud, 2001).

The results demonstrate a requirement for local discontinuities in the levels of Sca between cells during inhibitory signaling. This was also reported to be the case for eye development. Uniform expression of Sca under experimental conditions is unable to rescue the sca mutant phenotype. So the relative quantitative differences in the level of Sca between neighbouring cells is an essential feature of the spacing mechanism. Over-expression of Sca in a wild-type fly causes a phenotype opposite that of the loss-of-function phenotype. The distance between bristles actually increases and there are correspondingly fewer bristles in the domain of over-expression. In these flies, although exogenous Sca is uniformly distributed, regulation of the endogenous gene will provide local differences in levels of the protein. The greater distance between bristles suggests an extension in the range of the inhibitory signal. Scabrous is likely to be present in a gradient of decreasing concentration around each precursor. The molecule has been shown to have a very short half-life. Thus, it would decay rapidly after secretion and this would help maintain a graded distribution from the source. It is postulated that such a gradient could define the range of the inhibitory signal (Renaud, 2001).

A rescue of up to four cell diameters has been observed at the edges of clones of cells mutant for Dl. This suggests a signaling range that exceeds that required in wild-type flies, where bristles are usually only four to five cells apart. It is possible, however, that the signal range is longer than usual in this experimental situation because very large amounts of Sca are likely to be present in these clones due to the hyperplasia of precursors. It is, however, noteworthy that in other drosophilid species, such as D. ararama, the bristles are spaced by eight or nine cells, suggesting a possibly greater signaling range (Renaud, 2001).

One property of Sca is to modulate adhesive parameters of the epithelial cells surrounding the precursor. Discrete changes in the distribution of DE-cadherin, Discs large, and other junctional proteins are seen in the epidermis of sca mutants, that are associated with mislocalization of the adherens and septate junctions and some disruption of epithelial organization. The monolayered nature of the epithelium is retained, consistent with the fact that the morphogenetic changes of metamorphosis are not impaired in sca mutants. This phenotype is only observed in late third instar discs and early pupae, when bristle precursors are forming. If ac-sc activity is removed, the late third instar disc epithelium is wild type. In ac-sc mutants, the absence of Ac and Sc entails a loss of Sca, whose expression is dependent on Ac-Sc (Renaud, 2001).

The epithelial defects resulting from a lack of Sca protein thus coincide with ac-sc expression and the process of lateral inhibition. Scabrous may therefore be required to maintain normal epithelial integrity by counteracting the effects of a protein(s) activated during precursor segregation. It is therefore likely to act through association with a protein(s) whose expression is regulated by Ac and Sc. Notch is ubiquitously expressed in the epithelium, but is activated and probably up-regulated in future epidermal cells surrounding the precursors. Powell (2001) demonstrated that Sca binds N and as a result N is stabilized at the cell surface in S2 cells. Interestingly, in Nts1 mutants at 29°C, where the activity of N is strongly reduced, the distribution of DE-cadherin in the disc epithelium is also discretely altered and the epithelium appears similar, but not identical to that of sca. This suggests that the epithelial defects seen in sca mutants may be the result of a failure to stabilize N protein in epithelial cells. These results are consistent with the idea that Sca acts through N, and with earlier observations linking N to epithelial cell adhesion (Renaud, 2001).

scabrous is not required for inhibition of cells that are adjacent to the precursor. Furthermore, the requirement for sca in the fly is quite restricted: it is not expressed in many other tissues where Notch signaling takes place in its absence. While it is expressed in neural precursors in the embryo, loss of the protein there seems to be without consequence, perhaps because the interactions involve adjacent cells. This leads to the hypothesisis that the effects of Sca on cell adhesion and the stabilization of N may be specifically required when the levels of Dl are limiting (Renaud, 2001).

Delta is expressed in proneural domains and can still be detected in presumptive epidermal cells after precursor segregation. Nevertheless the amount of Dl remaining in presumptive epidermal cells appears to be insufficient, by itself, to repress the neural fate. In the absence of Sca, or in flies carrying hypomorphic alleles of Dl, the space between bristles is decreased. Furthermore, in epidermal cells, the transcription of Dl progressively declines due to repression of its regulators ac and sc following N activation, whereas in the bristle precursors the levels of Dl increase as the levels of Ac and Sc rise. This leaves two possibilities. One is that all of the signal originates in the precursor cell, in which case Dl must be transported, by some as yet unknown means, from the precursor to cells not in direct contact with the latter. The other, is that the concentration of Dl molecules remaining on the presumptive epidermal cells is insufficient to inhibit by itself, but can do so if helped by Sca. Dl from both groups of cells may participate in the wild type. In either case, stabilization of N may therefore be a means to increase the chances of receptor activation in the presence of limiting amounts of Dl (Renaud, 2001).

Changes in cell adhesion could also be the cause of the abnormal bristle organs seen in sca mutants, where two or more cells of the bristle organ lineage adopt the same fate at the expense of the others. In the wild type, spatial arrangements of the cells of the bristle organs are stereotyped as a result of the nonrandom orientation of the mitotic spindles at each division. In sca mutants, the cells are often randomly arranged and in some cases appear to drift apart from one another. This would be likely to prevent the precise cell-cell interactions, mediated by the N signaling pathway, necessary for the assignment of the correct cell fates (Renaud, 2001).

The precursor cells have a quite distinctive shape, reminiscent of neurons with a number of filopodial-like extensions that fan out in a planar orientation. It is not known whether, during bristle precursor segregation, the epidermal cells of the notum extend similar filopodia. Oriented epidermal outgrowths have been described in the epidermis of other insects and also in the wing pouch and peripodial membrane of Drosophila imaginal discs, where it has been suggested that they may function during signaling. Some of these structures project basally and others extend long, straight and polarized structures. Their morphology differs from that of the extensions observed (Renaud, 2001).

It is not known whether Dl from the precursor cell is able to reach cells that are not adjacent to the Dl-expressing precursor, but one means by which this could occur is via cytoplasmic extensions. This has been suggested for Lag-2 signaling in the nematode germ line. Indeed, the presence of Dl molecules can be detected on the filopodia. Although formation of filopodia will depend on properties of the neural precursor itself, subtle changes in junctional contacts and adhesion between surrounding epithelial cells may help to orient or stabilize these structures. The changes in the bristle density of both wild-type and sca flies that are seen after expression of a dominant negative form of DE-cadherin, suggest a role for adhesion molecules in bristle spacing. Preliminary observations indicate that Sca is not required for the extension of filopods. This is consistent with the nonautonomy of sca mutant cells. If filopodia are the means whereby nonadjacent cells are inhibited, and if Sca were to be required for extension of filopodia from the Sca-producing bristle precursors, then sca would be expected to behave autonomously (Renaud, 2001).

Further studies are necessary to determine the molecular basis of Sca function, but one possibility is that binding of Sca to N leads to discrete modifications in epithelial structure that allow Dl molecules on the cytoplasmic extensions to form stable ligand-receptor complexes. The colocalisation of Sca and Dl in cytoplasmic vesicles may indicate cellular trafficking of protein complexes that include Dl, N, and Sca (Renaud, 2001).

hephaestus encodes a polypyrimidine tract binding protein that regulates Notch signaling during wing development in Drosophila melanogaster

Drosophila hephaestus (heph) is required to attenuate Notch activity after ligand-dependent activation during wing development. The original male sterile heph allele was identified in a genetic screen for loci required for spermatogenesis. New lethal alleles of heph have been isolated that affect wing margin and wing vein pattern formation in genetic mosaics. Drosophila heph gene encodes the apparent homolog of mammalian polypyrimidine tract binding protein (PTB). PTB was first identified in vertebrates as a protein that binds to intronic polypyrimidine tracts preceding many 3' pre-mRNA splice sites. Many different functions have been identified for vertebrate PTB, including the control of alternative exon selection, translational control or internal ribosome entry site (IRES) use, mRNA stability and mRNA localization. PTB may also act as a transcriptional activator. The study of heph is the first genetic analysis of polypyrimidine tract binding protein function in any organism and the first evidence that such proteins may be involved in Notch signaling (Dansereau, 2002 and references therein).

Somatic clones lacking heph express the Notch target genes wingless and cut, induce ectopic wing margin in adjacent wild-type tissue, inhibit wing-vein formation and have increased levels of Notch intracellular domain immunoreactivity. Clones mutant for both Delta and hephaestus have the characteristic loss-of-function thick vein phenotype of Delta. These results led to the hypothesis that hephaestus is required to attenuate Notch activity following its activation by Delta (Dansereau. 2002).

How does heph regulate Notch activity? This study has linked together for the first time the PTB/hnRNPI RNA-binding proteins and the Notch signaling pathway. Given the strong sequence similarity shared between heph and vertebrate PTBs, it is probable that heph regulates the processing, stability or translation of a Notch pathway mRNA. However, the heph mosaic wing phenotypes most closely resemble the effects of low level ectopic Notch activation and cannot be easily correlated with an effect on any particular known element in the Notch pathway. The phenotypes of clones mutant for Delta and heph are most informative in explaining where heph acts in the Notch pathway. The epistasis of Dl over heph in double mutant clones indicates that the Notch activation in heph clones depends on Dl. This ligand dependency excludes the possibilities that Notch target genes are generally de-repressed, or that the Notch receptor is constitutively activated, in heph mutant cells. Rather, it suggests that in the absence of heph, existing Notch activity is amplified and/or maintained. Therefore, the favoured explanation is that heph is required to attenuate Notch activity after ligand-dependent activation (Dansereau. 2002).

The phenotypic consequences of heph are most prominent in the wing margin cells and wing vein cells. Both of these cell types require decreases in the levels of Notch activity during development and the heph phenotype results from persistent Notch activity in these cells. The wing margin cells lose Notch activation and wg expression during the refinement of wg and Dl/Ser expression during the late second and early third instar. During larval development, the cells that will ultimately give rise to the vein express low levels of Notch and Notch target genes such as E(spl)mß, indicating that these cells have low levels of Notch activation prior to the repression of Notch transcription in pupal vein cells. Although it is not certain how these cells normally lose Notch activation, one possibility is that NICD stability is tightly regulated in order for cells to change Notch activation states and that heph+ may be required for cells to degrade NICD following ligand activation of the Notch receptor (Dansereau. 2002).

The most intriguing possibility is that heph may negatively regulate the translation of E(spl)-C mRNAs. The E(spl) complex bHLH genes are transcribed in response to Notch signaling and this is counteracted by inhibition of translation by the 3'-UTR's of E(spl)-C mRNAs. This inhibition is presumably mediated through the binding of factors to conserved sequences found in most E(spl)-C mRNAs as well as in genes of the Bearded family, another group of Notch mediators. In this model, loss of heph function would increase the stability of E(spl)-C mRNAs, resulting in amplification of the effects of transcriptional activation by Notch signaling. Increased expression of E(spl)-C members has been demonstrated to inhibit wing vein differentiation, although the ectopic expression of individual E(spl)-C members has not been demonstrated to induce ectopic wing margin. However, it is possible that the stabilization of multiple E(spl)-C mRNAs could result in more dramatic effects on the wing margin. Furthermore, E(spl)-C members have different transcription patterns and may have divergent roles downstream of Notch. If heph were to regulate a subset of the E(spl)-C mRNAs, it would explain the limited requirement of heph in various Notch-mediated signaling events (Dansereau. 2002).

Combinatorial signaling in the specification of primary pigment cells in the Drosophila eye

In the developing eye of Drosophila, the EGFR and Notch pathways integrate in a sequential, followed by a combinatorial, manner in the specification of cone-cell fate. This study demonstrates that the specification of primary pigment cells requires the reiterative use of the sequential integration between the EGFR and Notch pathways to regulate the spatiotemporal expression of Delta in pupal cone cells. The Notch signal from the cone cells then functions in the direct specification of primary pigment-cell fate. EGFR requirement in this process occurs indirectly through the regulation of Delta expression. Combined with previous work, these data show that unique combinations of only two pathways -- Notch and EGFR -- can specify at least five different cell types within the Drosophila eye (Nagaraj, 2007).

Unlike photoreceptor R cells, cone cells do not express Delta at the third instar stage of development. However, these same cone cells express Delta at the pupal stage. In addition, correlated with this Delta expression, the upregulation of phosphorylated MAPK was observed in these cells. This is very similar to the earlier events seen in R cells during larval development, in which the activation of MAPK causes the expression of Delta. Also, as in the larval R cells, the pupal upregulation of Delta in cone cells is transcriptional. A Delta-lacZ reporter construct, off in the larval cone cell, is detected in the corresponding pupal cone cells. To determine whether EGFR is required for the activation of Delta in the pupal cone cells, the temperature-sensitive allele EGFRts1 was used. Marked clones of this allele were generated in the eye disc using ey-flp at permissive conditions and later, in the mid-pupal stages, shifted the larvae to a non-permissive temperature. Cells mutant for EGFR, but not their adjacent wild-type cells, showed a loss of Delta expression. However, both mutant and wild-type tissues showed normal cone-cell development, as judged by Cut (a cone-cell marker) expression. As supporting evidence, ectopic expression of a dominant-negative version of EGFR (EGFRDN) in cone cells using spa-Gal4 after the cells have already undergone initial fate specification also causes a complete loss of Delta expression without compromising the expression of the cone-cell-fate-specification marker (Nagaraj, 2007).

Gain-of-function studies further support the role of EGFR signaling in the regulation of Delta expression in cone cells. Although weak EGFR activation is required for cone-cell fate, activated MAPK is not detectable in cone-cell precursors of the third instar larval eye disc. When spa-Gal4 (prepared by cloning the 7.1 kb EcoRI genomic fragment of D-Pax2) is used to express an activated version of EGFR in larval cone cells, detectable levels of MAPK activation in these cells were found and the consequent ectopic activation of Delta in the larval cone cells occurred. Taken together, these gain- and loss-of-function studies show that, during pupal stages, EGFR is required for the activation of Delta. However, this Delta expression is not essential for the maintenance of cone-cell fate (Nagaraj, 2007).

In larval R cells, the activation of Delta transcription in response to EGFR signaling is mediated by two novel nuclear proteins, Ebi and Sno. To determine the role of these genes in wild-type pupal-cone-cell Delta expression, sno and ebi function were selectively blocked in the pupal eye disc. A heteroallellic combination of the temperature-sensitive allele snoE1 and the null allele sno93i exposed to a non-permissive temperature for 12 hours caused a significant reduction in Delta expression. Similarly, a dominant-negative version of ebi also caused the loss of Delta expression. Importantly, pupal eye discs of neither spa-Gal4, UAS-ebiDN nor snoE1/sno93i showed any perturbation in cone-cell fate, as judged by the expression of Cut. Thus, as in the case of larval R cells, the loss of ebi and sno in the pupal cone cells causes the loss of Delta expression without causing a change in cone-cell fate (Nagaraj, 2007).

To test whether the expression of Delta in pupal cone cells is required for the specification of primary pigment cells, Nts pupae were incubated at a non-permissive temperature for 10 hours during pupal development and pigment-cell differentiation was monitored using BarH1 (also known as Bar) expression as a marker. Loss of Notch signaling during the mid-pupal stages caused a loss of Bar, further demonstrating the requirement of Notch signaling in the specification of primary pigment-cell fate. Similarly, when the 54CGal4 driver line, which is activated in pigment cells, was used to drive the expression of a dominant-negative version of Notch, pupal eye discs lost primary pigment-cell differentiation, again suggesting an autonomous role for Notch in pigment-cell precursors. In neither the Nts nor the 54C-Gal4, UAS-NDN genetic background was perturbation observed in cone-cell fate specification. It is concluded that Delta activation mediated by EGFR-Sno-Ebi in pupal cone cells is essential for neighboring pigment-cell fate specification (Nagaraj, 2007).

Delta-protein expression in pupal cone cells is initiated at 12 hours and is downregulated by 24 hours of pupal development. To determine the functional significance of this downregulation, the genetic combination spa-Gal4/UAS-Delta was used, in which Delta is expressed in the same cells as in wild type, but is not temporally downregulated. Whereas, in wild type, a single hexagonal array of pigment cells surrounded the ommatidium, in the pupal eye disc of spa-Gal4, UAS-Delta flies, multiple rows of pigment cells were observed surrounding each cluster. Furthermore, in wild type, only two primary pigment cells were positive for Bar expression in each cluster, whereas, in spa-Gal4, UAS-Delta pupal eye discs, ectopic expression of Bar was evident in the interommatidial cells. Therefore, the temporal regulation of Notch signaling and its activation, as well as its precise downregulation, are essential for the proper specification of primary pigment-cell fate (Nagaraj, 2007).

By contrast to the autonomous requirement for Notch signaling in primary pigment cells, the function of the EGFR signal appears to be required only indirectly in the establishment of primary pigment-cell fate through the regulation of Delta expression in the pupal cone cells. When a dominant-negative version of EGFR was expressed using hsp70-Gal4 at 10-20 hours after pupation, no perturbation was observed in the specification of primary pigment cells, as monitored by the expression of the homeodomain protein Bar. By contrast, the expression of dominant-negative Notch under the same condition resulted in the loss of Bar-expressing cells. Thus, in contrast to Notch, blocking EGFR function at the time of primary pigment-cell specification does not block the differentiation of these cells. Importantly, blocking EGFR function in earlier pupal stages caused the loss of Delta expression in cone cells and the consequent loss of pigment cells. Based on these observations, it is concluded that, in the specification of primary pigment-cell fate, the Notch signal is required directly in primary pigment cells, whereas EGFR function is required only indirectly (through the regulation of Delta) in cone cells (Nagaraj, 2007).

The Runt-domain protein Lz functions in the fate specification of all cells in the developing eye disc arising from the second wave of morphogenesis. At a permissive temperature (25°C), lzTS114 pupal eye discs showed normal differentiation of primary pigment cells. lzTS114 is a sensitized background in which the Lz protein is functional at a threshold level. When combined with a single-copy loss of Delta, a dosage sensitive interaction caused the loss of primary pigment cells. By contrast, under identical conditions, a single-copy loss of EGFR function had no effect on the proper specification of primary pigment-cell fate. This once again supports the notion that the specification of primary pigment cells directly requires Lz and Notch, whereas EGFR is required only indirectly to activate Delta expression in cone cells (Nagaraj, 2007).

This study highlights two temporally distinct aspects of EGFR function in cone cells. First, this pathway is required for the specification of cone-cell fate at the larval stage, and EGFR is then required later in the pupal cone cell for the transcriptional activation of Delta, converting the cone cell into a Notch-signaling cell. Delta that was expressed in the cone cell through the activation of the Notch pathway functioned in combination with Lz in a cell autonomous fashion and promoted the specification of the primary pigment-cell fate (Nagaraj, 2007).

Studies using overexpressed secreted Spitz have shown that ectopic activation of the EGFR signal in all cells of the pupal eye disc results in excess primary pigment cells. This study shows that EGFR activation in the pupal eye disc is required for the transcriptional activation of Delta in cone cells, but that the loss of EGFR function at the time when primary pigment cells are specified does not perturb their differentiation. It is concluded that the ectopic primary pigment cells seen in an activated-EGFR background result from the ectopic activation of Delta, which then signals adjacent cells and promotes their differentiation into primary pigment cells. Indeed, it has been shown that excessive Delta activity results in the over specification of primary pigment cells. The results are also consistent with the previous observation that the EGFR target gene Argos is not expressed in primary pigment cells in pupal eye discs. Additionally, loss of EGFR function in pupal eye discs does not perturb the normal patterning of interommatidial bristle development, which develop even later than the primary pigment cells (Nagaraj, 2007).

The elucidation of the Sevenless pathway for the specification of R7 led to the suggestion that different cell types within the developing eye in Drosophila will require combinations of dedicated signaling pathways for their specification. However, studies from several laboratories have suggested that the Sevenless pathway seems to be an exception, in that cell-fate-specification events usually require reiterative combinations of a very small number of non-specific signals. Cone-cell fate is determined by the sequential integration of the EGFR and Notch pathways in R cells followed by the parallel integration of the EGFR and Notch pathways in cone-cell precursors. This study shows that the most important function of EGFR in the specification of primary pigment cells is to promote the transcriptional activation of Delta in cone cells through the EGFR-Ebi-Sno-dependent pathway. The sequential integration of the EGFR and Notch pathways, first used in the larval stage for Delta activation in R cells, is then reused a second time in cone cells to regulate the spatiotemporal expression of Delta, converting the cone cells at this late developmental stage to Notch-signaling cells. Delta present in the cone cell then signals the adjacent undifferentiated cells for the specification of primary pigment cells. For this process, the Notch pathway functions directly with Lz but indirectly with EGFR. Through extensive studies of this system it now seems conclusive that different spatial and temporal combinations of Notch and EGFR applied at different levels can generate all the signaling combinations needed to specify the neuronal (R1, R6, R7) and nonneuronal (cone, pigment) cells in the second wave of morphogenesis in the developing eye disc (Nagaraj, 2007).

The EGFR and Notch pathways are sequentially integrated, in a manner similar to that described here, in multiple locations during Drosophila development. In the development of wing veins, EGFR that is activated in the pro-vein cells causes the expression of Delta, which then promotes the specification of inter-vein cells. Similarly, these two pathways are sequentially integrated in the patterning of embryonic and larval PNS, and during muscle development. Indeed, there are striking similarities between the manner in which the EGFR and Notch pathways are integrated in the developmental program in the C. elegans vulva and the Drosophila eye. During vulval fate specification in the C. elegans hermaphrodite gonad, anchor cells are a source of EGFR signal (Lin3), which induces the specification of the nearest (P6) cell to the primary cell fate from within a group of six equipotent vulval precursor cells (VPC). This high level of EGFR activation induces the transcriptional activation of Notch ligands in the primary cells in what can be considered sequential integration of the two pathways - the Notch signal from the primary cell both inhibits EGFR activity in the VPCS on either side of P6.p and also promotes the secondary cell fate. Thus, the reiterative integration of these two signals, in series and in parallel, can be used successfully to specify multiple cell fates in different animal species. Given that the RTK and Notch pathways function together in many vertebrate developmental systems, it is likely that similar networks will be used to generate diverse cell fates using only a small repertoire of signaling pathways (Nagaraj, 2007).

Tumor suppressor gene l(2)giant discs is required to restrict the activity of Notch to the dorsoventral boundary during wing development

During the development of the Drosophila wing, the activity of the Notch signalling pathway is required to establish and maintain the organizing activity at the dorsoventral boundary (D/V boundary). At early stages, the activity of the pathway is restricted to a small stripe straddling the D/V boundary, and the establishment of this activity domain requires the secreted molecule Fringe (Fng). The activity domain will be established symmetrically at each side of the boundary between Fng-expressing and non-expressing cells. Evidence is presented that the Drosophila tumor-suppressor gene lethal giant discs (lgd), a gene whose coding region has yet to be identified, is required to restrict the activity of Notch to the D/V boundary. In the absence of lgd function, the activity of Notch expands from its initial domain at the D/V boundary. This expansion requires the presence of at least one of the Notch ligands, which can activate Notch more efficiently in the mutants. The results further suggest that Lgd appears to act as a general repressor of Notch activity, because it also affects vein, eye, and bristle development (Klein, 2003).

It has also been observed that wingless (wg) is expressed ectopically in the pouch of lgd mutants during wing development. Similar phenotypes are observed, if the Notch pathway is ectopically activated during wing development, raising the possibility that the lgd mutant phenotype could stem from the ectopic activation of the Notch pathway. The Notch pathway is indeed ectopically active in lgd mutants, and hyperactivation as well as ectopic activation of the pathway accounts for the lgd phenotype during wing development. In lgd mutants, the expression of Notch target genes along the D/V boundary is expanded, indicating that Lgd is required for the restriction of Notch activity to the D/V boundary. Furthermore, the mutant phenotype of lgd is suppressed by concomitant loss of Presenilin or Suppressor of Hairless function, indicating that the mutant phenotype is caused by the activation of the Notch pathway. Evidence is provided that the activity of fng and Serrate seem to be dispensable in lgd mutant wing disc and that Delta can activate Notch efficiently enough to maintain its activity during wing development. The presented results indicate that the negative regulation of Notch by Lgd is not restricted to wing development and occurs during several other developmental processes, such as vein, eye, and bristle development, suggesting that Lgd suppresses the activity of the Notch pathway in a variety of developmental processes (Klein, 2003).

Loss of lgd function leads to an overgrowth of the imaginal discs, clearly noticeable in the wing region of the wing disc, which becomes enlarged and flat (Bryant, 1971). wg expression is normally restricted to the D/V boundary of the wing pouch. In lgd mutants, wg is activated ectopically in a much broader domain that extends into the wing pouch. In addition, lgd mutant wing discs often develop a second wing pouch in the region of the anlage of the scutellum. Similar phenotypes are caused by gain-of-function alleles of N (for example, Abruptex) and are also observed upon expression of the activated intracellular form of Notch, Nintra, or expression of Notch ligands, such as Dl. The ectopic activation of wg can already be observed in early third instar wing discs and precedes the visible morphological changes that occur at later stages. The deficiency Df(2L) FCK-20 deletes the lgd locus, allowing the classification of the relative strength of the available alleles. The phenotype is always variable, but the overall phenotype of lgdd7 and lgdd10 in homozygotes and in trans over Df(2L)FCK-20 is very similar, indicating that these two alleles are strong, probably amorphic alleles. lgdd4 and lgdd1 are weaker alleles. All alleles display a qualitatively similar phenotype over the deficiency as in homozygotes, indicating that the observed phenotype is probably caused by the loss-of-function of the lgd gene (Klein, 2003).

The similarity between the loss of lgd function and ectopic N activation suggests that the phenotype of lgd could be caused by ectopic activation of the Notch pathway. To examine this possibility, the expression of E(spl)m8, cut, Dl, and Ser was monitored as well as the activity of the vg-boundary enhancer (vgBE) in mutant wing discs. The expression of all these markers is initiated in cells at the D/V boundary in a Notch-dependent manner. The vgBE is initially expressed along the D/V boundary of the wing, but late in the third instar, it is activated in an additional stripe along the anteroposterior compartment boundary (A/P boundary), which is also dependent on Notch activity. Both domains depend on the presence of a single Su(H) binding site in the enhancer. Similarly, the expression of cut and E(spl)m8 is initiated in cells at the boundary by the Notch-pathway, and E(spl)m8 is also dependent on the presence of Su(H) binding sites in its promoter. As described above, the expression of Dl and Ser is more complex but always dependent on the activity of Notch in cells at the D/V boundary. In lgd mutant wing discs, the vgBE as well as cut, Dl, Ser, and E(spl)m8 are activated ectopically within the wing pouch. The activation of the vgBE is dependent on the presence of the Su(H) binding site in the enhancer, since a version lacking it shows no ectopic activity in the mutants. As in the case of wg, the expression of the vgBE is already expanded in early third larval wing discs. Altogether, these results show that the loss of lgd function leads to the ectopic expression of Notch target genes. This suggests that the Notch pathway is ectopically activated in lgd mutants (Klein, 2003).

All tested Notch-target genes are ectopically activated in lgd mutant wing discs or lgd mutant cell clones. The ectopic activation of Notch target genes as well as the observed overproliferation of lgd mutants is abolished in lgd;Psn double mutants. In addition, Notch target gene expression is also abolished in Psn or Su(H) mutant clones generated in lgd mutant wing imaginal discs. These data suggest that the Notch pathway becomes ectopically active in the absence of lgd function. Furthermore, the fact that Delta alone seems to provide sufficient Notch activity to sustain wing development in lgd mutants indicates that the pathway can be activated more efficiently in the mutant background. The activation of Notch is a consequence of loss of lgd function also in other developmental processes, such as bristle, leg, and wing vein development. Thus, the presented data make lgd a good candidate gene that regulates activity of the Notch pathway during adult development of Drosophila (Klein, 2003).

Notch and planar polarity

The Drosophila eye is composed of several hundred ommatidia that can exist in either of two chiral forms, depending on position: ommatidia in the dorsal half of the eye adopt one chiral form, whereas ommatidia in the ventral half adopt the other. Chirality appears to be specified by a polarizing signal with a high activity at the interface between the two halves (the 'equator'), which declines in opposite directions towards the dorsal and ventral poles. Here, using genetic mosaics, it is shown that this polarizing signal is decoded by the sequential use of two receptor systems. The first depends on the seven-transmembrane receptor Frizzled (Fz) and distinguishes between the two members of the R3/R4 pair of presumptive photoreceptor cells, predisposing the cell that is located closer to the equator and having higher Fz activity towards the R3 photoreceptor fate and the cell further away towards the R4 fate. This bias is then amplified by subsequent interactions between the two cells mediated by the receptor Notch (N) and its ligand Delta (Dl), ensuring that the equatorial cell becomes the R3 photoreceptor while the polar cell becomes the R4 photoreceptor. As a consequence of this reciprocal cell fate decision, the R4 cell moves asymmetrically relative to the R3 cell, initiating the appropriate chiral pattern of the remaining cells of the ommatidium (Tomlinson, 1999).

The focus for the chirality choice maps to the presumptive R3/R4 photoreceptor pair; it maps specifically to the R3 cell. Although these two cells lie adjacent in the ommatidial precluster, fate-mapping experiments, together with histological analysis, indicate that they are initially distant from one another, separated along the equatorial axis by at least the remaining three cells that will enter the precluster. During normal development, the member of the pair that is closest to the equator invariably chooses to develop as an R3 cell, while the remaining member develops as an R4 cell. This asymmetric cell fate choice determines the chirality of the ommatidium. The R3/R4 cell fate decision is governed by the relative difference in activity of Frizzled (Fz) protein in the two cells comprising the presumptive R3/R4 pair. The cell with higher Fz activity becomes R3, while the remaining cell becomes R4. Since fz encodes a serpentine receptor-like protein, Fz is a candidate receptor for a factor X, consistent with the notion that the presumptive R3/R4 pair decodes the factor X gradient into a relative difference in Fz activity between the two cells. The difference in Fz activity between the two members of the presumptive R3/R4 pair biases a process of lateral specification between the two cells mediated by the receptor Notch (N) and its ligand Delta (Dl). In essence, the cell with higher Fz activity appears to be better at sending the Dl signal while the cell with lower Fz activity is better at receiving the signal. This difference is probably then amplified by a feedback mechanism in which receptor activation blocks ligand production in the same cell, while the loss of receptor activity leads to enhanced ligand production in the other cell. As a consequence, the N transduction pathway is fully induced in one cell but silenced in the other. The resulting disparity in N signal transduction is both necessary and sufficient to specify the reciprocal R3 and R4 cell fates and to determine the chirality of the ommatidia (Tomlinson, 1999).

Two models are presented for how Fz activity within the R3/R4 pair might bias the N-Dl interaction. In the first model, a scalar model, factor X activity positively regulates Fz activity, whereas in the second it negatively regulates Fz activity. A convention of factor X activity has been adopted; factor X is considered as being high at the equator and low at the poles. However, the results can be equally well explained if factor X has the opposite distribution, high at the poles and low towards the equator. In this scenario the positive and negative influences of factor X on Fz activity in the two models will be reversed. A difference in the levels of Fz activity between the two cells of the R3/R4 pair determines which cell will become R3 and which will become R4. Because the presumptive R3 cell lies closer to the equator than the presumptive R4, it will detect higher levels of factor X activity and consequentially will have a higher level of Fz activity. The higher levels of Fz activation then bias the subsequent N-Dl interaction so that the cell with greater Fz activity becomes a dedicated Dl signaling cell, while its partner, with less Fz activity, becomes a dedicated Dl receiving cell. For example, the level of Fz activity in each cell could govern the activity or level of expression of a component of the N-Dl signaling mechanism, such as N itself or Dl. Even a small difference in signaling capacity between the two cells would then bias the N-Dl system of feed-back regulation causing the cell with initially higher N transducing capacity to become a dedicated Dl receiving cell (and hence R4), while the remaining cell becomes a dedicated Dl sending cell (R3). Experimentally induced changes in Fz activity, which reverse the relative difference in Fz activity between the presumptive R3 and R4 cells, might cause a corresponding reversal in direction of N-Dl signaling and the R3/R4 cell fate decision. An issue raised by this model is whether the presumptive R3 and R4 cells have the capacity to meter accurately what are likely to be small differences in their absolute levels of factor X. Consider that up to 15 ommatidia can form along the equatorial-polar axis of each half of the eye, with the presumptive R3 and R4 cells located next to each other in each cluster and separated by several cell diameters from their counterparts in neighboring clusters. More than 75 cells are arrayed in each half of the eye along the Eq/Pl axis and any two neighboring cells at any position within that array would need to faithfully decode the factor X gradient. However, the results of fate-mapping analysis and histological studies suggest that the presumptive R3 and R4 cells are initially located at a distance from one another, separated along the Eq/Pl axis by the remaining three cells of the precluster (the presumptive R2, R8 and R5 cells). This raises the possibility that the presumptive R3 and R4 may meter factor X abundance when they are located at opposite ends of these cell lines before they come to lie next to each other. The physical separation between the two cells at this time would allow them to sample a broader segment of the factor X gradient, much as the separation between the two tips of the forked tongue of a snake facilitates the detection of odor gradients (Tomlinson, 1999).

In a second model, the vectoral model, each cell in the retina, and hence both the presumptive R3 and R4 cells, detects the gradient of factor X activity across its diameter and decodes it into a steeper gradient of Fz activity within the cell. Specifically, it is proposed that the factor X activity might negatively regulate Fz such that Fz activity is highest within each cell on its polar side and lowest on its equatorial side. Within the precluster, the presumptive R3 cell presents its polar face to the equatorial face of the presumptive R4 cell. The differential Fz activities across the two faces then bias the subsequent N-Dl interaction between the two cells. In this case, it would be argued that the bias is likely to be mediated through the direct and local modulation of a component of the N-Dl signaling apparatus, e.g. a post-translational modification of N or Dl activity along the surface of one of the two cells, where they abut. When Fz is overexpressed in the presumptive R4 cell, Fz activity is enhanced throughout the cell, including at the surface comprising the equatorial face where it abuts the polar face of the R3 cell. As a consequence, any influence of the polarizing factor X gradient would be subsumed, reversing the N-Dl signaling predisposition that would otherwise occur. Conversely, when Fz activity in the presumptive R3 cell is abolished, the polar face of the R3 cell would now have lower Fz activity than the adjacent, equatorial face of the R4 cell. As in the overexpression case, this change would reverse the direction of N-Dl signaling and hence reverse the reciprocal cell-fate choices made by the two cells (Tomlinson, 1999).

A key difference between the two models is that the scalar model requires cells to meter the absolute concentration of factor X via the level of activation of Fz protein, whereas the vectorial model requires them to detect and then amplify a relative difference of factor X concentration across the cell's diameter. Another difference is that factor X is predicted to have opposite effects on Fz activity, activating Fz in the scalar model and inhibiting Fz in the vectorial model. Hence, it is difficult to envisage how both mechanisms could work in concert, and as a consequence, they are viewed as mutually exclusive (Tomlinson, 1999).

One way in which a cell might amplify a difference in factor X abundance across its diameter would be to polarize the distribution of Fz itself in response to the ligand. Although the mechanisms are not yet clear, there are several precedents for the ability of cells to respond to shallow gradients of extracellular signals by polarizing the distribution of receptors and cytoskeletal components (e.g. during shmooing of yeast cells in response to mating pheromone, and the extension of pseudopods in Dictyostelium in response to cAMP). It is notable that Fz activity gradients have been implicated in the control of cell polarity in other epithelial tissues in the fly, particularly the wing. However, there is, at present, no indication that N signaling is involved in the establishment of cell polarity in this context. It is therefore suggested that Fz signaling generally mediates the establishment of cell polarity without requiring an N-Dl feedback amplification step. Hence, the involvement of N-Dl signaling in establishing ommatidial chirality may reflect a special attribute of this system, perhaps to allow the polarity of just two cells, the presumptive R3/R4 pair, to be used as a cue to control the pattern of a much larger ensemble of cells, the ommatidium (Tomlinson, 1999).

When Fz activity is absent throughout eye development, the Fz-dependent bias should be eliminated and each cell of the presumptive R3/R4 pair should have an equal chance of becoming either the signaling cell (R3) or the receiving cell (R4). Under these conditions, the choice of which cell becomes R3 and which becomes R4 would be determined by a stochastic variation, which gives one of the two cells a slight advantage that is then amplified by the N-Dl feedback mechanism. This explains why ommatidia in fz mutants 'choose' their chirality randomly when both cells have the same N gene dosage, but non-randomly when there is a 3:2 differential in N gene dosage. However, in fz minus eyes, approximately one third of the ommatidia are symmetrical, indicating that the R3/R4 distinction has not been resolved. One possible explanation is that the interaction between the R3 and R4 cells may be limited to only a few hours and this may be too short to ensure that a stochastic variation will arise and be amplified by the N pathway in all fz minus ommatidia. Timing may not be as critical in wild-type ommatidia, because the bias from Fz signaling is sufficient to ensure an appropriate resolution during this relatively brief interval (Tomlinson, 1999).

Barbu (Bbu), an alternative name applied to Twin of m4 (Tom: see E(spl) region transcript m4, Evolutionary Homologs section), can antagonize Notch signaling activity during Drosophila development. This gene functions to antagonize Notch signaling, as does E(spl) region transcript m4,. Ectopic expression studies with Barbu provide evidence that Barbu can antagonize Notch during lateral inhibition processes in the embryonic mesoderm, sensory organ specification in imaginal discs and cell type specification in developing ommatidia. Barbu loss-of-function mutations cause lethality and disrupt the establishment of planar polarity and photoreceptor specification in eye imaginal discs, which may also be a consequence of altered Notch signaling activities. Furthermore, in the embryonic neuroectoderm, Barbu expression is inducible by activated Notch. Taken together, it is proposed that Barbu functions in a negative feed-back loop, which may be important for the accurate adjustment of Notch signaling activity and the extinction of Notch activity between successive rounds of signaling events (Zaffran, 2000).

In the neuroectoderm, Bbu expression is excluded from the proneural clusters and neuroblasts in which these proneural proteins are expressed. In clusters from which single cells have been selected to maintain proneural gene expression, all cells except for the presumptive neuroblast express Barbu mRNA. These observations raised the possibility that the Notch signaling pathway, while repressing proneural genes, may activate Bbu in the presumptive non-neuroblast cells. Ectopic expression of activated Notch results in the overexpression of BBU mRNA. The presence of three putative Su(H) binding sites in the presumed Bbu promoter sequence indicates that activation of Bbu by Notch is possibly direct. One of these sequences ([-791]CGTGGG-AAA) matches exactly the previously reported consensus sequence for Su(H) sites [(C/T)GTG(G/A)GAA(C/A)]. Thus, the control of Bbu expression with respect to its dynamic 'filling in' of proneural clusters and exclusion from prospective neuroblasts seems to be a direct result of its activation by the Notch signaling pathway in the neuroectoderm. Additional experiments show that overepxression of Bbu in the mesoderm antagonizes Notch signaling giving rise to an increased number of Even-skipped expressing heart cells. Overexpression of Bbu also affects the development of lateral and ventral muscle progenitor muscles. Likewise, overexpression of Bbu increases the number of bristles in the adult (Zaffran, 2000).

Bbu mutants were induced by P-element excision. Although the presumed null allele Bbu99 confers embryonic lethality, no specific abnormalities could be detected in ectodermally or mesodermally derived tissues in homozygous embryos for this mutation. However, the phenotypic analysis of the hypomorphic alleles Bbu8 and Bbu105 proved to be informative. Both Bbu8 and Bbu105 confer pupal lethality and pharate adults homozygous for these mutations fail to undergo head eversion. Bbu hypomorphic alleles rescues the phenotype of homozygous Su(H) mutants, which are larval lethal with small eye discs. Bbu;Su(H) double mutants develop into the pupal stage with pharate adults displaying partially everted heads. Thus, reduced levels of Notch/Su(H) signaling can partially rescue the pupal phenotype caused by reduced levels of Bbu expression. Bbu105 gives rise to a small number of escapers when the flies are grown at low density. The bristles on the notum of these adults, particularly in the anterior portion, show defects in their polarity. Since both planar polarity and lateral inhibition involve the Notch signaling pathway, these results suggest that Bbu may act to adjust the levels of Notch signaling during these processes (Zaffran, 2000).

Bbu is the first member of this gene family to show a loss-of-function phenotype. While Bbu loss-of-function alleles cause lethality and developmental defects, null mutants for Brd and E(spl) m4 are homozygously viable and do not exhibit any detectable abnormalities. It is likely that the wild-type phenotypes of Brd and m4 null mutants are due to functional redundancy among genes of this family. Partial functional redundancy may also explain the failure to detect any phenotypes in Bbu null mutant embryos. Nevertheless, the observed pupal and adult phenotypes, as well as the genetic interactions of Bbu loss-of-function mutations with Su(H) mutations, are compatible with a Bbu role in the downregulation of Notch signaling activity. Prominent features that are shared with Notch gain-of-function phenotypes are polarity defects and disrupted cell specification during eye development. Similar to heat-shock induction of Nintra, reduction of Bbu activity results in increased levels of atonal expression anterior to the morphogenetic furrow and, in some instances, loss of ato expression in the prospective photoreceptor R8. No overt R3 to R4 transformations are seen in the mis-oriented ommatidia upon reduction of Bbu activity, although such cell fate transformations are observed upon ectopic Notch activation and thought to be a cause for polarity defects. Rather, randomly oriented ommatidia are observed with apparently normally specified photoreceptors, which is very similar to the defects reported for mutations in frizzled and its downstream effector gene dishevelled. Recent models for the origin of the ommatidial polarity have proposed the occurrence of reciprocal Notch/Delta signaling between the prospective R3 and R4 cells. This mutual interaction is thought to be biased by increased Frizzled/Dishevelled activities in R3, which downregulates Notch activity and upregulates the production of Delta in this cell. The observed Bbu mutant phenotype in ommatidia suggests that, upon the reduction of Bbu levels, this bias is removed and the decisions between R3 and R4 fates within each ommatidum become randomized. Thus, it is propose that, in the normal situation, Bbu is required together with activated Frizzled to antagonize Notch activity in the prospective R3 photoreceptor. While a model is preferred in which Bbu acts in parallel with Frizzled, perhaps by potentiating or stabilizing its negative effect on Notch, the alternative that Bbu acts in the Frizzled pathway upstream of Notch cannot be ruled out. Because Notch has been shown to participate in the specification of essentially all cell types of the eye disc, the reduced numbers of photoreceptors in Bbu null mutant clones may also be due to increased activities of the Notch pathway during different steps of eye development. Taken together, the combined data from overexpression and loss-of-function experiments make a strong case for a role of Bbu in downregulating Notch activity. However, by no means do they exclude the possibility that Bbu (and by extension, E(spl) m4, malpha, and Brd) acts in additional pathways that do not involve Notch signaling (Zaffran, 2000).

During patterning of the Drosophila eye, the Notch-mediated cell fate decision is a critical step that determines the identities of the R3/R4 photoreceptor pair in each ommatidium. Depending on the decision taken, the ommatidium adopts either the dorsal or ventral chiral form. This decision is directed by the activity of the planar polarity genes, and, in particular, higher activity of the receptor Frizzled confers R3 fate. Evidence is presented that Frizzled does not modulate Notch activity via Rho GTPases and a JNK cascade as previously proposed. The planar polarity proteins Frizzled, Dishevelled, Flamingo, and Strabismus adopt asymmetric protein localizations in the developing photoreceptors. These protein localizations correlate with the bias of Notch activity between R3/R4, suggesting that they are necessary to modulate Notch activity between these cells. Additional data support a mechanism for regulation of Notch activity that could involve direct interactions between Dishevelled and Notch at the cell cortex. In the light of these findings, it is concluded that Rho GTPases/JNK cascades are not major effectors of planar polarity in the Drosophila eye. A new model is proposed for the control of R3/R4 photoreceptor fate by Frizzled, whereby asymmetric protein localization is likely to be a critical step in modulation of Notch activity. This modulation may occur via direct interactions between Notch and Dishevelled (Strutt, 2002).

Frizzled tagged with green fluorescent protein (Fz-GFP) exhibits a dynamic subcellular distribution from early stages of ommatidial differentiation. Ommatidia are born behind the furrow in rows polarized in the anteroposterior axis. In row 4, Fz-GFP is enriched on the apical membranes of the newly recruited R3/R4 pair but excluded from the region where they contact R2/R5. No Fz-GFP enrichment is apparent around R2/R5, but it does accumulate on the posterior side of R8. By row 6, Fz-GFP is no longer enriched in R3, except at the boundary with R4 and sometimes at the boundary with the anterior cone cell. Conversely, R4 still has strong accumulation around its perimeter, except where it contacts R5. This accumulation around R4 persists through row 8, but accumulation fades elsewhere. Thus, Fz-GFP is initially in a symmetric pattern in R3/R4 but rapidly resolves into an asymmetric pattern that is visible by the time ommatidial rotation occurs in row 6. Using antibodies against Dsh and Fmi, these proteins were found to colocalize with Fz and show the same dynamic distribution (Strutt, 2002).

N is also at highest levels in apical membranes of cells posterior to the furrow and in rows 4 through 6 it overlaps with Fz-GFP at the R3/R4 boundary (but shows no asymmetry). The localization of Fz-GFP (and Fmi/Dsh) to the R3/R4 boundary is therefore consistent with Fz/Dsh being able to directly modulate N activity in this location. However, if Fz/Dsh are differentially regulating N activity, a crucial requirement is that these complexes should be preferentially localized on one side of the R3/R4 boundary. Since this cannot be distinguished by light microscopy, genetic mosaics were created in which both R3/R4 had sufficient fz activity for normal signaling and fate determination, but only one of the pair carried the Fz-GFP transgene. Using this approach, it was found that Fz-GFP is more highly enriched on the R3 side of the R3/R4 boundary in row 4 and more posteriorly is found exclusively on the R3 side of the boundary. Thus, about two rows prior to ommatidial rotation, Fz-GFP is asymmetrically distributed across the R3/R4 boundary. Since studies in the wing demonstrate that Dsh adopts the identical asymmetric localization to Fz (and indeed their asymmetric localization is mutually dependent), it is inferred that Dsh is also differentially localized on the R3 side of the R3/R4 boundary (Strutt, 2002).

The polarity gene stbm is required for R4 fate: whether Stbm protein also shows an asymmetrical localization in R3/R4 was investigated using a Stbm-YFP transgene. Stbm-YFP is apically localized in cells posterior to the furrow, and, subsequently, its distribution is similar but distinct from that exhibited by Fz-GFP. In row 4, a symmetric pattern is observed, with Stbm-YFP around R3/R4, except where these cells contact R2/R5, and enriched on the posterior face of R8. This symmetric pattern is maintained until the ommatidia are already rotated in row 6 and more posteriorly. Staining then fades around R3, except where R3 contacts R4. Mosaic analysis revealed that, in contrast to Fz-GFP, Stbm-YFP is enriched on the R4 side of the R3/R4 boundary from row 4 onward, i.e., Stbm is on the opposite side of the boundary with Fz (Strutt, 2002).

Therefore Fz, Dsh, Fmi, and Stbm localize to the apical region of the R3/R4 cell boundary, where they become asymmetrically distributed prior to or concomitant with R3/R4 fate determination. Normally, Fz/Dsh are enriched on one particular side of the cell boundary, in the presumptive R3 cell. However, in mosaic ommatidia where one or other cell is mutant for polarity genes, the assembly of the asymmetrical complexes can be reversed. In all conditions examined, the polarity of Notch signaling between R3/R4 is consistent with the polarity of the asymmetric complexes, with Notch activity being lowest in the cell where Fz/Dsh accumulate. Finally, evidence is provided that the domain of N, which is known to interact directly with Dsh, is required for efficient R3/R4 fate decisions (Strutt, 2002).

Considering these results together, it is proposed that an extracellular polarity signal leads to the asymmetric assembly of a complex of planar polarity proteins at the boundary between the R3/R4 cell pair. This asymmetric complex then leads to asymmetric N activity between the cell pair and thus determines cell fate. Since no evidence is found that this regulation occurs via the proposed signaling cascade downstream of Fz/Dsh (i.e., Rho GTPases/JNK) and since manipulation of Dl transcription does not perturb polarity of Notch signaling, it is concluded that there must be an alternative pathway by which asymmetrical Fz/Dsh affects Notch activity (Strutt, 2002).

One favored mechanism for the modulation of N/Dl activity is via local interactions between N and asymmetrically localized proteins and, in particular, between the intracellular domain of N and Dsh. Four lines of evidence support the proposal that the regulation occurs at the cell cortex: (1) Fz/Dsh are in the same subcellular domain as N at the apical R3/R4 boundary during the critical stages of development when the cell fate decision is made; (2) the appearance of the asymmetric Fz/Dsh complexes is shortly prior to or concomitant with the appearance of a bias in N/Dl activity and ommatidial rotation; (3) direct interactions between N and Dsh have been previously demonstrated and proposed to be important for patterning in other tissues, and these interactions have been found to be repressive, consistent with Fz/Dsh being required in R3, where N activity is lowest and (4) deletion of the domain of N required for interactions with Dsh leads to less-efficient R3/R4 fate decisions (Strutt, 2002).

The model whereby asymmetric Fz/Dsh localization leads to downregulation of N activity on the R3 side of the R3/R4 boundary is further supported by studies in the Drosophila leg, where loss of planar polarity gene activity leads to ectopic activity of Notch. However, there are still unexplained observations: if the only role of the polarity genes is to inhibit N in R3, mutations in fmi, fz, or dsh (which result in no apical Dsh localization) should have high N activity in both R3/R4, not the reduced activity that is detected (Strutt, 2002).

This discrepancy might be explained if there are two phases to polarity gene regulation of N activity. One would be an activation/derepression of N, which would require symmetric protein localization of Fz/Dsh in R3/R4. The second would be linked to asymmetric protein localization, when Fz/Dsh would in turn become repressors of N activity in R3 (Strutt, 2002).

The asymmetric localization of Fz, Dsh, and Fmi in the eye develops in a similar manner to that seen in the pupal wing. Thus, the R3/R4 cell boundary appears analogous to the proximodistal wing cell boundaries, with the R3 side of the boundary, where Fz and Dsh are localized, being equivalent to the wing cell distal edge. Another of the polarity gene products, Stbm, is localized on the R4 side of the boundary, which is consistent with the requirement for stbm function in R4. By analogy to the wing, it is likely that Fmi is present on both sides of the R3/R4 boundary and Pk-Sple/Sple is on the R4 side (Strutt, 2002).

The adoption of the asymmetric pattern occurs in two phases. The first involves symmetric apicolateral localization of Fz, Dsh, Fmi, and Stbm in R3/R4 (and in all other cells except R2/R5); this is evident in ommatidial row 4. As in the wing, the initial apical recruitment of Fz is dependent on Fmi, and the recruitment of Dsh is in turn dependent on Fz. Subsequently, the distribution evolves rapidly into an asymmetric pattern. Adoption of asymmetry requires the function of dsh, stbm, and the LIM domain protein Prickle-Spiny-legs (Pk-Sple), and if any of these are missing, Fz distribution remains symmetric in ommatidial rows 5/6, and ommatidial rotation is delayed. It is likely that the asymmetry evolves through the same mechanisms as in the wing, where it has been proposed that an extrinsic signal leads to a small bias in Fz/Dsh signaling on either side of the cell boundary, which subsequently becomes amplified by feedback loops that lead to Fz/Dsh becoming concentrated on one side of the interface and Pk-Sple/Stbm on the other (Strutt, 2002).

One notable difference between the eye and the wing is that asymmetric Fz/Dsh distribution is eventually observed in stbm and pk-sple eye discs, but in both cases it occurs with a random bias and is delayed by about one to two ommatidial rows. This correlates well with the fact that the adult phenotypes of stbm and pk-sple exhibit a low incidence of achiral ommatidia. Conversely, in fmi, fz and dsh, negligible asymmetric protein localization occurs, and there is a relatively high proportion of 'achiral' ommatidia in the adult eye, suggesting that achirality is a result of poor asymmetric complex formation. In general, the aquisition of asymmetry also correlates with mDelta0.5 activity, particularly in pk-sple and sple mutations where its expression usually resolves into a single cell by row 10 (Strutt, 2002).

In the pupal wing, asymmetric localization of Fz/Dsh/Pk-Sple is proposed to involve a signaling feedback loop that amplifies an initially small bias in Fz/Dsh activity across the axis of each cell. In the eye, the N/Dl feedback loop was proposed to perform a similar function, amplifying an initially small difference in Fz/Dsh activity between R3/R4. With the observation that Fz/Dsh are also distributed in asymmetric complexes in the eye, it appears that both mechanisms are operating in R3/R4, although it is not clear why both would be required, since either alone should be sufficient to amplify small biases in signaling activity (Strutt, 2002).

One possible explanation is that use of both mechanisms increases the speed and robustness of the R3/R4 fate decision. A fast fate decision may be necessary because of the dynamic nature of eye patterning, in which the R3/R4 decision is only part of a complex series of events involving cell recruitment and movement to generate the final polarized ommatidium. It is also possible that a rapid decision is required because the extrinsic polarity cue is transient in nature. It is noted that the rapidity of the decision would be further enhanced if N/Dl signaling also influenced Fz/Dsh localization. While there is no direct evidence for this, it could explain the eventual, randomly polarized, asymmetric protein localization seen in stbm and pk-sple backgrounds in the eye. In this case, the inability of Fz/Dsh to efficiently localize asymmetrically in the absence of Stbm/Pk-Sple might lead to N/Dl making a stochastic decision that then leads to Fz/Dsh asymmetry. Conversely, in the pupal wing, where N/Dl are not active in planar polarity decisions, Stbm/Pk-sple activity would be absolutely required, since their absence would not be compensated for by the N/Dl feedback loop (Strutt, 2002).

A number of lines of evidence have previously suggested that Rho/Rac GTPases and the JNK cascade are required for ommatidial polarity decisions and, in particular, the R3/R4 fate decision. These include the following: overexpression of Fz or Dsh in the eye gives a polarity phenotype that is dominantly suppressed by RhoA, bsk, hep, and Djun; RhoA clones or expression of dominant-active/negative RhoA or Rac1 gives ommatidial polarity phenotypes; overexpression of dominant-active/negative JNK pathway components and human Jun elicits ommatidial polarity defects, and expression of a Dl enhancer trap is altered by overexpression of either fz or dsh or by activated human Jun, Hep, RhoA, or Rac1. These observations led to the hypothesis that higher levels of Fz/Dsh signaling in R3 result in higher activation of Dl transcription in R3 via a Rho GTPase/JNK cascade, biasing the N/Dl feedback loop to produce high N in R4 (Strutt, 2002).

Taken together, the phenotypic evidence from loss-of-function studies does not support a primary role for Rho GTPases/JNK cascades in the R3/R4 fate decision. But the weight of genetic evidence does support a secondary role for some of the proposed pathway components, possibly in the augmentation of polarity decisions driven largely by asymmetric localization of polarity proteins and direct repression of N activity. In addition, the observation that RhoA mutations result largely in defects in ommatidial rotation supports the hypothesis that RhoA acts downstream of the planar polarity genes in regulating this aspect of ommatidial polarity (Strutt, 2002).

Lethal giant larvae acts together with Numb in Notch inhibition and cell fate specification in the Drosophila adult sensory organ precursor lineage

The tumor suppressor genes lethal giant larvae (lgl) and discs large (dlg) act together to maintain the apical basal polarity of epithelial cells in the Drosophila embryo. Neuroblasts that delaminate from the embryonic epithelium require lgl to promote formation of a basal Numb and Prospero crescent, which will be asymmetrically segregated to the basal daughter cell upon division to specify cell fate. Sensory organ precursors (SOPs) also segregate Numb asymmetrically at cell division. Numb functions to inhibit Notch signaling and to specify the fates of progenies of the SOP that constitute the cellular components of the adult sensory organ. In contrast to the embryonic neuroblast, lgl is not required for asymmetric localization of Numb in the dividing SOP. Nevertheless, mosaic analysis reveals that lgl is required for cell fate specification within the SOP lineage; SOPs lacking Lgl fail to specify internal neurons and glia. Epistasis studies suggest that Lgl acts to inhibit Notch signaling by functioning downstream or in parallel with Numb. These findings uncover a previously unknown function of Lgl in the inhibition of Notch and reveal different modes of action by which Lgl can influence cell fate in the neuroblast and SOP lineages (Justice, 2003).

The discovery that lgl function is required to specify cell fate within the SOP lineage, but does not affect asymmetric segregation of Numb, suggests that Lgl function is distinct from Dlg function in the SOP. Lgl function is most likely required after polarization of the SOP and somehow contributes to the selective inhibition of Notch activity that specifies the fate of the pIIb cell. How might Lgl fulfill this function? Lgl is a WD repeat-containing protein conserved in eukaryotes ranging from yeast to man. Similar to many other WD repeat-containing proteins, Lgl likely interacts with multiple partners in a dynamic manner. It binds type II myosins and t-SNAREs on the plasma membrane and is known to be involved in exocytosis in yeast and Drosophila by presumably targeting vesicles to the plasma membrane and thereby inserting membrane proteins at specific zones along the apical-basal axis of epithelial cells and releasing extracellular signaling molecules such as Dpp. The requirement for Lgl function, however, is not restricted to membrane proteins and secreted proteins that require vesicular transport. For example, formation of the basal crescent in neuroblasts involves cytoplasmic and cortical movements of globular proteins, such as Numb, Pon, Prospero, and Miranda, that attach to the cytoplasmic side of the membrane via lipid modifications or association with membrane proteins. One plausible scenario for the role of Lgl in mediating basal Numb crescent formation in neuroblasts is that Lgl and motor proteins form a complex that mediates basal transport of determinants. Such Lgl-containing adaptor complexes in the SOP must differ from those in embryonic neuroblasts under this scenario, given that anterior Numb crescent formation in the SOP is independent of Lgl (Justice, 2003).

A scenario is imagined in which Lgl is required to deliver components of the machinery required for Numb-mediated inhibition of Notch cannot be excluded. Alternatively, Lgl could directly participate in such a mechanism and could perhaps target endocytic vesicles containing Numb and Notch to the lysosome for degradation. A direct role for Lgl in the Notch pathway is supported by studies suggesting that vesicle trafficking of Notch and Delta plays a critical role during Notch pathway signaling. Lgl might bring Notch inhibitors to the plasma membrane or traffic endocytic vesicles in an inhibitory mechanism with Numb and alpha-Adaptin that specifies cell fates in the SOP lineage (Justice, 2003).

Lgl regulates Notch signaling via endocytosis, independently of the apical aPKC-Par6-Baz polarity complex

The Drosophila melanogaster junctional neoplastic tumor suppressor, Lethal-2-giant larvae (Lgl), is a regulator of apicobasal cell polarity and tissue growth. Previous studies have shown in the developing Drosophila eye epithelium that, without affecting cell polarity, depletion of Lgl results in ectopic cell proliferation and blockage of developmental cell death due to deregulation of the Hippo signaling pathway. This study shows that Notch signaling is increased in lgl-depleted eye tissue, independently of Lgl's function in apicobasal cell polarity. The upregulation of Notch signaling is ligand dependent and correlates with accumulation of cleaved Notch. Concomitant with higher cleaved Notch levels in lgl- tissue, early endosomes (Avalanche [Avl+]), recycling endosomes (Rab11+), early multivesicular bodies (Hrs+), and acidified vesicles, but not late endosomal markers (Car+ and Rab7+), accumulate. Colocalization studies revealed that Lgl associates with early to late endosomes and lysosomes. Upregulation of Notch signaling in lgl- tissue requires dynamin- and Rab5-mediated endocytosis and vesicle acidification but is independent of Hrs/Stam or Rab11 activity. Furthermore, Lgl regulates Notch signaling independently of the aPKC-Par6-Baz apical polarity complex. Altogether, these data show that Lgl regulates endocytosis to restrict vesicle acidification and prevent ectopic ligand-dependent Notch signaling. This Lgl function is independent of the aPKC-Par6-Baz polarity complex and uncovers a novel attenuation mechanism of ligand-activated Notch signaling during Drosophila eye development (Parsons, 2014).

This study demonstrates a novel function for the cell polarity regulator lgl in regulation of endocytosis and Notch signaling. In the developing Drosophila eye epithelium that (1) Notch targets, E(spl)m8, CycA, and Rst, are upregulated in lgl- tissue; (2) Notch upregulation contributes to the lgl- mosaic adult eye phenotype; (3) Notch upregulation in lgl- clones is ligand dependent and requires endocytosis; (4) Lgl colocalizes with intracellular Notch and endocytic markers; (5) lgl- tissue accumulates Avl+ EEs, Hrs+MVBs, Rab11+ REs, endocytic compartments, and acidified vesicles, but not LE markers, Rab7, and Car; (6) Notch upregulation in lgl- clones is independent of the ESCRT-0 complex (Hrs/Stam) or Rab11, but it requires Rab5 function and acidification of endosomes; and (7) Notch upregulation in lgl- clones is independent of aPKC-Baz-Par6. Altogether, these data reveal a novel role for Lgl in attenuating ligand-activated Notch signaling via restricting the acidification of endocytic compartments, independently of its role in regulating the aPKC-Baz-Par6 cell polarity complex (Parsons, 2014).

The data have revealed that increased vesicle acidification in lgl- tissue is responsible for elevated Notch signaling. Because S3 cleavage (by γ-secretase) of Notch extracellular domain (Next) to form the intracellular domain (Nicd) depends on acidification of endosome, this suggests that increased vesicle acidification in lgl- tissue leads to aberrant γ-secretase activity and cleavage of Next to Nicd, resulting in upregulation of Notch signaling (Parsons, 2014).

The precise endocytic compartment in which the Notch receptor undergoes γ-secretase-mediated S3 proteolytic processing is controversial. In Drosophila epithelial tissues, V-ATPase function is implicated in Notch activation in the EEs or the MVBs; however, whether this is ligand dependent or independent is unclear. In contrast, ligand-independent generation of Nicd in the LE and/or lysosome compartment has been described. Because increased Notch signaling in lgl- tissue depends on Rab5 EE activity, but Rab7 and Car LE compartments were not perturbed in lgl- tissue, a model is favored in which Lgl restricts vesicle acidification and activation of Notch signaling in the EE and/or early MVB compartments (Parsons, 2014).

It is speculated that Lgl regulates Notch signaling by two possible mechanisms: (1) via direct regulation of vesicle acidification or (2) via regulation of endosomal maturation, which indirectly affects vesicle acidification. In the first model, Lgl might inhibit V-ATPase activity by regulating levels and/or subunit composition or the association and/or dissociation of the V-ATPase complex. In the second model, Lgl might regulate endosomal maturation, and alterations in this process subsequently lead to accumulation of acidic vesicles and ectopic Notch activation. The data showing that lgl- tissue accumulates EEs (Avl+), REs (Rab11+), and early MVBs (Hrs+), but not LEs (Rab7+ or Car+), suggest that Lgl regulates a specific step in endosome maturation after early MVB formation. Further studies are required to determine whether Lgl controls vesicle acidification by affecting the V-ATPase or endosome maturation to regulate Notch signaling (Parsons, 2014).

Previous work has revealed that alterations in components of endocytic compartments, such as Rab5 overexpression or mutation of tsg101/ept or vps25, disrupt epithelial cell polarity and upregulate Notch signaling. However, in these cases, it is unclear whether perturbation of endocytosis alters Notch signaling via cell polarity disruption or whether changes in endocytic compartments directly impact on Notch. This study shows that without cell polarity loss, lgl- tissue displays altered endocytic compartments and upregulates Notch signaling, indicating that changes in endocytosis alone are sufficient to upregulate Notch pathway activity. Moreover, this study shows that reducing aPKC-Baz-Par6 complex activity does not rescue Notch pathway upregulation in lgl- tissue, revealing that Lgl's roles in regulating cell polarity and endocytosis are separable. Interestingly, the Crb polarity protein also has separable roles in the regulation of cell polarity and Notch signaling and endocytosis, via different mechanisms to Lgl (Parsons, 2014).

The discovery that Lgl depletion increases Notch activation without cell polarity loss has implications for tumorigenesis. In the developing eye epithelium, increased Notch signaling results in upregulation of the cell cycle regulator, CycA, and the cell survival regulator, Rst, which in lgl- tissue, is expected to contribute to increased cell proliferation and survival, concomitant with impaired Hippo signaling. Because elevated Notch signaling is associated with various human cancers, the finding that Lgl regulates Notch signaling warrants investigation of whether elevated Notch signaling in human cancer is associated with Lgl depletion. Notably, Lgl1 knockout in the mouse brain induces hyperproliferation and decreased differentiation, associated with increased Notch signaling, and mutation of zebrafish Lgl disrupts retinal neurogenesis, dependent on increased Notch signaling. Moreover, the finding that Lgl plays a novel role in regulating endosomal acidification and the striking suppressive effect of chloroquine on the adult lgl- mosaic phenotype reveal the importance of acidification in tumor growth, perhaps by also modulating Hippo signaling. The data, together with evidence that many cancers show higher acidity due to increased V-ATPase activity, which contributes to tumorigenesis, posit the question of whether Lgl dysfunction might contribute to acidification defects in human cancer (Parsons, 2014).

Evidence that stem cells reside in the adult Drosophila midgut epithelium: Involvement of Notch in regulating cell proliferation

Adult stem cells maintain organ systems throughout the course of life and facilitate repair after injury or disease. A fundamental property of stem and progenitor cell division is the capacity to retain a proliferative state or generate differentiated daughter cells; however, little is currently known about signals that regulate the balance between these processes. A proliferating cellular compartment has been characterized in the adult Drosophila midgut. Using genetic mosaic analysis it has been demonstrated that differentiated cells in the epithelium arise from a common lineage. Furthermore, reduction of Notch signalling leads to an increase in the number of midgut progenitor cells, whereas activation of the Notch pathway leads to a decrease in proliferation. Thus, the midgut progenitor's default state is proliferation, which is inhibited through the Notch signalling pathway. The ability to identify, manipulate and genetically trace cell lineages in the midgut should lead to the discovery of additional genes that regulate stem and progenitor cell biology in the gastrointestinal tract (Micchelli, 2006).

The adult Drosophila midgut can be identified on the basis of two anatomical landmarks along the anterior-posterior axis of the gastrointestinal tract: the cardia and pylorus. The inner surface of the midgut is lined with a layer of cells that project into the gut lumen. These cells exhibit apical-basal polarity; staining for F-actin reveals the presence of a distinct striated border on their lumenal surface. This observation is consistent with the suggestion that the midgut is lined by a cellular epithelium (Micchelli, 2006).

Wild-type midguts were stained with 4,6-diamidino-2-phenylindole (DAPI) to reveal the distribution of cell nuclei within the tissue. Nuclei of the midgut display a distinct distribution and fall into two main categories. The most prominent cells lining the midgut contain large oval nuclei that stain strongly with DAPI. These cells exhibit a region of the nucleus that does not stain with DAPI, giving the nucleus a hollow appearance. This unstained region may correspond to the large nucleolus characteristic of differentiated cells. A second population of cells containing small nuclei can be detected at a basal position within the tissue. The small nuclei are distant from the gut lumen and often lie in close apposition to the two layers of overlying visceral muscle that surround the gut. On the basis of nuclear size, position and morphology two general populations of midgut cells can, therefore, be distinguished (Micchelli, 2006).

Previous studies in Drosophila have led to conflicting views over the existence of cell proliferation in the adult gastrointestinal tract. Early reports suggested that somatic stem cells were present in the adult because of morphological similarity to certain larval cells and by analogy to different insect species. In contrast, 3H-thymidine labelling experiments detected DNA synthesis in the adult Drosophila midgut, but no mitotic figures were observed in a large sample analysed. On the basis of these observations, it was concluded that no somatic cell division occurs during the lifetime of Drosophila. To distinguish between these possibilities, a series of three independent assays was used to test whether cell proliferation can be detected in the adult midgut. In the first assay genetically marked wild-type cell lineages were used to identify dividing cells. The production of marked clones after mitotic recombination depends upon subsequent cell division and is, therefore, a direct means to assay proliferation. In these experiments, wild-type lineages were positively marked in adult flies using the MARCM system. Mitotic recombination was induced by heat shock and green fluorescent protein (GFP)-marked clones could be detected in the midgut. Similar results were obtained when adults were heat shocked up to 10 days after eclosion. This suggests that the ability to generate clones is not transient, and probably persists throughout the entire life of the animal (Micchelli, 2006).

Under the experimental conditions used, the MARCM system produced some background GFP signal that could be detected in control animals. To quantify the background signal, the number of GFP-labelled cells was compared in control and experimental animals. A greater than sixfold increase in the number of GFP-labelled cells was detected after heat shock. A second independent clone marking method was used that did not rely on either Gal4 or Gal80. In these experiments, clones were marked by the loss of a ubiquitously expressed GFP and similar results were observed. It is concluded that a population of actively dividing somatic cells is present in the adult Drosophila midgut (Micchelli, 2006).

To extend these findings, 5-bromodeoxyuridine (BrdU) incorporation studies were constructed. Both large and small BrdU-labelled midgut cells were detected. Large nuclei adjacent to each other can be differentially labelled, suggesting asynchrony in the timing or extent of DNA synthesis over the course of the labelling period. This is consistent with the notion that the large nuclei are endoreplicating. However, both endoreplication and the canonical cell cycle require new DNA synthesis. To distinguish endoreplicating from dividing cells in the midgut the tissue was stained with an antibody raised against phospho-histone H3. Careful examination revealed that very low levels of phospho-histone H3 staining could be detected in all cells. However, double staining with DAPI revealed that elevated levels of phospho-histone H3 indicative of mitosis could be detected only among the population of cells with small nuclei. Thus, cells in the midgut seem to have two distinct cell cycles; whereas both large and small nuclei undergo DNA synthesis, only the cells with small nuclei undergo cell division (Micchelli, 2006).

In order to characterize further the small cell population, an expression screen was conducted to identify cell-specific molecular markers. Three markers expressed in small cells were identified: escargot (esg), a transcription factor that belongs to the conserved Snail/Slug family; prospero (pros), a conserved homodomain transcription factor, and Su(H)GBE-lacZ, a transcriptional reporter of the Notch signalling. Simultaneous detection of esg expression (esg-Gal4, UAS-GFP), anti-Pros, Su(H)GBE-lacZ expression and DAPI has demonstrated that small cells can be subdivided into the following classes on the basis of differential gene expression: esg-positive (esg+), pros-positive (pros+), esg-negative pros-negative (esg- pros-), esg-positive Su(H)GBE-lacZ-positive [esg+ Su(H)GBE-lacZ+] and esg-positive Su(H)GBE-lacZ-negative [esg+ Su(H)GBE-lacZ-]. esg+ and pros+ expression define distinct cell populations, whereas Su(H)GBE-lacZ expression subdivides the esg+ class into esg+ Su(H)GBE-lacZ+ and esg+ Su(H)GBE-lacZ- subpopulations. Quantification reveals that each cell type is present in the midgut in different proportions. The ability to distinguish different cell types using molecular markers enabled determination of the cell lineage relationships in this tissue. If the large and small nuclei are lineally distinct then marked clones should be restricted to one or the other cell type. However, if a common stem cell progenitor exists in the adult midgut, then marked lineages should contain both large and small nuclei within a clone. To distinguish between these possibilities positively marked MARCM clones were generated and nuclei were labeled using DAPI. Lineage analysis shows that marked clones generated in the adult contain both large and small nuclei. In addition, both esg expression and anti-Pros-labelled cells could be detected within the clones. These lineage-tracing experiments suggest that a stem cell progenitor exists and is sufficient to generate the distinct cell types of the adult midgut. This cell is referred to as the adult intestinal stem cell (ISC) (Micchelli, 2006).

esg expression in diploid cells has been shown to be necessary for the maintenance of diploidy. In addition, the distribution of esg messenger RNA has been used as a marker for male germline stem cells. Together, these observations raise the hypothesis that esg expression may also mark a population of progenitors in the midgut. It was therefore asked whether esg expression correlates with markers of cell proliferation. Simultaneous staining with anti-BrdU and DAPI reveals that esg-expressing cells are among the population of cells that are also positively labelled by BrdU. To ask whether esg-expressing cells also undergo cell division, the midgut was double stained to detect both esg expression and phospho-histone H3. High levels of phospho-histone H3 can be detected specifically in esg-expressing cells. These results demonstrate that esg expression marks a population of proliferating progenitor cells in the midgut (Micchelli, 2006).

However, the esg+ cell population can be divided on the basis of Su(H)GBE-lacZ expression. To distinguish functionally the two esg+ populations, the consequences of altering Notch signalling in the adult midgut were examined. The effect of globally reducing Notch signalling was tested using the conditional Notch temperature-sensitive (Nts) mutant. Nts flies were first crossed to an allelic series that included N55e11, N264.47, Nts1 and Nnd.1. The strongest loss of function combinations (Nts/N55e11 and Nts/N264.47) failed to generate viable adult flies even at the permissive temperature, often dying as pharate adults. Nts/Nts flies produced viable adults at the permissive temperature with midguts similar to wild type. Nts/Nts flies shifted to the non-permissive temperature led to a mild increase in the number of small cells. The weakest allelic combination, Nts/Nnd.1, also produced viable adults at the permissive temperature but showed no detectable phenotype when shifted to the non-permissive temperature (Micchelli, 2006).

The requirement of N only in esg+ progenitor cells was tested. To obtain both spatial and temporal control over transgene expression in esg-expressing cells, the temperature-sensitive Gal80 inhibitor, Gal80ts was combined with the esg-Gal4 transcriptional activator. To verify that the Gal80ts transgene functions in the midgut, the temporal and spatial induction of a UAS-GFP transgene was characterized. Adult esg-Gal4,UAS-GFP, tub-Gal80ts flies grown at the permissive temperature showed no detectable GFP expression in their midguts In contrast, when these flies were shifted to the non-permissive temperature they showed high levels of GFP expression that were detectable after 1 day and maximal by 2 days (Micchelli, 2006).

The requirement of Notch was then tested in esg+ cells using a UAS-NRNAi transgene, to reduce Notch signalling. In control experiments, UAS-NRNAi; esg-Gal4,UAS-GFP, tub-Gal80ts flies grown at the permissive temperature appear to have wild-type midguts and show no detectable GFP expression, suggesting that under these conditions UAS transgenes are efficiently suppressed. In contrast, UAS-NRNAi; esg-Gal4,UAS-GFP, tub-Gal80ts flies shifted to the non-permissive temperature show an increase in the number of small cells (19 out of 20 midguts). Notably, the presence of esg-Gal4, UAS-GFP in this experiment enabled a determination that the increased number of small cells were also esg+. When these guts were co-stained with anti-Pros antibody ectopic small cells were observed that also expressed pros, and these cells were often associated with lower levels of esg expression. Taken together these experiments suggest that Notch signalling in esg+ cells is necessary to restrict proliferation (Micchelli, 2006).

The effect of Notch activation was tested in esg+ cells using Nintra, a constitutively active form of Notch. In control experiments, esg-Gal4,UAS-GFP, tub-Gal80ts; UAS-Nintra flies grown at the permissive temperature appear to have wild-type midguts and show no detectable GFP expression. In contrast, esg-Gal4,UAS-GFP, tub-Gal80ts; UAS-Nintra flies shifted to the non-permissive temperature showed a decrease in phospho-histone H3 staining compared to controls that were not shifted. In addition, although some esg+ cells appear to be wild type, a region-specific decrease was observed in the levels of esg expression and a concomitant increase in nuclear size similar to that of midgut epithelial cells. These observations demonstrate that Notch activation is sufficient to limit proliferation of esg+ cells and suggests that Notch may also be sufficient to promote early steps of epithelial cell differentiation (Micchelli, 2006).

This characterization of the adult Drosophila midgut suggests that a population of adult stem cells resides within this tissue. This analysis of the Notch signalling pathway in esg+ cells suggests that esg+ Su(H)GBE-lacZ- cells mark a population of dividing progenitors and that Notch is necessary and sufficient to regulate proliferation. A model is proposed in which esg+ Su(H)GBE-lacZ- progenitors generate at least two different types of daughter cells depending on the level of Notch activation. Under conditions of reduced Notch function an expansion of both esg+ progenitor cells and pros+ cells is observed. These observations suggest that esg+ cells give rise to pros+ cells in a Notch-independent manner. Under conditions of Notch activation a decrease is observed in the proliferation and promotion of epithelial cell fate differentiation, while the number of pros+ cells remains unaffected (Micchelli, 2006).

Several lines of evidence suggest that pros+ cells correspond to gut enteroendocrine cells. Previous studies show that prox1, the vertebrate pros homologue, is associated with post-mitotic cells and early steps of differentiation in the central nervous system. Furthermore, in Drosophila, pros is thought to be a pan-neural selector gene that is both necessary and sufficient to terminate cell proliferation. Finally, although vertebrate enteroendocrine cells arise from endodermal origins they are known to express neural-specific markers. Therefore, pros+ cells probably define a population of enteroendocrine cells in the midgut (Micchelli, 2006).

Studies of stem cell compartments in Drosophila have led to the characterization of two types of progenitor cells in the germ line. The first is referred to as the germline stem cell and is sufficient to give rise to the respective cells of either the male or female germ line. The second type of progenitor cell described is called the cystoblast in female germ line and gonialblast in the male germ line. Although the cystoblast and gonialblast both have the capacity to generate the differentiated cells of their respective tissues, they are thought to be more restricted in their fate than the germline stem cells. On this basis, it is suggested that an analogous progenitor may also exist in the adult Drosophila midgut; this cell is referred to as the enteroblast (EB). The population of esg+ Su(H)GBE-lacZ- progenitor cells, which has been described, displays characteristics of both the ISC and the EB; therefore, additional experiments will be necessary to distinguish unambiguously these alternatives (Micchelli, 2006).

Distinct levels of Notch activity for commitment and terminal differentiation of stem cells in the adult fly intestine

Tight regulation of self-renewal and differentiation of adult stem cells ensures that tissues are properly maintained. In the Drosophila intestine, both commitment, i.e. exit from self-renewal, and terminal differentiation are controlled by Notch signaling. This study shows that distinct requirements for Notch activity exist: commitment requires high Notch activity, whereas terminal differentiation can occur with lower Notch activity. The gene GDP-mannose 4,6-dehydratase (Gmd), a modulator of Notch signaling, was identified as being required for commitment but dispensable for terminal differentiation. Gmd loss results in aberrant, self-renewing stem cell divisions that generate extra ISC-like cells defective in Notch reporter activation, as well as wild-type-like cell divisions that produced properly terminally differentiated cells. Lowering Notch signaling using additional genetic means, further evidence was provided that commitment has a higher Notch signaling requirement than terminal differentiation. This work suggests that a commitment requirement for high-level Notch activity safegards the stem cells from loss through differentiation, revealing a novel role for the importance of Notch signaling levels in this system (Perdigoto, 2011).

These results show that the self-renewal/commitment decision has a specific requirement for high Notch signaling levels. Using loss of Gmd activity, loss of Ofut1 catalytic activity, mutants of rumi, which encodes a O-glucosyltransferase that adds glucose to EGF repeats, at 21 and 22°C, and using RNAi against the Notch receptor at 22°C, self-renewal/commitment could be uncoupled from the enteroendocrine cell/enterocyte cell (ee/EC) terminal differentiation decision. It is proposed that after ISC division, one of the two ISC daughter cells first receives a high, GDP-fucose-dependent, Notch commitment signal to exit the self-renewal program and that terminal differentiation into ee or EC can occur via lower Notch activity. It is hypothesized that a subset of cells lacking Gmd could activate Notch signaling to some extent but not to high enough levels to commit. These cells would therefore express detectable Su(H)GBE-lacZ reporter though fail to commit, and would re-enter self-renewal and cell division. It has also been proposed that the EC fate, but not the ee fate, is dependent on Notch signaling. The current data could also be consistent with this model, although the data suggest that precise control of commitment, requiring high-level Notch signaling, is at least indirectly important for specification of both terminal differentiated cell types, as failure to commit produces supernumerary multipotent ISCs (Perdigoto, 2011).

The finding that the reliable commitment of one ISC daughter at each cell division relies on high-level Notch receptor activation is somewhat surprising in light of previous work suggesting that different levels of Notch signaling dictate the terminal fate of EBs. How then can a level-based Notch signaling be used to instruct the terminal differentiation status of the same cell that has received a high level of Notch signaling to exit self-renewal? It is speculated that regulation of Delta ligand levels in the ISC after the commitment decision may then control further activation of Notch transcriptional output, thereby promoting terminal differentiation choice. This would be consistent with both current findings and earlier finding, in which a correlation of Delta levels with recent daughter terminal cell fate was observed (Perdigoto, 2011).

The Drosophila intestine represents an important system in which to understand the interplay between cell fate decisions and tissue homeostasis. It is currently unclear when and how the ee and EC terminal differentiation fate choices are made. There are three possibilities (Perdigoto, 2011).

  1. The state of the ISC prior to its cell division (such as expression levels of Delta or another factor) could dictate the outcome of the daughter terminal differentiation decision occurring after division. Thus, the choice between ee and EC fate of the daughter cell terminal has already been made at the time of cell division.

  2. Alternatively, the state of the ISC may be plastic and have the capacity to be altered after cell division (perhaps via levels of Delta) to impact the terminal fate of its neighboring sister cells. In this model, the terminal differentiation decision is not pre-made, but can be modulated in the ISC after cell division.

  3. Finally, it is possible that the presumptive EB is the recipient of signals both from the ISC (like Delta) but also parallel input that could communicate the needs of the tissue necessary to decide between ee and EC fates.

The findings do not fit well with a model in which levels of Delta ligand in the ISC prior to division would dictate daughter terminal cell fate. Indeed, it is not clear how a low level of Delta ligand (promoting ee cell specification) would provide high enough levels of Notch activation for commitment to occur. A model postulating that the ISC is plastic in state and could be modified to accommodate the needs of the tissue and dictate the terminal differentiation of its sister cell would best fit the data. Importantly, the current findings that the self-renewal/commitment step can be affected without impacting the terminal differentiation process or the relative abundance of the ee and EC cell types suggest that commitment does not simply arise from activation of the terminal differentiation process, at least in the genetic contexts of the current experiments (Perdigoto, 2011).

It is noted that the current findings raise the possibility that stem cell numbers in the intestine may be controlled through modulation of Notch signaling levels, which could affect self-renewal without affecting terminal differentiation. Understanding how each step of cell fate is acquired in the relatively simple Drosophila ISC system may provide significant insight in to more complicated mammalian lineages that may use similar strategies in which stem cells can choose between three or more cell fates (Perdigoto, 2011).

Interestingly, the 'mistakes' in commitment seen either in the Gmd mutant or in the rumi mutant at 21 and 22°C did not occur at each and every ISC division, but the EB cells, when produced, could appropriately terminally differentiate. What might be the source of this variability and is it inherent to the commitment decision? One possibility could be stochastic cell-to-cell variability (such as in gene transcription, chromosomal domain replication, protein expression, signaling network and/or cell cycle kinetics). Thus, in a stochastic manner, individual daughter cells with weakened Notch pathway activity maybe able to reach levels of pathway output required for commitment or not depending other components of the Notch signaling network. Could the commitment decision be inherently more susceptible variation to signaling levels? One possibility is that the timing of the commitment decision is made during a shorter time window, perhaps in the G1 phase of the cell cycle, than that of the terminal differentiation decision, necessitating signaling modulators to boost signaling that are not required for terminal fate decisions with a longer time window (Perdigoto, 2011).

A requirement of high-level signaling for commitment choices could represent a strategy whereby stem cells must first cross a high signaling barrier for commitment to occur prior to the terminal differentiation process and could provide insurance that the stem cell pool is not lost. In some instances, it may be preferable to gamble having too many stem cells in a tissue, possibly putting the tissue in jeopardy for development of a cancer-like state, rather than risk the depletion of tissue specific stem cells that may have more immediate detrimental consequences on the tissue. Consequently, commitment decisions may need specific modifiers of signaling networks that may not be required for generalized function of these signaling networks. It will be important to determine whether this model is a more general feature of stem-daughter cell decisions (Perdigoto, 2011).

Drosophila midgut homeostasis involves neutral competition between symmetrically dividing intestinal stem cells

The Drosophila adult posterior midgut has been identified as a powerful system in which to study mechanisms that control intestinal maintenance, in normal conditions as well as during injury or infection. Early work on this system has established a model of tissue turnover based on the asymmetric division of intestinal stem cells (ISC). From the quantitative analysis of clonal fate data, this study shows that tissue turnover involves the neutral competition of symmetrically dividing stem cells, mediated by Dl/N signaling. This competition leads to stem-cell loss and replacement, resulting in neutral drift dynamics of the clonal population. As well as providing new insight into the mechanisms regulating tissue self-renewal, these findings establish intriguing parallels with the mammalian system, and confirm Drosophila as a useful model for studying adult intestinal maintenance (de Navascues, 2012).

This study has analysed long- and short-term lineage progression by size, survival and composition, and shows that Drosophila midgut is maintained by population asymmetry. This contrasts with a general notion in the field that homeostasis is based on the fate asymmetry of the ISC offpsring. The results reveal that ISCs divide symmetrically in response to the differentiation and subsequent loss of a neighbouring ISC (or vice versa), which leads to neutral drift of the clonal population. It is estimated that, in a context of fast turnover (two or three divisions per day), 2 in 10 divisions result in stem-cell loss and replacement. The results further indicate that the fate of the ISC daughters might not be specified on division, but rather resolved through competition between proximate cells following division (de Navascues, 2012).

The observation of neutral competition calls for the elucidation of its underlying molecular mechanism. By the nature of the process of neutral competition in this system, the associated mechanism must play a fundamental role in ISC self-renewal and enteroblast (EB) commitment and be able to implement, non-cell autonomously, the stochastic resolution of binary fate decisions. Dl-N signalling fulfils both requirements: numerous experiments underpin its central role in the choice of commitment versus self-renewal in the midgut, and studies of its function in neurogenesis have established an intrinsic ability to resolve stochastically binary choices of cell fate through the process of 'lateral inhibition', which involves reciprocal signalling between equivalent cells (de Navascues, 2012).

The model of population asymmetry involves neutral competition between proximate cells (therefore based on extrinsic signals), which implies that ISC daughters are intrinsically equivalent at birth, and that fate is resolved after ISC division. Therefore, the ISC daughters are functionally equivalent and a good substrate for lateral inhibition, This is compatible with the observations that ISC daughter cells express N and they segregate Dl symmetrically. Furthermore, evidence is provided that this situation leads to mutual signalling. The excess of ISCs found in null and hypomorphic conditions for N could be interpreted, in the framework of a unidirectional Dl signal from the ISC towards the EB, as a failure to implement the EB fate and a lapse into the 'default' ISC fate. However, this interpretation cannot account for the increased number of cells committing to EB fate in +/l(1)NB and +/NMCD1 midguts. These alleles exhibit Dl-dependent increased N signalling which, in a scenario of unidirectional signalling, should not affect the balance of EB commitment. Rather, their phenotype suggests that both ISC daughters can receive Dl signal and reach the threshold of N activity for commitment. This is further highlighted by the parallel between the effects of these alleles on the ISC/EB ratio and on the development of peripheral nervous system (PNS) of Drosophila, a classical model for lateral inhibition. In the PNS, a reduction of N activity, as in +/N55e11, leads to neurogenic phenotypes with supernumerary sensory bristles, whereas excess of N activity, as in +l(1)NB and +/NMCD1, results in antineurogenic phenotypes, with reduced number of bristles and more cells adopting the epidermal fate. It is noteworthy that the molecular machinery that participates in Dl/N-mediated lateral inhibition in other contexts is also involved in ISC/EB fate decision. With these elements, it is proposed that ISCs divide symmetrically, and the fate of the progeny is resolved through lateral inhibition mediated by Dl/N signalling (de Navascues, 2012).

Lateral inhibition thus provides a straightforward way of implementing neutral competition. In many cases, Dl/N interaction will be restricted to sibling cells, the progeny of a single ISC division, and these cases will resolve into an ISC/EB asymmetric pair. However, if in the course of tissue turnover the division of two ISCs occurred in close proximity, non-sibling cells could interact via Dl/N and engage in competition for the ISC fate through lateral inhibition. This in turn may lead to sibling cells adopting identical fates and therefore in ISC loss and replacement. Such behaviour would translate precisely to the coupled events of loss and replacement implicit in the model, with the value of 2r weighting, combined, the chance of this contact and of its resolution into ISC loss and replacement. In this regard, the rich variety of esg+ cell nest composition in EB and ISC cells, as well as the frequent proximity of ISC pairs (19% of nearest pairs), suggests that such encounters are possible in space. This may require nests to drift in position, but it is significant that, unlike other Drosophila adult stem cells, ISCs are not associated with niches having hub-like anatomical properties (de Navascues, 2012).

Although the current observations can, in principle, be explained solely as a result of lateral inhibition, other mechanisms cannot be ruled out. In particular, the tissue could allow for a combination of either symmetric or intrinsically asymmetric divisions such that part of the ISC divisions that result in asymmetric fate could derive from either neutral competition or an intrinsic regulatory process. However, to conform with the observation that ISCs are equipotent, the stem-cell progeny of an intrinsically determined asymmetric cell division will have the same chance for loss and replacement in subsequent divisions as any other ISC. In other words, when facing their next division, all ISCs irrespective of their lineage history, would have to decide, stochastically, whether to undergo intrinsically asymmetric division, and, if they do not, the fate outcome of the division would be resolved again, stochastically, by extrinsically driven neutral competition. It is difficult to conceive of a scenario for the molecular regulation of such a system (de Navascues, 2012).

Although the model provides an excellent fit to the early time course, the departure of the model at day 16 is significant. Although the clone size distribution conforms closely with the predicted scaling form, consistent with the same underlying pattern of ISC fate, the overall average size of the surviving clones is smaller than predicted by a simple extrapolation of the fits to the earlier time point data with the same ISC loss/replacement rate. The departure of theory and experiment may reflect a breakdown of homeostasis due to ageing. In particular, at this point of the clone chase the flies are 21-23 days old, an age at which ageing-related non-homeostatic phenotypes can be detected in the midgut. Indeed, such behaviour might be explained by a shift towards uncompensated loss of terminally differentiated cells, consistent with the fact that the clone density continues to fall, and in a manner consistent with theory. Alternatively, it is also possible that the heat shock, which does not produce damage leading to detectable alteration of the tissue size or cell density, instead triggers the acceleration of tissue turnover, an effect that would last at least 8 days, but less than 16. This effect could be similar, but milder, to that observed in the recently described Drosophila gastric adult stem cells, which show a sharp activation of homeostatic turnover in response to heat shock. This view is supported by the observation that the mitotic index in the posterior midgut is, after heat-based clonal induction, higher than in untreated organs (de Navascues, 2012).

There are similarities between the Drosophila midgut and mammalian intestine at the levels of cell biology and genetics. This study adds a new parallel from the perspective of their strategy for homeostatic maintenance. In humans, studies of methylation patterns point at stem-cell loss and replacement in the intestinal crypt and in the mouse, using an approach similar to that employed in this study, recent studies have shown that the intestinal crypt is maintained by an equipotent Lgr5+ stem-cell population in which the loss of cells from the stem-cell compartment is compensated by the symmetric multiplication of neighbours. Further evidence suggests that stem-cell competence in the small intestine is ensured by proximity to Paneth cells, which aggregate throughout the crypt base. As tissue is turned over, Lgr5+ cells undergo neutral competition resulting in a progression towards crypt monoclonality. At longer timescales, crypts undergo fission, leading to a further 'coarsening' of the clonal population. It is speculated that in Drosophila, the esg+ nests fulfil a function analogous to intestinal crypts, playing host to a much smaller equipotent cell population, and undergoing fission with a far greater frequency. However, in contrast with intestinal crypt, it appears that ISC competence in Drosophila does not require a separate niche-supporting cell (de Navascues, 2012).

In summary, these studies show that, in Drosophila, adult midgut homeostasis follows a pattern of population asymmetry involving an equipotent population of ISCs. ISC division may result in any of all three possible fate outcomes leading to asymmetric fate (ISC/EB), symmetric duplication (two ISCs), or symmetric differentiation (two EBs), the latter two balanced in frequency. These findings point at a mechanism involving lateral inhibition and provide a natural framework to explain regeneration following injury or infection (de Navascues, 2012).

Drosophila adult muscle precursors form a network of interconnected cells and are specified by the rhomboid-triggered EGF pathway

In Drosophila, a population of muscle-committed stem-like cells called adult muscle precursors (AMPs) keeps an undifferentiated and quiescent state during embryonic life. The embryonic AMPs are at the origin of all adult fly muscles and, as is demonstrated in this study, they express repressors of myogenic differentiation and targets of the Notch pathway known to be involved in muscle cell stemness. By targeting GFP to the AMP cell membranes, it was shown that AMPs are tightly associated with the peripheral nervous system and with a subset of differentiated muscles. They send long cellular processes running along the peripheral nerves and, by the end of embryogenesis, form a network of interconnected cells. Based on evidence from laser ablation experiments, the main role of these cellular extensions is to maintain correct spatial positioning of AMPs. To gain insights into mechanisms that lead to AMP cell specification, a gain-of-function screen was performed with a special focus on lateral AMPs expressing the homeobox gene ladybird. The data show that the rhomboid-triggered EGF signalling pathway controls both the specification and the subsequent maintenance of AMP cells. This finding is supported by the identification of EGF-secreting cells in the lateral domain and the EGF-dependent regulatory modules that drive expression of the ladybird gene in lateral AMPs. Taken together, these results reveal an unsuspected capacity of embryonic AMPs to form a cell network, and shed light on the mechanisms governing their specification and maintenance (Figeac, 2010).

In late Drosophila embryos, each abdominal hemisegment features six AMPs at stereotypical positions associated with differentiating muscle fibres. To better characterize these cells, tests were performed to see whether the Notch pathway, which is known to be required for generation of satellite cells from muscle progenitors and for keeping them ready to engage in muscle regeneration, is also active in AMPs. Analysis of a GFP reporter line, E(spl)M6-GFP, described as a read-out of the Notch pathway in Drosophila, revealed that it is co-expressed with Twist in AMPs. Also, transcripts of another Notch target, Him, specifically accumulated in AMPs. By testing several mesodermal cell markers, it was found that, in addition to Twist, two other transcription factors, Zfh1 and Cut, are expressed in all AMPs. Zfh1 expression in embryonic AMPs has been reported previously, whereas cut has been used to reveal a subset of AMPs associated with larval wing and leg imaginal discs. Despite expressing common markers, the AMPs are heterogenous and differ by the expression of muscle identity genes. For example, slouch (S59) and Pox meso are specifically expressed in ventral (V) AMPs whereas ladybird (lb) and Kruppel (Kr) display lateral (L) AMP-specific expression (Figeac, 2010).

To gain insights into AMP cell shapes and their behaviour, an E(spl) M6-GAL4 line was generated that recapitulates M6-GFP expression, and it was used it to drive a membrane-targeted GFP. It has been previously reported that AMPs are associated with the larval peripheral nervous system (PNS) and that in daughterless mutant embryos lacking all the larval sensory system, the final pattern of AMPs is deranged. This study showed that all embryonic AMPs are closely associated with both the PNS and the differentiated muscles, sitting either at the top of muscle fibres [LAMPs and dorsal (D) AMPs] or on their internal face [dorsolateral (DL) AMPs and VAMPs)]. In late embryos, the AMPs form a network of cells displaying irregular shapes and are interconnected by long cellular processes aligning PNS nerves. Connections between the AMPs initially form within the parasegments, but the AMPs very quickly send filopodia posteriorly and make contact with DLAMPs of the adjacent segment, thus interlinking all AMPs. In addition to the interconnected M6+/twi+ AMPs, a population of morphologically distinct M6+/twi- cells of unknown fate, located more internally in central and posterior regions of the abdomen, was identified (Figeac, 2010).

It has been reported that a subset of muscle progenitors divides asymmetrically and gives rise to numb-positive founder cells that undergo differentiation and to Notch-expressing AMPs. Through this pathway, six AMPs are born in each abdominal hemisegment. In contrast to founders, AMPs express the Notch target Holes in muscle (Him) and Zfh1, the Drosophila homolog of ZEB, both of which are able to counteract Mef2-driven myogenic differentiation. Interestingly, another general AMP marker, E(spl)M6, also corresponds to a Notch target, suggesting that Notch signalling could play an evolutionarily conserved role in muscle cell stemness. It operates not only in vertebrate satellite cells but also, as shown in this study, in Drosophila AMPs. Finally, it is reported that, similar to muscle progenitors, the AMPs are heterogenous and express different muscle identity genes, such as lb or slou. This strongly suggests that AMPs acquire a positional identity that makes them competent to form a given type of muscles during adult myogenesis. For example, the lateral AMPs expressing lb are at the origin of all lateral body wall muscles of the adult fly. In support of the specific positional identities of AMPs comes also the analysis of lame duck (lmd) mutant embryos known to be devoid of fusion-competent myoblasts (FCMs). In this mutant context, the number of Twi-positive and Zfh1-positive AMP-like cells is highly increased, while the number of Lbe- and Twi-positive LAMPs committed to the lateral lineage remains unchanged. Thus in the absence of lmd, some presumptive FCMs can adopt the AMP-like fate but they do not carry positional information transmitted by the identity genes such as lb (Figeac, 2010).

Based on the premise that the AMPs correspond to a novel population of transient stem cells, their shapes and behaviour were analyzed in living embryos carrying M6-GAL4 and UAS-GAP-GFP transgenes. Surprisingly it was found that shortly after their specification, the AMPs start to send cellular processes that align along the nerves of the PNS, with the result that, by the end of embryogenesis, all AMPs become linked together. Interestingly, the intersegmental connections are made via an intermediary M6+ twi- cell of unknown fate. In addition to this particular cell, which ensures the intersegmental link between AMPs, the embryos also contained other M6+ twi- non-neural cells of rounded morphology located more internally that were unconnected to the AMP cell network. The origin and identity of these cells remain unknown (Figeac, 2010).

Exploiting the possibility of following AMPs in vivo, test were performed to see how AMPs would behave if their connections were broken. Since the AMPs separated from the network by laser ablation changed shape and lost their normal positions, it is concluded that one important reason for which AMPs form a cell network is to keep precise spatial positioning. Based on the observation that AMPs send long cellular processes along the peripheral nerves, it is probable that nerves serve as a support for extending AMP cell protrusion. This possibility is supported by the abnormal pattern of AMPs observed in daughterless mutant embryos lacking the PNS and in embryos in which the PNS was affected by the Elav-GAL4 driven expression of the inducer of apoptosis, Reaper. PNS nerves might also represent a source of signals for AMPs such as Delta in order to maintain Notch activity. However, analysis of the lateral domain revealed that Delta expression was associated with the segment border muscle (SBM) precursor but not with the PNS neurons, indicating that Notch activity in lateral AMPs is regulated by Delta produced in the SBM rather than in nerves (Figeac, 2010).

Taking advantage from the restricted number of embryonic AMPs and the genetic tools available in Drosophila, a large-scale gain-of-function screen was performed to identify the genes involved in AMP specification. rho and other components of the EGF signalling pathway were found to be crucially required for both specification and maintenance of AMPs. Importantly, as reported by Krejci (2009), several components of EGF signalling are direct targets of Notch in AMPs, thus creating a link between the two signalling pathways. The high number of AMPs in EGFRCA and RAS gain-of-function contexts provides evidence that RAS signalling not only promotes muscle founder specification, but is also crucial for specifying AMPs when induced by EGF signals. Further support for a key role of the EGFR pathway is the identification of cells sending EGF to lateral AMPs and the demonstration of their role in AMP cell maintenance. It also turns out that the anti-apoptotic role of the EGFR pathway in Drosophila AMPs described in this study is conserved across evolution, since EGF signalling also promotes survival of vertebrate satellite cells (Figeac, 2010).

The evidence for a major role of the EGFR pathway in the specification and maintenance of AMPs raises important questions about EGF targets operating in these muscle-committed stem-like cells in Drosophila. lb genes have been shown to be required for specification of LAMPs, making them candidate targets of EGF signalling in the lateral region. This study shows that lb regulatory modules contain binding sites for ETS factors that act as EGFR effectors and goes on to demonstrate their crucial role in AMP enhancer activity. The proximity of the ETS binding sites and homeodomain binding sites in the AMP element suggests that an adapted spatial conformation of interacting factors is important in allowing simultaneous binding and thus maintenance of the lineage-restricted activity of this enhancer. Interestingly, the main difference between regulatory modules driving expression in differentiated muscle lineages versus regulatory modules that act in non-differentiated AMPs is the responsiveness of the latter category to extrinsic EGF signals. In opposition to this, this study found that intrinsic Mef2 inputs are sufficient to drive expression in differentiated muscle lineage. The ETS and Mef2-driven expression of these two distinct regulatory modules is positively regulated by lb, which is known to play a pivotal role in the specification of muscle lineages in the lateral domain. The specific expression of lb in a subset of AMP cells and of its ortholog Lbx1 in activated satellite cells suggests that similarities in genetic control of Drosophila and vertebrate muscle stem cells might extend beyond those discussed here (Figeac, 2010).

The muscle pattern of the Drosophila abdomen depends on a subdivision of the anterior compartment of each segment

In the past, segments were defined by landmarks such as muscle attachments, notably by Snodgrass, the king of insect anatomists. This study shows how an objective definition of a segment, based on developmental compartments, can help explain the dorsal abdomen of adult Drosophila. The anterior (A) compartment of each segment is subdivided into two domains of cells, each responding differently to Hedgehog. The anterior of these domains is non-neurogenic and clones lacking Notch develop normally; this domain can express stripe and form muscle attachments. The posterior domain is neurogenic and clones lacking Notch do not form cuticle; this domain is unable to express stripe or form muscle attachments. The posterior (P) compartment does not form muscle attachments. In vivo films indicate that early in the pupa the anterior domain of the A compartment expresses stripe in a narrowing zone that attracts the extending myotubes and resolves into the attachment sites for the dorsal abdominal muscles. The tendon cells were mapped precisely, and it was show that all are confined to the anterior domain of A. It follows that the dorsal abdominal muscles are intersegmental, spanning from one anterior domain to the next. This view is tested and supported by clones that change cell identity or express stripe ectopically. It seems that growing myotubes originate in posterior A and extend forwards and backwards until they encounter and attach to anterior A cells. The dorsal adult muscles are polarised in the anteroposterior axis: the hypothesis is disproved that muscle orientation depends on genes that define planar cell polarity in the epidermis (Krzemien, 2012).

All the dorsal muscles of a typical segment of the abdomen, including the larval persistent muscles and the MOL, attach only to a small subregion: part of the anterior (A) compartment of each adult segment. This discussion first considers the mechanisms responsible for the muscle pattern and then the general significance of what was found (Krzemien, 2012).

It appears that the pupal A compartment contains two distinct types of cells even before Hh arrives to pattern it. In an anterior domain of the A compartment, the cells develop normally without N and do not form bristles and, above a certain level, Hh specifies a1 cuticle. By contrast, when presumptive a3 cells of the posterior domain of the A compartment receive Hh, they now make a4-a6 cuticle as well as the appropriate types of bristles. Here, it was confirmed that epidermal cells of this posterior domain absolutely depend on N activity as N- clones do not form cuticle in that territory and instead make clusters of neurons. More evidence is added for the two domains: the anterior domain appears to be competent to express sr and to form muscle attachments whereas the posterior domain does neither. Also, in the thorax of Drosophila, there is earlier evidence that sensory bristles and tendon cells cannot be both formed by the same progenitors. If sr is overexpressed in the thorax, bristle formation is inhibited. Reciprocally, expression of achaete-scute in the sr domain leads to impairment of the flight muscles. This antagonism between achaete-scute and sr is conserved in many dipteran groups. These findings provide a genetic correlate with the two domains of the A compartment (Krzemien, 2012).

In general, the pattern of attachment of muscles is related to the patterned expression of sr in the ectoderm. In the embryo, as soon as the myotubes arrive nearby, they become attracted to the sr-expressing cells and contact them. This is crucial; only the epidermal cells that establish contact with a myotube can maintain a high level of sr expression and can mature properly. In the pupal abdomen, as only the anterior domain of the A compartment can express sr, one would expect muscle attachments to be limited to those cells, as was found. The P compartment is a special case; N- clones form normally showing the cells to be non-neurogenic, consistent with an absence of bristles and sensilla in P compartments. However, muscle attachments do not form there either, perhaps because the action of en on cell identity abrogates the expression of sr (Krzemien, 2012).

Consistently, it was find that ectopic expression of either srA or srB bypasses the original positional identity, even P compartment cells will attach muscles if they express ectopic sr. In these circumstances, muscles may lose their normal anteroposterior orientation and bend to reach the clones expressing sr. This is more evidence that the orientation of the dorsal abdominal muscles is a response to an Sr-dependent signal and not a response to polarity information in the epidermis (Krzemien, 2012).

The pattern of muscle attachments therefore depends on information specified in the ectoderm, raising the question of how far the mesodermal founder cells are also programmed. In pupal myogenesis, the myoblasts and histoblasts develop in close association and the movies illustrate this in vivo suggesting that they interact. The most concrete evidence for some interaction between the ectoderm and the mesoderm comes from the 'muscle of Lawrence' (MOL). The MOL is longer than other adult muscles, extending further both at the front and at the back, even though both ends attach within the tendon-competent zones. The peculiar attachment sites and size of the MOL depend entirely on the intervention of a neuron that is active only in the fifth abdominal segment of males; without this intervention, these myotubes form the same attachment sites as the normal dorsal abdominal muscles (Krzemien, 2012).

This study presents some additional evidence that signals are sent from the epidermis to the developing myotubes. Clones in the epidermis with changed cell identities were induced in third instar larvae, before the formation of myotubes. These clones affected the growing myotubes: if the tendon-competent cells of the anterior domain of the A compartment were replaced by tendon-incompetent cells of the posterior domain, the muscles passed over the transformed cells to attach to anterior domain cells further away, suggesting that a signal emanating from these tendon-competent (sr-expressing) cells had attracted the extending myotubes (Krzemien, 2012).

Films show that precise anteroposterior orientation of the developing myotubes is maintained from the beginning. How is this achieved? One possibility is that polarity information originating in the epidermis might orient the muscle migration but this is shown to be not correct, at least with regard to the PCP genes. The films also show that, just as the myotubes are forming, a band of cells in the anterior region of each A compartment expresses sr. Probably, as in the embryo, these sr-expressing cells of the pupa attract and orient the growing myotubes. Later still, sr expression is increased at the actual sites of attachment, presumably as a result of short-range interaction between the specified myoblasts and the epidermal cells; again, as in the embryo (Krzemien, 2012).

For many decades, the definition of a segment was contentious -- scientists argued using anatomical criteria about the homology of parts of one species with similar parts of another. Most had been content with Snodgrass' perspective that the integumentary plates of harder cuticle (tergites in the dorsal abdomen of Drosophila) form the essential segments and these are separated by more flexible cuticle he called the intersegmental membranes (Snodgrass, 1935). Muscle attachments were traditionally used as landmarks for the borders of segments, particularly in juvenile and primitive soft-bodied insects. Using these criteria, it was concluded that most of the longitudinal muscles were 'intrasegmental'. But, in adults or in more advanced arthropods, Snodgrass noted that most muscles spanned from one integumentary plate to the next, and he therefore described those muscles as 'intersegmental'. He concluded that the muscle attachments could shift during development and evolution. Following Brenner, one could call this rather vague picture an 'external description' of a segment. However, a different way of defining a metamere came from studies of cell lineage during development and from mapping domains of gene expression, and these methods redefined a segment as the sum of two precisely defined group of cells: the A and the P compartments. This definition amounted to an 'internal description' (Brenner, 1999; Krzemien, 2012 and references therein)

This study shows that the dorsal abdominal muscles of the abdomen can attach to a limited anterior domain of the A compartment at the front of one segment and, as they extend, they cross over the posterior domain of the A compartment as well as the P compartment to reach the anterior domain of the next segment. It follows that all the three types of muscles of the dorsal abdomen that were studied are intersegmental in extent. It is likely that these same fundamental subdivisions of the segments are conserved in other insects and, if so, would confine most muscle attachment sites to particular locations, thereby constraining the range of muscle patterns that can be built by evolution (Krzemien, 2012).

Regulation of epithelial stem cell replacement and follicle formation in the Drosophila ovary

Though much has been learned about the process of ovarian follicle maturation through studies of oogenesis in both vertebrate and invertebrate systems, less is known about how follicles form initially. In Drosophila, two somatic follicle stem cells (FSCs) in each ovariole give rise to all polar cells, stalk cells, and main body cells needed to form each follicle. One daughter from each FSC founds most follicles but that cell type specification is independent of cell lineage, in contrast to previous claims of an early polar/stalk lineage restriction. Instead, key intercellular signals begin early and guide cell behavior. An initial Notch signal from germ cells is required for FSC daughters to migrate across the ovariole and on occasion to replace the opposite stem cell. Both anterior and posterior polar cells arise in region 2b at a time when approximately 16 cells surround the cyst. Later, during budding, stalk cells and additional polar cells are specified in a process that frequently transfers posterior follicle cells onto the anterior surface of the next older follicle. These studies provide new insight into the mechanisms that underlie stem cell replacement and follicle formation during Drosophila oogenesis (Nystul, 2010).

The Drosophila ovary is a highly favorable system for studying epithelial cell differentiation downstream from a stem cell. New follicles consisting of 16 interconnected germ cells surrounded by an epithelial (follicle cell) monolayer are continuously produced during adult life and develop sequentially within ovarioles (see Prefollicle cells associate with cysts in an ordered fashion downstream from follicle stem cells). Follicle formation begins in the germarium, a structure at the tip of each ovariole that houses 2-3 germline stem cells (GSCs) and 2 follicle stem cells (FSCs) within stable niches. Successive GSC daughters known as cystoblasts are enclosed by a thin covering of squamous escort cells and divide asymmetrically four times in sucession to produce 16-cell germline cysts, comprising 15 presumptive nurse cells and a presumptive oocyte. At the junction between region 2a and region 2b, cysts are forced into single file as they encounter the FSCs, lose their escort cell covering, and begin to acquire a follicular layer. Follicle cells derived from both FSCs soon mold them into a 'lens shape' characteristic of region 2b. Under the influence of continued somatic cell growth, cysts and their surrounding cells round up, enter region 3 (also known as stage 1), and bud from the germarium as new follicles that remain connected to their neighbors by short cellular stalks (Nystul, 2010).

A complex sequence of signaling and adhesive interactions between follicular and germline cells is required for follicle budding, oocyte development, and patterning. However, the mechanisms orchestrating the initial association between follicle cells and cysts within the germarium are less well understood. While lineage analysis indicates the presence of two FSCs, low fasciclin III (FasIII) expression has been claimed to specifically mark FSCs, leading to the conclusion that more FSCs are present under some conditions (Nystul, 2010).

The differentiation of polar cells at both their anterior and posterior ends is required for normal follicle production, and depends on Notch signals received from the germline. Subsequently, anterior polar cells send JAK-STAT and Notch signals that specify stalk cells. While the source of these signals and their effects are clear, the timing of polar cell specification and its dependence on cell lineage are not. Some anterior and posterior polar cells (but not stalk cells) were inferred by lineage analysis to arise and cease division within region 2b. In contrast, on the basis of marker gene expression it was concluded that anterior polar cells are specified later, in stage 1, and posterior polar cells in stage 2. Up to four polar cells may eventually form, but apoptosis reduces their number to a single pair at each end by stage 5. Moreover, polar and stalk are believed to arise exclusively from 'polar/stalk' precursors that separate from the rest of the FSC lineage and these cells were proposed to invade between the last region 2b cyst to affect follicle budding (Nystul, 2010).

This study analyzes the detailed behavior of FSCs and their daughters in the germarium. No evidence of polar/stalk precursors was observed, and it was shown that the first anterior and posterior polar cells are specified in region 2b, prior to the previously accepted time of follicle cell specialization. Additional polar cells are also formed later during stages 1 and 2. Follicle cell differentiation appears to be independent of cell lineage, but is orchestrated by sequential cell interactions, and in particular by Notch signaling. These results reveal the sophisticated, self-correcting behavior of an epithelial stem cell lineage at close to single-cell resolution (Nystul, 2010).

The data provide a much clearer picture of the follicle stem cell lineage than was previously available. They suggest that key aspects of FSC regulation depend on mechanisms that move cysts into a single file and program the loss of their escort cells precisely as they encounter FSCs and enter region 2b. Contact with incoming region 2a cysts likely induces FSC divisions, ensuring that cysts acquire a daughter cell from each stem cell as they stretch out to span the width of the germarium at the region 2a/2b junction. The asymmetry in cyst organization exposes FSC daughter cells to different cyst faces and, therefore, potentially to different signals. The FSC and daughter located on the same side as the entering cyst are exposed to the posterior face of the cyst while it is still in region 2a, covered by escort cells. In contrast, the opposite FSC and daughter contact the anterior face of the cyst as it migrates into 2b, at a time when the cyst is shedding its escort cell layer and exposing the Delta signal on the germ cell surface. Since region 2a cysts tend to interdigitate in forming a single file, cyst entry will usually alternate sides as successive cysts pass, causing FSC daughters arising from the same side to alternate migration paths. An advantage to this system may be its flexibility, allowing follicles to form normally even if multiple cysts enter from the same side in succession (Nystul, 2010).

Notch signaling in early FSC daughters promotes a 'prefollicle' state by blocking follicle cell differentiation. Consistent with this, it was observed that FSCs and their early daughters have much lower levels of differentiation markers such as FasIII and IMP-GFP. This developmental delay may prevent prefollicle cells from immediately incorporating into the differentiated follicular epithelium, allowing them to instead retain a more mesenchymal character conducive to cross-migration, and may also contribute to their ability to compete with the resident FSC for niche occupancy. Notch mutant daughters did not replace wild-type FSCs, most likely because they were unable to migrate into proximity. A role for Notch in suppressing differentiation downstream from the FSC might also explain why cells expressing activated Notch failed to migrate posteriorly (Nystul, 2010).

Follicle cell fates are specified by intercellular signals rather than lineage: The two FSC daughters and their descendants, with few exceptions, continue to associate with the cyst they first contact at the 2a/2b boundary throughout subsequent development. Their division rate increases briefly, because daughters divide four times in the time it takes to generate three new cysts. Despite their growing number, however, all the cells retain the ability to produce main body, stalk, and polar cells for at least the first two to three divisions (8- to 16-cell stage). In contrast to previous reports, no evidence was found that polar and stalk cells derive from a lineage-restricted polar/stalk precursor population. Claims of a polar/stalk fate were based on experiments using higher rates of clone induction than in the experiments reported in this study. While many clones were also observed in these studies that contained both polar and follicle cells or both stalk and follicle cells, they were discounted as double clones (Nystul, 2010).

By examining clones induced at low frequency (more than threefold lower than in previous studies) it was possible to minimize the need for statistical correction for double clones. Furthermore, by studying clones induced at multiple times downstream from the FSC, overweighting small clones induced just as the polar and stalk cell fates are being determined by signaling within small cell groups was avoided. This has the effect of increasing the proportion of clones containing only one or two cell types even in the absence of any lineage restriction. At early, intermediate, and late times in somatic cell development in the germarium, clones that included all combinations of cell fates were always observed, indicating that follicle cells are multipotent prior to polar or stalk specification. This fits well with recent studies showing that many additional cells in the germarium can be induced to take on a polar cell fate by strong Notch signaling, while high levels of JAK-STAT signaling can induce more stalk cells. In contrast, no mechanism, time, or location where putative polar/stalk precursor cells are specified has ever been documented. Previous models also did not explain how these cells would preferentially arrive in the zone of cells separating regions 2b and 3 or what would become of the many extra cells that can sometimes be found in this region beyond the number needed for these fates (Nystul, 2010).

The finding that polar cells are initially specified in region 2b suggests that more spatial information is available within region 2b follicles than has been detected in earlier experiments. It was found that the first anterior and posterior polar cells are specified when cysts are associated with 8- to 16-cell follicle cells, in mid-to-late region 2b. This agrees closely with previous studies, which found that polar cells were first specified at the 14-cell stage. The early polar cells are detected by lineage because they cease dividing; however, no gene expression markers specific for these cells have been identified. Consequently, it remains uncertain where they are located at the time of induction or whether they function while remaining in region 2b. Since evidence was observed of Notch signal reception within individual follicle cells located at the anterior and posterior regions of stage 2b cysts, the simplest model is that these polar cells are induced by Delta signaling from the germline in a normal anterior/posterior (A/P) orientation. Although no Upd expression was detected at this time, these cells may nonetheless signal to the surrounding somatic cells to establish the graded levels of cadherin that define the initial anterior/posterior axis of the cyst (Nystul, 2010).

Where does the information come from that allows a small number of polar cells to be specified at this time? One possibility is a 'signal relay' from more posterior follicles. Highly accurate timing of polar cell formation relative to the signaling events during follicle budding might help to further test this model. However, the observation of localized Notch signal reception and polar cell specification in region 2b follicles suggests that the germline at this stage is already sufficiently polarized to signal in a limited manner along the future A/P axis. Some of this information may come from the inherent asymmetry within the germline cyst whose cells differ systematically in their fusome content, organelle content, and microtubule organization. The future oocyte and its sister four-ring canal cell are always located in the center of the region 2b cysts and hence might be one source for this inductive signal. Alternatively, there may be additional differences within this region of the germarium that have yet to be detected and that may contribute (Nystul, 2010).

These studies confirm previous conclusions that additional polar cells are formed during the process of budding and provide new insight into the budding process itself. Anterior-biased clones were almost always confined to a single follicle, but a significant fraction of posterior-biased clones (~33%) extended onto the next older follicle where they encompassed both an anterior polar cell and 2-30 anterior follicle cells. This suggests that cells at the posterior of the nascent follicle outgrow their cyst as it rounds up and are forced into the space between the posterior 2b cyst and the budding cyst. The origin of these cells has long been a mystery. A fraction of the interstitial cells likely contact and move onto the anterior of the downstream cyst where those that happen to lie adjacent to the existing polar cells are induced as new polar cells and stalk cells. Any remaining interstitial cells likely rejoin the main body of follicle cells as budding is completed or are eliminated by apoptosis as the stalk resolves to its final size (Nystul, 2010).

This study of early follicle cell development provides a rare opportunity to analyze how epithelial cells behave downstream from a stem cell. Most characterized Drosophila stem cell daughters receive information asymmetrically from their mother stem cell and differentiate rapidly. Germline stem cells and their niches ensure that cystoblasts receive an asymmetric fusome segment as well as differential environmental signals that program exactly four stereotyped divisions prior to entering meiosis. Under nonstressed conditions, intestinal stem cells utilize Notch signals to specify their daughters as either enterocytes or enteroendocrine cells and to terminate subsequent division. Neuroblasts program a stereotyped sequence of daughter cell fates by differential division and signaling. In contrast, FSC daughters undergo eight to nine divisions and differentiate independently of lineage over the course of several divisions and are capable of producing normal follicles even when the usual pattern of cellular interactions is altered. The increased resolution of follicle cell behavior afforded by these studies provides a valuable opportunity to study how epithelial cells are able to robustly bring about defined outcomes in the absence of the precise early programming (Nystul, 2010).

Several mechanisms are likely to contribute to successful follicle formation. First, genes characteristic of a polarized epithelium turn on slowly downstream of the FSC. The cross-migrating cell and several other cells frequently lacked such gene expression, but instead expressed genes characteristic of escort cells, suggesting that follicles are able to maintain some germline-soma interactions while completely replacing their somatic coverings. The early differentiation of polar cells may help guide subsequent cell behavior. In conjunction with the intrinsic asymmetric structure of germline cysts, differential adhesive interactions between germ and somatic cells across the follicle, differential pressures resulting from cell growth, and the resistive forces of the ovariolar wall, signals from these cells may be sufficient to ensure that the oocyte moves to the posterior and that cysts begin to round (Nystul, 2010).

These characteristics of the FSC lineage, although unique among well-studied stem cells in Drosophila, may be closer to those governing the epithelial lineages within many mammalian tissues. Thus, the mechanisms that give FSCs and their daughters their developmental flexibility and robustness are likely to be both widespread and medically relevant (Nystul, 2010).

The microRNA pathway regulates the temporal pattern of Notch signaling in Drosophila follicle cells

Multicellular development requires the correct spatial and temporal regulation of cell division and differentiation. These processes are frequently coordinated by the activities of various signaling pathways such as Notch signaling. From a screen for modifiers of Notch signaling in Drosophila the RNA helicase Belle, a recently described component of the RNA interference pathway (Ulvila, 2006; Zhou, 2008), was identified as an important regulator of the timing of Notch activity in follicle cells. Loss of Belle delays activation of Notch signaling, which results in delayed follicle cell differentiation and defects in the cell cycle. Because mutations in well-characterized microRNA components phenocopied the Notch defects observed in belle mutants, Belle might be functioning in the microRNA pathway in follicle cells. The effect of loss of microRNAs on Notch signaling occurs upstream of Notch cleavage, as expression of the constitutively active intracellular domain of Notch in microRNA-defective cells restored proper activation of Notch. Furthermore, evidence is presented that the Notch ligand Delta is an important target of microRNA regulation in follicle cells and regulates the timing of Notch activation through cis inhibition of Notch. This study has uncovered a complex regulatory process in which the microRNA pathway promotes Notch activation by repressing Delta-mediated inhibition of Notch in follicle cells (Poulton, 2011).

The strict regulation of important cellular processes, such as the temporal activity of signaling pathways like Notch, is an essential point of control in guiding the development of multicellular organisms. Cells have therefore evolved a complex array of mechanisms to regulate signaling pathways. miRNA regulation of gene expression has rapidly emerged as one of the most important of these regulatory mechanisms. This study has shown that the correct timing of Notch activity in follicle cells requires the miRNA pathway and the newly identified RNAi component Bel. The data suggest that one important target of miRNA-based regulation of Notch signaling in follicle cells is Delta, in which Delta acts as a repressor of Notch activity (Poulton, 2011).

These findings that two core components of miRNA production are required to properly initiate the mitotic-to-endocycle switch in follicle cells by promoting Notch signaling describe a novel mechanism by which the miRNA pathway regulates this key developmental event. Interestingly, the miRNA pathway appears to control the overall timing of Notch activity, as disruption of the miRNA pathway results in a delay of Notch activation and inactivation in follicle cells. Previous work has shown that certain miRNAs, known as heterochronic miRNAs, regulate the timing of important developmental processes on a wide biological scale, from changes in cell cycle to the transition from juvenile to adult. This research identifies a new example of heterochrony mediated by miRNAs, in which cell cycle switches and differentiation are shifted in time as a result of delayed Notch signaling activity (Poulton, 2011).

Bel is a DEAD-box RNA helicase that was recently identified in two Drosophila cell culture screens as necessary for effective siRNA knockdown (Ulvila, 2006; Zhou, 2008). Precisely how Bel functions in this process is unknown, although data from the Zhou screen suggest that Bel acts downstream of siRNA production and loading. Interestingly, although Bel did not significantly disrupt miRNA-based assays in that screen, Bel was found to be in a complex with components of both the miRNA and siRNA pathways, and Bel immunoprecipitation pulled down both miRNAs and siRNAs, suggesting that Bel might be involved in both pathways. The similarities described between the bel mutant phenotype and the phenotypes of the miRNA pathway components Dicer (Dcr-1) and pasha imply that Bel might function in the miRNA pathway. Attempts were made to test the role of Bel in the miRNA pathway more directly using the GFP-tagged Delta 3'UTR sensor line, the expression of which is regulated by miRNA activity, but the results of these experiments were inconclusive. Although Bel appears to function in the siRNA pathway, this study found that the siRNA pathway is not involved in regulating Notch in follicle cells. A few reports have also identified several phenotypes associated with disruption of bel that indicate that Bel functions in the G1/S transition in the eye disc by affecting Hedgehog signaling and Dacapo expression (Ambrus, 2010; Ambrus, 2007), as well as identifying a role for Bel with the zinc-finger protein Zn72D in regulating the splicing and translation of maleless transcripts (Worringer, 2009). It will be interesting to determine whether the function of Bel in these other important cellular processes is also related to a role in RNAi pathways (Poulton, 2011).

Notch can be both activated and inhibited by its ligands. In oogenesis, it is known that Delta from the germline cells functions in trans to activate Notch in the surrounding follicle cells. This study found that Delta expressed in the follicle cells operates in its repressive capacity to prevent premature activation of Notch. Because Delta is actually upregulated in the germline by stage 5/6, well before the expression of Notch target genes at stage 7, and in light of the data on the inhibitory role of follicle cell Delta, it is likely that the presence of Delta from the germline alone is not what determines the precise timing of Notch activity. Instead, a model is favored in which the timing of Notch activity is determined by a titration of trans-activating germline Delta relative to the cis-inhibitory effects of follicle cell Delta. Therefore, loss of follicle cell Delta, as in the Delta mutant clone experiments, allows earlier activation of Notch by the lower levels of Delta presented by the germline before stage 7, as well as higher levels of Notch activity relative to wild-type cells in mid-oogenesis. This antagonistic relationship between germline and follicle cell Delta suggests that there must be a precise balance between these two populations of Delta that determines exactly when Notch is activated during oogenesis; analysis of the miRNA pathway suggests that miRNAs might help to fine-tune this balance (Poulton, 2011).

The conclusion that Delta is a relevant target of miRNA-based control of Notch activity in follicle cells is supported by the following observations. First, expression of NICD is sufficient to restore proper activation of Notch in the Dcr-1 mutant, indicating that the relevant miRNA target functions upstream in the Notch pathway (prior to ligand-induced Notch cleavage). Because ligand-based inhibition by Delta acts upstream of Notch cleavage, Delta is a logical candidate of miRNA regulation. Second, Delta,Dcr-1 double-mutant analysis strongly suggests that Delta is an important target of miRNAs. Specifically, in Dcr-1 single-mutant clones, Notch signaling is delayed, yet removal of Delta along with loss of Dcr-1 leads to premature activation of Notch, as seen in Delta single-mutant clones. This indicates that the inhibitory effects on Notch signaling caused by loss of miRNAs requires the presence of Delta. However, the possibility cannot be ruled out that the activating effects of loss of Delta on Notch might be stronger than the inhibitory effects of loss of miRNAs on repressing Notch activity through some other miRNA target. Third, Delta is an apparent direct target of the miRNA pathway, as indicated by experiments demonstrating that follicle cell clones of Dcr-1 and pasha result in increased Delta protein and increased expression of a Delta 3'UTR sensor. Together, the ectopic expression of Delta protein and of the Delta 3'UTR sensor in the Dcr-1,pasha clones, in conjunction with the Delta,Dcr-1 double-mutant analysis, strongly suggest that the miRNA pathway regulates Notch activity by repressing Delta protein levels (Poulton, 2011).

Cis inhibition of Notch has also been described for Ser, raising the possibility that Ser might be functioning in follicle cells in a similar capacity to that which was discovered for Delta. However, Ser mutant follicle cell clones possess no defects in Notch activity markers. To determine whether Ser is repressed by the miRNA pathway in follicle cells, Ser protein levels were examined in follicle cells double mutant for Dcr-1 and pasha, and no changes were observed in Ser expression, which in the wild type was essentially undetectable. It is concluded that Ser does not play a role in regulating Notch activity in follicle cells (Poulton, 2011).

More than two dozen miRNAs are predicted to target Delta mRNA. Owing largely to a lack of readily available mutants to conduct a thorough loss-of-function screen for the miRNA(s) involved, it remains unknown which miRNAs are important in governing the timing of Notch signaling in follicle cells. Both loss of function and overexpression of miR-1, which has been previously demonstrated to regulate Delta in Drosophila heart development, were tested; however, neither produced any phenotype consistent with the described Notch defects. As the genetic tools available to investigate the roles of specific miRNAs improve, and the ability to predict which miRNAs target certain transcripts also improves, it should be possible to identify the relevant miRNAs involved in this process (Poulton, 2011).

These findings describe a complex system by which developing egg chambers regulate the timing of several key events, including cell cycle programs and differentiation. Mechanistically, it was found that the miRNA pathway controls the temporal pattern of Notch activity, apparently by limiting Delta protein levels in follicle cells, in which Delta exerts an inhibitory effect on Notch. The data support a model in which the timing of Notch activation is determined not just by the expression of germline Delta, but also by a multi-layered regulatory system in which follicle cell Delta prevents premature Notch activation, while miRNAs serve to counter this inhibitory effect by limiting Delta expression. Such a model of miRNA function in follicle cells fits well with the developing theme that miRNAs commonly serve to fine-tune developmental processes by subtle regulation of key regulators. It will be interesting to determine whether miRNAs also regulate Notch signaling in other tissues of the fly through a similar mechanism of ligand-mediated inhibition of Notch, and it will be particularly exciting to investigate whether this regulatory network is utilized in other animals (Poulton, 2011).

DRP1-dependent mitochondrial fission initiates follicle cell differentiation during Drosophila oogenesis

Exit from the cell cycle is essential for cells to initiate a terminal differentiation program during development, but what controls this transition is incompletely understood. This paper demonstrates a regulatory link between mitochondrial fission activity and cell cycle exit in follicle cell layer development during Drosophila melanogaster oogenesis. Posterior-localized clonal cells in the follicle cell layer of developing ovarioles with down-regulated expression of the major mitochondrial fission protein DRP1 had mitochondrial elements extensively fused instead of being dispersed. These cells did not exit the cell cycle. Instead, they excessively proliferated, failed to activate Notch for differentiation, and exhibited downstream developmental defects. Reintroduction of mitochondrial fission activity or inhibition of the mitochondrial fusion protein Marf-1 in posterior-localized DRP1-null clones reversed the block in Notch-dependent differentiation. When DRP1-driven mitochondrial fission activity was unopposed by fusion activity in Marf-1–depleted clones, premature cell differentiation of follicle cells occurred in mitotic stages. Thus, DRP1-dependent mitochondrial fission activity is a novel regulator of the onset of follicle cell differentiation during Drosophila oogenesis (Matra, 2012).

The Drosophila follicle cell layer encapsulates egg chambers containing 15 nurse cells and one oocyte. The follicle cells comprising this cell layer progress through different developmental stages. During stages 1-5 (S1-5), most follicle cells undergo mitotic divisions, with a few cells exiting the mitotic cycle under Notch activation to form stalk cells separating consecutive egg chambers. During S6-8, all follicle cells exit the mitotic cycle in response to Notch activation and differentiate into an endocycling, polarized epithelium patterned into posterior follicle cells (PFCs), main body cells (MBCs), and anterior follicle cells (AFCs). To examine the effect of inhibiting mitochondrial fission activity in this system, Drosophila follicle cell clones where generated mozygous for a functionally null allele of DRP1 called drp1KG. Clones were identified by lack of a ubiquitin promoter-GFP (UbiGFP) label in their nucleus. The potentiometric dye tetramethylrhodamine ethyl ester (TMRE), which incorporates into the mitochondrial matrix, was used to label mitochondria (Mitra, 2009).

In an S10 egg chamber, nonclonal cells containing a nuclear UbiGFP label have mitochondrial elements widely distributed. Microirradiation at a single point within mitochondria of these cells triggers depolarization (i.e., loss of fluroescent TMRE signal) only at the irradiated site, with little loss of TMRE outside the microirradiated site. This suggested the mitochondrial network of these cells is discontinuous. In drp1KG clones (no UbiGFP label), mitochondria were tightly clustered in a small region of each cell. Single-point microirradiation of mitochondria in a drp1KG clone depolarizes the cell's entire mitochondrial cluster, with complete loss of TMRE signal in 5 s. This indicated that mitochondria in drp1KG clones are highly fused. Reduced mitochondrial fission in drp1KG clones, therefore, causes normally fragmented mitochondrial elements in follicle cells to hyperfuse into a tight cluster (Matra, 2012).

Next, weather presence of drp1KG clones affects follicle epithelial layer organization was examined. In S6-8 egg chambers, follicle cells normally form a single epithelial monolayer. The presence of drp1KG clones, however, disrupts this monolayer arrangement. The effect is most striking in the PFC region, in which drp1KG clones massively overproliferate. The overpopulated clones undergo mitotic cycling even at S10 or later: they incorporate BrdU, demonstrating that they synthesize DNA, and stain with pH3 antibody, indicating that they transit through mitosis. Surrounding heterozygous tissue and drp1KG MBC clones, in contrast, are postmitotic: they neither incorporate BrdU nor stain for pH3. DRP1 depletion thus prevents cell cycle exit primarily in drp1KG PFC clones, leading to their overpopulation in postmitotic egg chambers (Matra, 2012).

As cell cycle exit is a prerequisite for initiating differentiation, whether the drp1KG PFCs are prevented from differentiating was examined. Follicle cells in S6-8 egg chambers normally undergo cell cycle exit to differentiate under the influence of the homeodomain gene Hindsight (Hnt). Notably, clones of drp1KG in the PFC region marked by CD8GFP fail to express Hnt, unlike surrounding nonclonal cells. 95% of drp1KG PFC clones show this phenotype, whereas no drp1KG MBC clonal cells do. Thus, drp1KG PFC clones fail to differentiate (Matra, 2012).

Hnt expression is rescued in all drp1KG PFC clones generated in the background of HA-DRP1 and in 43% of drp1KG PFC clones with DRP1 reintroduced into them. In both conditions, DRP1 expression prevented the clustered mitochondrial phenotype. Lack of differentiation in drp1KG PFC clones, therefore, results from loss of DRP1 activity (Matra, 2012).

Down-regulation of Marf-1, the Drosophila homologue of mitofusins (Deng, 2008), combined with DRP1 down-regulation in drp1KG PFC clones causes 22% of the clones to now partially express Hnt. Because Marf-1 RNAi expression causes mitochondrial fragmentation when expressed alone or in drp1KG PFC clones, it was concluded that fragmentation of mitochondria reverses the differentiation block in drp1KG PFCs. Therefore, DRP1-driven mitochondrial fission is required for PFCs to differentiate. Loss of function of the inner mitochondrial membrane fusion protein OPA1 caused cell death in this system (Matra, 2012).

Differentiation of Drosophila follicle cells requires Notch receptor activation. Upon ligand binding, the Notch receptor is cleaved to release the Notch intracellular domain (NICD), which redistributes into the nucleus to activate genes required for differentiation. To investigate whether DRP1-driven mitochondrial fission activity acts upstream or downstream of Notch activation in driving PFC differentiation, whether NICD is cleaved and released from the plasma membrane was examined in drp1KG PFC clones. Significant NICD levels are retained on the plasma membrane in drp1KG PFC clones marked by CD8GFP relative to nonclonal cells in S6-8 egg chambers. The Notch extracellular domain (NECD) is also retained on the plasma membrane in these clones, confirming that Notch is inactive. In addition, Cut down-regulation, which occurs in response to Notch activation, does not occur in drp1KG PFC clones. DRP1-driven mitochondrial fission activity thus acts upstream of Notch activation to drive PFC differentiation (Matra, 2012).

NICD loss from the membrane (indicative of Notch activation) increases by 28.2% in drp1KG PFC clones after Marf-1 down-regulation. This suggested that Notch inactivation in drp1KG PFC clones is related to mitochondria being highly fused, with mitochondrial fission a prerequisite for Notch receptor activation in the PFCs. Importantly, expression of an activated Notch (N-Act) domain in drp1KG PFC clones partially overrides the differentiation block in 53% of drp1KG PFC clones, resulting in Hnt expression in these clones. As this occurs without the fused mitochondrial morphology of drp1KG PFC clones changing, the data confirmed that DRP1's role in triggering PFC differentiation is upstream of Notch (Matra, 2012).

Why is DRP1's role in triggering follicle cell differentiation specific to PFCs? Indeed, drp1KG MBC clones show no differentiation block, as Notch activation still occurs in drp1KG MBC clones. Higher levels of bound DRP1 was found in PFCs compared with MBCs after cell permeabilization with digitonin, which may reflect different mitochondrial morphology between PFCs and MBCs. Supporting this, in S6-8 ovarioles it was found that mitochondria in PFCs exist as dispersed fragments both apically and basolaterally, whereas mitochondria in MBCs are tightly clustered at the lateral side of the nucleus. After S9, no observable differences were seen in mitochondrial morphology (Matra, 2012).

Fluorescence loss in photobleaching (FLIP) experiments in follicle cells of S6-8 egg chambers revealed that the dispersed mitochondria of PFCs have less matrix continuity relative to the fused mitochondrial cluster of MBCs. Furthermore, single-point microirradiation caused a 44% loss in TMRE mitochondrial signal per MBC compared with a 12% loss per PFC. The rapid loss of mitochondrial TMRE signal in MBCs was similar to drp1KG clonal cells, with mitochondrial morphology in wild-type MBCs indistinguishable from that of drp1KG MBC clones. Together, the observed differences in mitochondrial organization and bound DRP1 levels in PFCs and MBCs suggested greater DRP1-driven mitochondrial fission activity occurs in PFCs relative to MBCs. This corroborates findings that PFCs, unlike MBCs, differentiate under the influence of DRP1 (Matra, 2012).

PFCs are known to be specified by EGF receptor (EGFR) signaling. In egfrt1/egfrt1 egg chambers (hypomorphic allele of EGFR), mitochondria in PFCs are primarily clustered to one side of the nucleus, in contrast to those in wild-type or egfrt1/+ egg chambers, in which mitochondria are dispersed throughout cells. A similar clustering of mitochondria occurs when a dominant-negative (DN) form of EGFR (EGFR-DN) is clonally expressed in the PFC population. Because PFC mitochondria cluster/fuse in the absence of EGFR signaling, the data suggest that EGFR activation in PFCs promotes mitochondria fragmentation in these cells. This could explain why MBCs, which do not receive the EGFR signal, have fused mitochondria. The underlying basis for how EGFR signaling influences mitochondrial dynamics (by altering fission or fusion components) requires further investigation (Matra, 2012).

Interestingly, PFCs expressing EGFR-DN did not escape differentiation in spite of having clustered mitochondria. This may imply that a highly fused mitochondrial cluster may only allow escape from differentiation in the context of activated EGFR signaling. Indeed, EGFR-DN expression in drp1KG PFC clones (with fused mitochondria) partially induces differentiation (i.e., Hnt expression) in 40% of the clonal cells compared with no Hnt expression in drp1KG PFC clone. Expression of an activated form of EGFR (EGFR-Act) did not induce differentiation in drp1KG PFC clones. This explains why MBCs, which are not exposed to the EGFR ligand, do not proliferate under DRP1 down-regulation. Thus, cross talk exists between mitochondria and the EGFR signaling pathway in postmitotic PFCs, which helps cells decide whether to differentiate or continue in the mitotic cycle (Matra, 2012).

Whether DRP1 activity is important for regulation of cell cycle exit of mitotic follicle cells to allow onset of differentiation was investigated. The majority of follicle cells in S1-5 (during which all cells are mitotic) have fragmented mitochondria, suggesting that DRP1-dependent fission activity is high. drp1KG follicle cell clones introduced into the mitotic follicle cell layer and lacking UbiGFP harbor characteristic mitochondrial clusters. Clones also contain more pH3-positive cells and have qualitatively greater incorporation of BrdU relative to nonclonal tissue, with Cut expression unaltered. Without DRP1, therefore, S1-5 follicle cells undergo faster mitotic cycling (Matra, 2012).

To test whether DRP1 activity is necessary for mitotic cells to differentiate, Marf-1 RNAi was expressed to allow unopposed DRP1 activity in S1-5 egg chambers. Strikingly, Marf-1 RNAi expressing follicle cell clones (marked by CD8GFP) show premature expression of Hnt, whereas neighboring nonclonal mitotic follicle cells do not. The effect is not restricted to any stage or region of the mitotic follicle cell layer. The Marf-1 RNAi follicle cell clones exhibit increased mitochondrial mass as assessed by HSP-60 staining and MitoTracker loading, similar to that reported previously from mitofusin knockout mice . Importantly, drp1KG follicle cell clones expressing Marf-1 RNAi do not show premature differentiation; Hnt and HSP-60 expression levels are comparable with wild-type cells. Therefore, the premature differentiation of Marf-1 RNAi clones is dependent on DRP1. This indicates that DRP1-driven mitochondrial fission activity is required for mitotic follicle cells to exit the cell cycle and initiate their differentiation regimen (Matra, 2012).

Because of DRP1's role in differentiation, lack of DRP1 should generate developmental defects. Consistently, DRP1 down-regulation in early follicle cells in the germarium inhibits stalk cell formation, required to separate consecutive egg chambers. The missing stalk cells in egg chambers, encapsulated by early drp1KG follicle cell clones, leads to fused egg chambers containing pH3-labeled drp1KG clonal cells that lack UbiGFP. FasIII-enriched polar cells, known to induce stalk cells, are seen in wild-type ovarioles but are absent in the drp1KG clonal follicle cell population. Lack of polar cells is not the basis of cell proliferation of drp1KG PFCs because FasIII-positive polar cells appear in the surrounding heterozygous tissue. In addition, compound egg chambers with drp1KG follicle stem cell clones frequently arise, including egg chambers with 30 nurse cells and two oocytes (Matra, 2012).

Down-regulation of DRP1 also causes developmental defects in the postmitotic follicle cell layer. There, in 22% of the cases, drp1KG PFC clones fail to trigger migration of the oocyte nucleus toward the anterior. The postmitotic stage drp1KG phenotypes resemble loss of function of the Hippo-Salvador-Warts pathway, which has tumor suppressor effects in higher organisms, including mice (Matra, 2012).

The observed link between cell differentiation and mitochondrial fission state during oogenesis could relate to cyclin E, which controls S-phase entry. Indeed, inhibition of mitochondrial ATP synthesis in a cytochrome oxidase mutant promotes specific degradation of cyclin E (but not other cyclins) and blocks S-phase entry in Drosophila. In fibroblasts, cyclin E levels increase under conditions of DRP1 inhibition. In Drosophila follicle cells cyclin E levels were found to increase when DRP1 is down-regulated and decrease when Marf-1 is down-regulated. This suggests that DRP1-driven mitochondrial fission activity may cause cell cycle exit by lowering cyclin E levels to allow differentiation (Matra, 2012).

The results support a model in which mitochondrial fission/fusion dynamics regulates cell differentiation across the follicle cell layer of the Drosophila ovariole (see A model for mitochondria's role in cell fate determination). In mitotic stages, increased DRP1-driven mitochondrial fission is required for cell cycle exit as noted in premature DRP1-dependent differentiation of Marf-1 RNAi clones and enhanced proliferation of drp1KG clones. During postmitotic transition, activation of EGFR in the posterior region causes mitochondrial fragmentation. This, in turn, permits cell cycle exit and Notch activation, which drives PFC differentiation. In drp1KG PFC clones with fused mitochondria, therefore, Notch remains inactive, and cells proliferate. In the main body region, not exposed to the EGFR ligand, postmitotic differentiation and patterning occur in the absence of DRP1. Thus, cell proliferation/differentiation mechanisms have an intimate relationship to mitochondrial morphology and function during follicle layer development (Matra, 2012).

Bunched sets a boundary for Notch signaling to pattern anterior eggshell structures during Drosophila oogenesis

Organized boundaries between different cell fates are critical in patterning and organogenesis. In some tissues, long-range signals position a boundary, and local Notch signaling maintains it. How Notch activity is restricted to boundary regions is not well understood. During Drosophila oogenesis, the long-range signals EGF and Dpp regulate expression of bunched (bun), which encodes a homolog of mammalian transcription factors TSC-22 and GILZ. This study shows that bun establishes a boundary for Notch signaling in the follicle cell epithelium. Notch signaling is active in anterior follicle cells and is required for concurrent follicle cell reorganizations including centripetal migration and operculum formation. bun is required in posterior columnar follicle cells to repress the centripetal migration fate, including gene expression, cell shape changes and accumulation of cytoskeletal components. bun mutant clones adjacent to the centripetally migrating follicle cells showed ectopic Notch responses. bun is necessary, but not sufficient, to down-regulate Serrate protein levels throughout the follicular epithelium. These data indicate that Notch signaling is necessary, but not sufficient, for centripetal migration and that bun regulates the level of Notch stimulation to position the boundary between centripetally migrating and stationary columnar follicle cells (Dobens, 2005).

Previous work demonstrated that opposing gradients of EGF and BMP signals established a sharp boundary for expression of one of the Bun transcripts, BunB, and that the resultant protein Bun1 can repress the operculum fate (Dobens, 2000). The data presented in this study indicate that bun is required around the circumference of the FC to prevent changes in cellular architecture that are associated with centripetal migration. These include changes in α-spectrin subcellular localization, non-muscle myosin accumulation and cell shape. Consistent with the model that bun regulates centripetal migration, rare, small, anterior clones of bun4230 have been observed where the mutant cells elongate and pinch into the oocyte. Taken together, these data indicate that bun maintains the columnar FC fate and prevents both centripetal migration and operculum fate determination. However, the data from bun clonal analysis indicate that bun function is position-dependent, suggesting that it regulates competence to respond to additional signals. Strong mutant phenotypes occurred only in columnar bun FC clones that contacted the centripetal migrating FC (Dobens, 2005).

The data indicate that concurrent Notch signaling is required for centripetal migration and that bun blocks this signaling in columnar FC at the time of centripetal migration. Altogether, these data suggest that bun organizes a boundary for morphogenesis, in part by regulating the level of Notch signaling. Thus, fine patterning of the anterior eggshell fates involves the action of three independent signals, Dpp, EGF and Notch, coordinated through expression and activity of bun (Dobens, 2005).

Previously, the role of Notch signaling in anterior FC patterning has been attributed to the early requirement for Notch in patterning the egg chamber termini. Notch signaling is required for differentiation and/or survival of the polar FC, which are necessary for anterior/posterior patterning of both follicle cells and the underlying oocyte. Building on this model, it was recently proposed that centripetal migrating FC are one of several fates that are specified by graded levels of JAK/STAT signaling prior to stage 6, stimulated by Upd produced from anterior polar cells. While JAK/STAT signaling appears necessary for gene expression in centripetally migrating FC, other aspects of the Upd morphogen gradient model are controversial (Dobens, 2005).

Notch signaling is repeatedly required during FC development. Notch is important for initial formation of the two FC lineages, the stalk/polar cell lineage and the epithelial FC lineage. Subsequently, during stage 6, Notch is required throughout the FC epithelium to switch from mitotic cell divisions to endoreplication. It has been proposed that Notch signaling at stage 6 initiates differentiation of the epithelial FC and that all subsequent phenotypes in Nts egg chambers are due to failed differentiation. Indeed, Notch protein levels are high throughout the epithelial FC prior to stage 7 and then decrease substantially by stage 8. By this model, defective anterior morphogenesis would be a deferred consequence of failed Notch signaling during stages 6 and 7 (Dobens, 2005).

However, this study shows that Notch signaling is active in anterior FC during centripetal migration and subsequent morphogenesis of the operculum and dorsal appendages. Multiple Notch target reporters -- Dl-lacZ, 12XSu(H)BS-lacZ and E(spl)mβ7-CD2 -- are expressed in anterior FC at stages 10-14, where Notch, Delta and Serrate are detected during stages 9-14. Furthermore, a temperature-sensitive allele of Notch could be used to disrupt anterior FC morphogenesis without significant perturbation of endoreplication. These observations indicate that late Notch signaling is required for centripetal migration (Dobens, 2005).

An unusual feature of this late Notch activity in anterior FC is the activation of responses in cells that co-express both ligand and receptors. Over-expression studies suggest that high levels of Delta or Serrate can block signaling by Notch from the same cells, which is thought to be important for spatial localization of Notch signaling during wing margin development. This inhibitory effect can be observed in the FC with gratuitous expression of high levels of Serrate. In contrast, low levels of ligand appear to stimulate target gene expression in the same cell, during ectopic expression or under endogenous conditions in proneural clusters and pairs of photoreceptors in the developing eye. Although two ligands are present during anterior FC morphogenesis, the levels for each protein were near the limit of detection for immunofluorescence in wild type FC. Target gene expression levels were also low (Dobens, 2005).

The spatial and temporal restrictions observed for Notch responses suggest that endogenous levels of either Delta or Serrate alone are insufficient to stimulate Notch signaling for anterior morphogenesis. Manipulation of bun activity has strong effects on Serrate accumulation but only weak effects on Delta. Delta gene expression responds to experimental manipulation of Notch activity. It is proposed that stimulation of Notch signaling is localized to centripetally migrating cells through this dual regulation. Serrate accumulates at higher levels in FC with low bun activity, and Delta expression is increased by Notch signaling through a positive feedback loop. Notch signaling in anterior FC may be initiated by Delta signaling from the nurse cells. However, during eggshell deposition, FC expression of ligands is needed to maintain Notch signaling in FC that overlie the oocyte. Detailed analysis of the requirements for Delta and Serrate will be needed to validate the model that both ligands are needed for activation of Notch signaling in centripetally migrating FC. The data presented in this study demonstrate that bun prevents the Notch stimulus from spreading inappropriately into the columnar FC (Dobens, 2005).

The molecular mechanism for bun regulation of Notch signaling is unknown. Two types of non-autonomous effects were observed, suggesting that regulation of Notch stimulation is indirect. First, mutant cells that contact bun+ cells at the edge of the clone often had wild type phenotype. Thus, contact with a wild type cell can rescue these phenotypes in bun mutant cells. Consistent with this, in wild type egg chambers, a single-cell gap between high Notch levels and bun-lacZ expression was observed in ventro-lateral FC during late oogenesis. Second, it appears that high levels of Serrate accumulation can be stimulated by the presence of neighboring cells with higher levels of the Bun1 transcription factor. This non-autonomous effect suggests that even regulation of Serrate levels is indirect. Although numerous genes can modulate Notch signaling, few have been tested for cell-autonomous function. Additional bun target genes are being sought that may modulate Notch signaling (Dobens, 2005).

At present, it is not known if bun regulates Notch activity in other tissues. bun mutant phenotypes occur in the peripheral nervous system, eye, wing margin and denticle belt patterning, all regions that require Notch signaling. In chickens, the bun-related TSC-22 is expressed in developing feather buds at a time coincident with Notch signal activity. Further work will be required to determine whether this is a general function for TSC-22/DIP/BUN family members (Dobens, 2005).

Transforming Growth Factor β/activin signalling induces epithelial cell flattening during Drosophila oogenesis

Although the regulation of epithelial morphogenesis is essential for the formation of tissues and organs in multicellular organisms, little is known about how signalling pathways control cell shape changes in space and time. In the Drosophila ovarian epithelium, the transition from a cuboidal to a squamous shape is accompanied by a wave of cell flattening and by the ordered remodelling of E-cadherin-based adherens junctions. This study shows that activation of the TGFβ pathway is crucial to determine the timing, the degree and the dynamic of cell flattening. Within these cells, TGFβ signalling controlled cell-autonomously the formation of Actin filament and the localisation of activated Myosin II, indicating that internal forces were generated and used to remodel AJ and to promote cytoskeleton rearrangement. TGFβ signalling controlled Notch activity and its functions were partly executed through Notch. Thus, the cells that underwent the cuboidal-to-squamous transition produced active cell-shaping mechanisms, rather than passively flattening in response to a global force generated by the growth of the underlying cells. Thus, this work on TGFβ signalling provides new insights into the mechanisms through which signal transduction cascades orchestrate cell shape changes to generate proper organ structure (Brigaud, 2015: PubMed).

Border of Notch activity establishes a boundary between the two dorsal appendage tube cell types

Boundaries establish and maintain separate populations of cells critical for organ formation. Notch signaling establishes the boundary between two types of post-mitotic epithelial cells, the Rhomboid- and the Broad-positive cells. These cells will undergo morphogenetic movements to generate the two sides of a simple organ, the dorsal appendage tube of the Drosophila egg chamber. The boundary forms due to a difference in Notch levels in adjacent cells. The Notch expression pattern mimics the boundary; Notch levels are high in Rhomboid cells and low in Broad cells. Notch mutant clones generate an ectopic boundary: ectopic Rhomboid cells arise in Notch+ cells adjacent to the Notch mutant cells but not further away from the clonal border. Pangolin, a component of the Wingless pathway, is required for Broad expression and for rhomboid repression. It is further shown that Broad represses rhomboid cell autonomously. These data provide a foundation for understanding how a single row of Rhomboid cells arises adjacent to the Broad cells in the dorsal appendage primordia. Generating a boundary by the Notch pathway might constitute an evolutionarily conserved first step during organ formation in many tissues (Ward, 2006).

At the boundary, cells with high Notch express rhomboid, whereas cells with lower Notch express Broad. A new boundary is established at Notch mutant clone borders, where Notch+ cells adjacent to Notch cells ectopically express rhomboid and do not express Broad. Thus, in the dorsal anterior, when two cells with different Notch levels are adjacent to one another, the cell with higher Notch levels simultaneously represses Broad and promotes rhomboid expression. broad cells ectopically express rhomboid, indicating that Broad normally represses rhomboid expression. It is inferred that cells with higher Notch levels repress Broad, thereby allowing rhomboid expression. It is now proposed that when cells with different levels of Notch are located next to each other, the cells with high Notch repress Broad, allowing rhomboid expression. In contrast, cells with low Notch express Broad and therefore repress rhomboid expression (Ward, 2006).

Notch, an important modulator of boundary function in other tissues, establishes the boundary that defines the Rhomboid and the Broad dorsal appendage cell types. When Notch is removed from cells that should span the boundary, rhomboid is not expressed, and Broad is ectopically expressed. Thus, at the boundary, Notch regulates the patterning of both Rhomboid and Broad cell types. When Notch activity is removed from Region 1, ectopic Rhomboid cells (Notch+) arise adjacent to Notch (Broad) cells, thus resembling the normal Notch border. It is proposed that these Notch mutant clones produce ectopic borders of differential Notch activity, which in turn generate ectopic boundaries between Rhomboid and Broad domains (Ward, 2006).

Normally, Rhomboid cells arise all along the high–low Notch boundary in each dorsal appendage primordium. Based upon this observation, one might expect that Rhomboid cells would surround the Notch clones. In the current studies, however, it was found that only those cells close to the normal boundary turned on ectopic rhomboid. Two factors probably contribute to this result. First, other signaling pathways, most notably EGFR and DPP, are involved in specifying and positioning the Rhomboid and Broad cell populations within the follicular epithelium. Presumably, these other signaling pathways influence Broad/rhomboid expression in cells adjacent to Notch clones. Second, the ectopic Notch borders generated by Notch clones arise within the Broad domain, which normally has low levels of Notch. Therefore, many cells at the ectopic border may not have sufficient Notch activity to repress Broad and activate rhomboid (Ward, 2006).

Within the domain that would normally express Broad, loss of Notch causes the loss of Broad non-cell autonomously in adjacent cells and the appearance of ectopic rhomboid in these same cells. Furthermore, Notch clones spanning the boundary ectopically express Broad and do not express rhomboid. These findings are consistent with previous results demonstrating that dorsal appendage cells express either rhomboid or Broad, but never both markers. This work shows that broad cells ectopically express rhomboid, suggesting that one function of Broad in the follicular epithelium is to directly or indirectly repress rhomboid expression. Such regulation must occur (at least in part) in the 2.2-kb fragment that drives lacZ expression in a reporter construct. CONSITE software detects twenty Broad binding sites clustered together in this region; all four zinc-finger isoforms have the potential to bind. Thus, high levels of Broad could directly regulate rhomboid in Region 1. Additional work is needed to test this hypothesis (Ward, 2006).

Other factors must also regulate rhomboid expression in Region 2. Within clones spanning the boundary, ectopic expression of Broad prevents rhomboid expression. In cells adjacent to Notch clones, loss of Broad expression allows ectopic rhomboid expression. Nevertheless, the simple absence of Broad is insufficient to induce rhomboid expression, since the majority of cells in Region 2 lack Broad expression and do not express rhomboid. Presumably, high levels of EGFR and DPP signaling prevent rhomboid expression in these cells (Ward, 2006).

The Notch loss- and gain-of-function data, as well as the Notch expression pattern, all suggest that juxtaposition of two cells with different Notch levels is critical for establishing the boundary between Rhomboid and Broad cell types. How, then, is Notch protein level regulated? The restricted pattern of Notch in the dorsal anterior follicle cells suggests that Notch expression is determined by a combination of patterning instructions from DPP along the anterior/posterior axis and EGFR signaling along the dorsal/ventral axis (Ward, 2006).

The importance of regulating Notch protein levels is underscored by data showing that overexpression of full-length Notch represses Broad expression throughout the follicular epithelium. Since the full-length Notch receptor must be bound by ligand to initiate Notch signaling, a Notch ligand is either present throughout the follicular epithelium or is presented to the follicle cells by the underlying germ line. The Drosophila genome encodes two known Notch ligands, Delta and Serrate, and several potential ligands, such as CG9138. The absence of both Delta and Serrate in the follicular layer did not affect Broad or rhomboid expression. The function of other potential ligands in follicle cells is not currently known. It is also possible that the ligand for this process is present in the germ line. Delta is expressed in the germ line at the appropriate time and functions in the germ line to regulate follicle cell processes, such as the pinching-off of egg chambers in the germarium and the mitotic-to-endocycle transition at stage 7. Additionally, previous work demonstrates that egghead and brainiac, which encode modulators of Notch function, act in the germ line to pattern the dorsal anterior follicle cells. Regardless of the tissue distribution of the ligand, however, the ability to uniformly activate the Notch pathway throughout the follicle cell layer is note-worthy. This observation suggests that Notch levels, rather than spatial location of a ligand (or ligand modulator), determines where or how Notch signals in follicle cells of late stage egg chambers (Ward, 2006).

One of the most surprising aspects of the work presented here is that Notch clones act in a non-cell-autonomous manner to regulate Broad and rhomboid expression in adjacent cells. While surprising, non-cell-autonomous Notch activity occurs in the embryo, and most notably, at the D/V boundary in the wing disc. In the third-instar wing disc, Wingless is expressed in a 3- to 6-cell wide stripe spanning the D/V boundary, which separates the dorsal and ventral portions of the future wing blade. In this system, wingless-lacZ is repressed both within and adjacent to Notch clones. Thus, Notch clones act non-cell autonomously in two different tissues where boundaries act to distinguish different cell types (Ward, 2006).

What is the nature of the non-autonomous signal from the Notch clones? It is proposed two potential mechanisms to explain this process. First, Notch itself measures Notch levels in adjacent cells, either directly through homophilic adhesion or indirectly through interaction with Notch-binding proteins. When a Notch clone occurs in the dorsal anterior, adjacent cells sense the absence of Notch and respond as wild-type cells do when high-Notch cells neighbor low-Notch cells; they either repress Broad directly, or they repress Broad indirectly by affecting Pangolin (or some other component of the Wingless signaling pathway). Pangolin is needed to express Broad and therefore down-regulate rhomboid throughout the follicle cell layer. A second possibility is that when cells have little or no Notch activity, they might secrete an inhibitor of the Pangolin pathway that only affects cells with high Notch. The first mechanism is favored for its simplicity in accounting for rhomboid expression only at the border between high- and low-Notch-expressing cells (Ward, 2006).

The establishment of a border between Rhomboid and Broad cells is important for preventing intermingling of these cell types during tube formation (Ward, 2005). It is not clear, however, what mechanism separates the Broad and Rhomboid cells from each other at the border. In some situations, the non-transcriptional branch of the Notch pathway regulates F-actin (Major, 2005), which creates a “fence” that could help separate the two cell types from each other in the border. In dorsal anterior follicle cells, however, the canonical Notch pathway acts through the transcription factor Su(H). It is possible that in this cell type, the Notch pathway transcriptionally regulates a cell adhesion molecule or other component of an actin-binding protein complex, which in turn coordinates the cytoskeleton, thereby maintaining a separation between the Rhomboid cells and the Broad cells. Unlike cells at other boundaries in which an actin fence is evident, the Rhomboid and Broad cells undergo dramatic morphological changes and reorganize their actin networks to produce these effects. A fence that could maintain the separation of these cells during apical constriction, directed elongation, and convergent extension would be critical during these processes. One such Notch-interacting candidate gene that links to actin filaments is Echinoid. Future experiments will define whether Echinoid plays a role during border formation between Rhomboid and Broad cells (Ward, 2006).

Animals have a wide variety of organs containing different cell types arranged in a stereotypical manner. While the general morphogenesis of most organs has been described, little is known about the molecular mechanisms required to specify boundaries between diverse cell types and direct their subsequent reorganization to produce a functional structure. This study has shown that canonical Notch signaling is necessary to establish a boundary between the Broad and Rhomboid cells, which will form the dorsal and ventral portions of the dorsal appendage tube. Notch is also required in the vertebrate hindbrain for rhombomere boundary formation. Thus, in simple and more complex organs, Notch specifies boundaries between distinct cell populations needed for organ formation. Generating a boundary through Notch signaling could be an evolutionarily conserved first step during organ formation in many tissues. The next challenge is to define the molecular nature of the physical power that keeps the two different cell types separated from each other in the border (Ward, 2006).

Notch signaling links interactions between the C/EBP homolog slow border cells and the GILZ homolog bunched during cell migration

In the follicle cell (FC) epithelium that surrounds the Drosophila egg, a complex set of cell signals specifies two cell fates that pattern the eggshell: the anterior centripetal FC that produce the operculum and the posterior columnar FC that produce the main body eggshell structure. The long-range morphogen DPP represses the expression of the bunched (bun) gene in the anterior-most centripetal FC. bun, which encodes a homolog of vertebrate TSC-22/GILZ, in turn represses anterior gene expression and antagonizes Notch signaling to restrict centripetal FC fates in posterior cells. From a screen for novel targets of bun repression, the C/EBP homolog slow border cells (slbo) has been identified. At stage 10A, slbo expression overlaps bun in anterior FC; by stage 10B they repress each other's expression to establish a sharp slbo/bun expression boundary. The precise position of the slbo/bun expression boundary is sensitive to Notch signaling, which is required for both slbo activation and bun repression. As centripetal migration proceeds from stages 10B-14, slbo represses its own expression and both slbo loss-of-function mutations and overexpression approaches reveal that slbo is required to coordinate centripetal migration with nurse cell dumping. It is proposed that in anterior FC exposed to a Dpp morphogen gradient, high and low levels of slbo and bun, respectively, are established by modulation of Notch signaling to direct threshold cell fates. Interactions among Notch, slbo and bun resemble a conserved signaling cassette that regulates mammalian adipocyte differentiation (Levine, 2007).

bunched refines a DPP activity gradient by antagonizing Notch signaling to establish the posterior edge of the operculum-forming centripetal FC. This study reveals that bunched is part of an intricate switch reliant on Notch activation of slbo to direct alternate FC fates. These observations contribute to a model in which bunched connects long-range morphogen cues to short range, cell contact-dependent signaling. Together with recent work on the bunched homologue GILZ in mammalian cell culture, these data suggest that this family of proteins is part of a conserved signaling cassette regulating cell fate decisions, as detailed below (Levine, 2007).

In different contexts cells migrate either as integrated sheets, such as during convergent extension, or as small groups of cells, such as during neural crest migration. During border cell migration from stages 8-10, a subset of anterior FC transiently loses epithelial polarity, delaminates and rounds into a small semi-polarized cell cluster that migrates through the nurse cell complex. In contrast, during centripetal migration from stages 10-14 a ring of anterior follicle cells changes shape and squeezes through the oocyte/nurse cell complex in a process coordinated with rapid nurse cell dumping. Marker gene expression indicates that the centripetal FC stretch to cover the anterior of the oocyte and retain epithelial contacts with the anterior and posterior nurse cell FC and columnar FC groups, respectively, throughout this mass cell ingression. While unique genetic pathways likely regulate these distinct cell migrations, because both the border cells and the centripetal FC coordinately migrate through the germ line cyst and arrive in the same vicinity at the anterior of the egg, it is unsurprising that common components are involved in both processes. Non-muscle myosin (zipper) and DE-cadherin (shotgun) are expressed and required for migration in both cell types. As well, it has been shown that slbo itself is required for DE-cadherin accumulation during both border cell and centripetal FC migrations, an observation consistent with the role for slbo function in the centripetal FC that are demonstrated in this study. Recently, screens for border cell-specific gene expression have identified many transcripts expressed in both tissues (Levine, 2007).

Comparing the role and regulation of slbo during the centripetal FC sheet and border cell cluster migrations reveals both shared and unique requirements. Weak slbo mutations, which completely block border cell migration, have no discernable effect on centripetal FC migration, which is disrupted only in stronger allelic combinations. While early slbo mutant clones reduced DE-cadherin accumulation in the dorsal anterior FC and in the border cells, late slbo mutant clones in the nurse cell FC and centripetal FC are difficult to recover and properly stage. These clones result in several effects on late stage egg chambers. First, these resulted in increased levels of DE-cadherin and decreased levels of DLG consistent with changes in epithelial polarity and adhesion. Second, large anterior slbo mutant clones are associated with a failure of centripetal FC ingression to coordinate with nurse cell dumping. It is noted that slbo mutant phenotypes are distinct from DE-cadherin shotgun (shg) mutants, which result in ectopic centripetal migration between posterior nurse cells. slbo mutants do resemble dlg mutant phenotypes associated with defects in FC shape and epithelial invasiveness. And third, ectopic slbo-lacZ expression associated with disintegration of the follicular epithelia and egg chamber collapse which are likely connected to defects in epithelial maintenance. Thus previous reports that the strong slbo allele has no effects on centripetal FC migration may result from difficulties recovering and staging these highly aberrant and friable late stage mutant egg chambers (Levine, 2007).

The mechanism of slbo regulation in the border cells and centripetal FC is also distinct. It has been shown that post-transcriptional regulation of slbo protein levels is critical to proper border cell migration but does not occur in the centripetal FC. This study shows that in both cell groups, Notch initiates slbo expression and slbo is necessary and sufficient to repress its own expression as centripetal migration proceeds. SLBO protein can bind to a DNA sequence element located near the start site of its own promoter, and several matches to the canonical C/EBP binding site occur as well in the sequence of the slbo2.6 element that is sufficient to mediate autorepression, so this regulation is likely direct. Thus slbo adopts two strategies to fine-tune its levels: post-transcriptional regulation specifically in the border cell and transcriptional autoregulation in the both cell groups, as shown in this study (Levine, 2007).

It has been shown that DPP establishes the position of the bun expression boundary in the anterior FC and this boundary coincides with the posterior edge of the operculum eggshell structure. This study shows that as this boundary forms, slbo and bun expression patterns initially overlap and subsequently slbo and bun repress each other's expression to resolve respective expression patterns into two distinct cell groups. Notch signaling plays a central role in these interactions: Notch activates slbo expression in the centripetal FC and bun is required to antagonize Notch activation in posterior cells adjacent to the boundary (Levine, 2007).

The position of the boundary is highly sensitive to Notch activity so that increased Notch signaling leads to increased slbo2.6 expression both in the centripetal FC and, surprisingly, in adjacent columnar FC. Ectopic slbo expression in Nintra-expressing columnar FC at stage 10B is not associated with changes in FC proliferation and thus the spread of Notch activity likely relies on cell–cell signaling. This may arise either from (1) Notch activation of slbo expression in a large group of centripetal FC precursors that is not subsequently downregulated to a more narrow domain or (2) a Nintra-dependent activation of Notch signaling in adjacent columnar FC leading to cell contact-dependent posterior spread of slbo expression. The latter explanation is preferred because slbo2.6GAL4 expression expanded to almost all columnar FC in many egg chambers. In this way the position of the DPP-dependent cell fate boundary that defines the operculum is quite flexible but always drawn sharply by Notch activation (Levine, 2007).

While several canonical bun and Suppressor of Hairy [Su(H)] binding sites are located in the slbo2.6 element indicating slbo regulation by bun1 and Notch signaling, respectively, might be direct, several observations indicate slbo regulation at the boundary by bun is likely more complex. It has been noted previously that: (1) high levels of Notch and Notch target gene expression occur in anterior FC, with slightly reduced levels in centripetal FC in contact with bun-expressing cells and (2) increased levels of Notch targets occur in all cells of bun mutant clones at the centripetal FC boundary except those that contact bun+ cells. A parallel relationship is observed between bun and the Notch target slbo: (1) reduced levels of slbo occur in cells adjacent to bun-expressing cells in WT egg chambers, and (2) slbo expression occurs in bun mutant clones located at the centripetal FC boundary, with lower slbo levels in bun cells in contact with bun+ cells. Thus while bun may repress slbo directly, bun also antagonizes Notch activation of slbo in a non-cell autonomous manner. Consistent with this, bun clones removed from the centripetal FC do not lead to increased slbo expression and bun1 is not sufficient to block Nintra activation of slbo2.6 in the centripetal FC (Levine, 2007).

Notch modulation of slbo expression may be indirect as well. Because the Nts; slbo01310/slbo01310 double mutant egg chambers retain strong slbo-lacZ expression throughout the FC compared to Nts; slbo01310/+ egg chambers stained in parallel, it is hypothesized that Notch blocks SLBO protein's ability to repress its own expression. In this scenario, which must be further tested, the rapid reduction in slbo expression as centripetal migration proceeds results from both (1) decreasing Notch activation of slbo via Su(Hw) sites in the slbo promoter and (2) relief of a block on slbo autorepression. Consistent with rapid changes in Notch levels in the migrating centripetal FC, as slbo levels decrease a corresponding increase is seen in the levels of Cut protein, a key target of Notch repression in these cells. Because reduced dorsal appendages and opercula are seen in Nintra-expressing egg chambers, it is likely that rapid reduction in Notch levels is critical to permit the further patterning of anterior structures (Levine, 2007).

Dynamic interactions among bun, slbo and Notch signaling tightly regulate DE-cadherin levels in the centripetal FC. bun mutant clones lead to increased Notch signaling and DE-cadherin accumulation and Nintra is sufficient to increase DE-cadherin levels in the FC. slbo mutant clones lead to loss of DE-cadherin expression early and ectopic DE-cadherin levels late. Thus a recurring theme is that tight modulation of DE-cadherin levels is required in the FC at late oogenesis for epithelial transitions including border cell migration, centripetal FC migration and dorsal appendage elongation (Levine, 2007).

Recently, it has been shown that the bun homolog GILZ antagonizes the ability of C/EBP to activate expression of the key fat cell master regulator gene PPARγ2 (Peroxisome Proliferator Activator γ2) in adipogenic mesenchymal stem cells (Shi, 2003). GILZ binds a promoter element required for C/EBP-mediated activation and recruits HDAC1 (Histone Deacetylase 1) to repress PPARγ2 expression and promote the osteogenic cell fate. GILZ can also directly bind to C/EBP in vitro. Shi (2003) proposes that a balance of GILZ repressor and C/EBP activator in precursor mesenchymal cells regulates levels of PPARγ2, the master fat cell regulator. The similarities between these pathways are striking and it is proposed they constitute a conserved signaling cassette required for cell fate commitment. In support of a role for Notch in both, it has been shown that Notch signaling promotes adipogenesis in tissue culture , although the specific role of Notch in adipogenesis has been questioned. Targets may be conserved as well: expression of a gene homologous to PPARγ2 in the centripetal FC has been noted. While a connection between border cell specification and adipogenesis has been noted, slbo has no role in fly fat body formatio. However, bun expression hduring fat body formation has been detected suggesting that portions of this fly signaling cassette may operate in a general pathway required for storage cell differentiation (Levine, 2007).

Insulin levels control female germline stem cell maintenance via the niche in Drosophila

Stem cell maintenance depends on local signals provided by specialized microenvironments, or niches, in which they reside. The potential role of systemic factors in stem cell maintenance, however, has remained largely unexplored. This study shows that insulin signaling integrates the effects of diet and age on germline stem cell (GSC) maintenance through the dual regulation of cap cell number (via Notch signaling) and cap cell-GSC interaction (via E-cadherin) and that the normal process of GSC and niche cell loss that occurs with age can be suppressed by increased levels of insulin-like peptides. These results underscore the importance of systemic factors for the regulation of stem cell niches and, thereby, of stem cell numbers (Hsu, 2009).

The stem cell microenvironment (niche) controls stem cells, and niche aging leads to stem cell decline. The Drosophila germline stem cell (GSC) niche includes terminal filament cells, cap cells, and escort stem cells, and GSC fate and activity require direct contact with cap cells and exposure to niche-derived signals. GSCs also respond to systemic signals, such as Drosophila insulin-like peptides (DILPs), which directly modulate their proliferation. Increased age leads to decreased niche size and signaling and GSC loss. The molecular basis for age-dependent changes in the niche, however, remains poorly understood (Hsu, 2009).

Because diet influences aging, its effects on GSC maintenance were examined, exploiting the fact that GSCs can be unambiguously identified by their anteriorly anchored fusome (a membranous cytoskeletal structure) and by their juxtaposition to cap cells. A decrease was observed in GSC numbers in well-fed females over time. In females on a poor diet, however, the rate of GSC loss was significantly increased (Hsu, 2009).

Insulin secretion and signaling respond to diet and diminish in aging humans. Using a phosphoinositide 3-kinase reporter, reduced insulin signaling was found in older ovaries. To address if GSC maintenance requires insulin signaling, GSC numbers were measured in Drosophila insulin receptor (dinr) mutants. The dinr339/dinrE19 females contain slightly fewer GSCs at eclosion and lose them significantly faster than controls. GSC death was not observed in dinr339/dinrE19 or control germaria, suggesting that GSC loss results from differentiation (Hsu, 2009).

The chico1 homozygotes, which lack insulin receptor substrate, a major insulin pathway component, also show increased GSC loss. Thus, insulin signaling controls GSC maintenance. Next, whether DILP expression in germarial somatic cells could counteract the wild-type age-dependent GSC loss was tested. The c587-GAL4 driver was used to express a UAS-dilp2 transgene, encoding the DILP most closely related to human insulin, and thereby increase the local levels of insulin-like signals. GSC loss on rich and poor diets was significantly suppressed by DILP2 overexpression, although this was less pronounced in 4-week-old females on a poor diet. The less effective rescue on a poor diet could potentially be attributable to lower expression of the c587-GAL4 driver, to the actions of additional diet-dependent signals, or to a combination thereof. Nevertheless, these results suggest that the normal GSC loss observed in wild-type females as their age increases results largely from reduced insulin signaling (Hsu, 2009).

DILPs control GSC division directly, leading to a cell-autonomous dinr requirement. It was therefore asked whether dinr is required within GSCs for their maintenance. In genetic mosaics, homozygous dinr339 or dinrE19 GSCs are not lost at a higher rate than control GSCs, demonstrating that DILPs do not promote GSC maintenance directly (Hsu, 2009).

It was next hypothesized that insulin signaling may regulate GSC fate via the niche. Indeed, expression of wild-type dinr in somatic cells of dinr339/dinrE19 germaria rescued GSC loss. To examine dinr339/dinrE19 niche structure, terminal filament and cap cells were counted. Terminal filament cell numbers in dinr339/dinrE19 and control females are similar. In contrast, dinr339/dinrE19 females eclose with fewer cap cells and also lose them faster over time, suggesting that insulin signaling controls cap cell number during development and adulthood. Moreover, DILP2 overexpression suppresses the wild-type age-dependent cap cell number decrease. It is concluded that DILPs control GSC niche size and that the reduced cap cell numbers observed with increased female age at least in part reflect low insulin signaling levels (Hsu, 2009).

It was next asked whether DILPs control cap cell number directly. In mosaic germaria containing β-gal-negative dinr339 or control cap cells, the distribution (and average number) of β-gal-negative cap cells was indistinguishable, indicating that dinr does not control cap cell number cell autonomously. It is possible that a second cell type, such as terminal filament cells, produces an intermediate factor; alternatively, cap cells themselves may control their own maintenance via paracrine signaling (Hsu, 2009).

Notch signaling controls cap cell number during niche formation and in adults. Notch hyperactivation during development forms ectopic cap cells, leading to excess GSCs. Conversely, defective Notch signaling reduces niche size and GSC number. Notch activation is strongly detected in larval terminal filament and cap cells and is also detected in adult cap cells. Notch signaling was examined in dinr mutants using the E(spl)mβ-CD2 reporter. Every control germarium had strong CD2 expression in both terminal filament and cap cells. In contrast, CD2 levels were severely reduced in dinr339/dinrE19 germaria, indicating that insulin signaling controls Notch activation in the niche (Hsu, 2009).

It was next asked if the reduced cap cell number in dinr mutants was attributable to impaired Notch signaling. Weak hypomorphic dinrE19/dinr353 females have no reduction in GSC or cap cell number. Similarly, Notch heterozygotes (half the Notch dosage) have normal GSC and cap cell numbers. In contrast, dinrE19/dinr353 females heterozygous for Notch have significantly reduced GSC and cap cell numbers. A decrease in small cap cell number has been reported for Notch heterozygotes; this discrepancy may reflect slightly reduced insulin signaling in the latter study attributable to diet (Hsu, 2009).

To determine if Notch signaling is sufficient to rescue dinr defects, an activated form of Notch was expressed in the somatic cells of dinr339/dinrE19 germaria, and the GSC and cap cell loss phenotypes were rescued. These results and the genetic interaction between dinr and Notch are consistent with the insulin pathway acting upstream or in parallel to Notch. Nevertheless, the reduced Notch reporter levels in dinr mutants favor the model that insulin signaling leads to Notch activation, thereby controlling cap cell number and, indirectly, GSC maintenance (Hsu, 2009).

GSCs and terminal filament cells express the Delta ligand for Notch, and removal of Delta function from GSCs has been reported to affect niche activity. It was reasoned that dinr could be required in GSCs, terminal filament cells, the cap cell population, or a combination thereof to control Delta production and Notch activation. dinr mosaic germaria were examined in which all GSCs were dinr339 homozygous, and the number of cap cells in those germaria was indistinguishable from control numbers, suggesting that dinr is not required in GSCs for Notch signaling. DILPs may instead regulate Delta within terminal filament or cap cells or, alternatively, act via other intermediate signals to regulate Notch activation within the niche (Hsu, 2009).

Cap cell and GSC numbers correlate. Indeed, in germaria containing control β-gal-negative cap cells (control C1), total cap cell and GSC numbers are roughly proportional. Remarkably, despite similar cap cell numbers, a significant fraction of germaria in which dinr mutant cap cells are present contains fewer GSCs relative to control C1 or C2 (i.e., germaria without cap cell clones from dinr mosaics). Thus, although dinr does not control cap cell number per se autonomously, it is required within cap cells either for the optimal production and/or secretion of a GSC maintenance factor(s) or to promote GSC attachment (Hsu, 2009).

Niche-derived bone morphogenetic protein (BMP) signals directly stimulate GSCs to repress differentiation. To test if insulin signaling controls BMP pathway activation in GSCs, the Dad-lacZ reporter was used. Dad-lacZ levels in dinr339/dinrE19 and control females are indistinguishable, showing that dinr does not control BMP signaling. Insulin signaling in cap cells must therefore control another GSC maintenance signal and/or the cap cell-GSC association (Hsu, 2009).

To investigate if dinr controls the physical interaction between cap cells and GSCs, the percentage of dinr339 versus control cap cells directly contacting GSCs was measure in mosaic germaria. Indeed, 21% of dinr339 cap cells contact GSCs, compared with 50% of control cap cells, indicating that dinr339 cap cells have significantly reduced attachment to GSCs. These results suggest that insulin signaling in cap cells controls their association with GSCs. Alternatively, insulin signaling may regulate the production of a short-range GSC maintenance signal, such that only GSCs in contact with dinr mutant cap cells are affected (Hsu, 2009).

E-cadherin-mediated adhesion between cap cells and GSCs is required for retaining GSCs in the niche. Therefore E-cadherin levels were measured at the GSC-cap cell junction. In controls, it was found that E-cadherin levels vary with changes in the fusome, a membranous cytoskeletal structure. When the fusome is round, its predominant morphology, there is a higher intensity of E-cadherin at the junction, although when the fusome is elongated, the intensity is lower. The intensity of E-cadherin at the junction of cap cells with GSCs displaying elongated fusomes in dinr339/dinrE19 mutants is similar to that of control. In contrast, the round fusome GSC-cap cell junctions contain significantly lower E-cadherin levels in dinr mutants than in controls. These results suggest that insulin signaling influences E-cadherin levels at the GSC-cap cell junction and may explain the age-dependent E-cadherin reduction that contributes to GSC loss (Hsu, 2009).

These studies demonstrate that systemic insulin-like signals integrate inputs from diet and age to regulate GSC maintenance via the niche. Specifically, it is proposed that DILPs control cap cell number via Notch and also E-cadherin- mediated GSC retention within the niche. Because diet and insulin signaling control GSC proliferation, it is also likely that the proliferation decline reported in older females results from reduced insulin signaling. These results also provide insights into recent findings that systemic factors from young mice can restore Notch activation and skeletal muscle progenitor proliferation and regenerative capacity to old mice in heterochronic parabiotic pairings. Finally, the results are intriguing in light of the well-established connection between low insulin signaling, restricted diet, and extended lifespan and of studies in C. elegans suggesting that GSCs may have a negative effect on longevity. It is conceivable that excessive stem cell activity in general is deleterious and that slight decreases in stem cell number or activity with age as a result of reduced insulin signaling may actually promote longevity (Hsu, 2009).

Regulation of epithelial stem cell replacement and follicle formation in the Drosophila ovary

Organisers control the patterning and growth of many tissues and organs. Correctly regulating the size of these organisers is crucial for proper differentiation to occur. Organiser activity in the epithelium of the Drosophila ovarian follicle resides in a pair of cells called polar cells. It is known that these two cells are selected from a cluster of equivalent cells. However, the mechanisms responsible for this selection are still unclear. This study presents evidence that the selection of the two cells is not random but, by contrast, depends on an atypical two-step Notch-dependent mechanism. This sequential process begins when one cell becomes refractory to Notch activation and is selected as the initial polar cell. This cell then produces a Delta signal that induces a high level of Notch activation in one other cell within the cluster. This Notch activity prevents elimination by apoptosis, allowing its selection as the second polar cell. Therefore, the mechanism used to select precisely two cells from among an equivalence group involves an inductive Delta signal that originates from one cell, itself unable to respond to Notch activation, and results in one other cell being selected to adopt the same fate. Given its properties, this two-step Notch-dependent mechanism represents a novel aspect of Notch action (Vachias, 2010).

Controlling the number of final polar cells (PCs) is essential in limiting the activity of the organizer, which triggers the differentiation of the neighbouring cells. Indeed, increasing or decreasing the PC number affects the number of border cells (population of about eight cells in wild type immediately adjacent to the PC). As supernumerary PC are quite rare in wild type, a robust mechanism must exist to guarantee the selection of only two cells from among a group of cells with similar developmental histories and similar developmental potential. At least three models can be invoked to explain such a selection: (1) the two cells are chosen randomly; (2) two cells acquire similar selective properties; or (3) two cells acquire selective properties that are different from each other, as well as from the others. The data rule out the first two, as it was demonstrated in several ways that the polar-fated cells (pfcs) are no longer equivalent at the time of selection, and that the two selected cells display different properties. By contrast, the results fit and even improve upon, the third model by revealing that the selection of the two cells with the same eventual fate occurs sequentially, and that the selection of the second depends on a signal from the first. It is proposed that among the cluster, one pfc becomes unable to respond to the N pathway and also resistant to apoptosis by a mechanism that remains to be determined. In turn, this pfc produces a Dl signal that activates the N pathway in a second pfc and prevents it from being eliminated by apoptosis. In parallel, this signalling also promotes an efficient apoptotic process that eliminates all of the other pfc. Cells other than the N-refractory pfc (follicle cells, stalk cells, germline cells and/or other pfc) could participate to some extent in the specific N activation in the second selected pfc. Nevertheless, the data show that the role of the N-refractory cell in producing Dl is preponderant. This is in agreement with multiple studies in which it has been shown that cells that do not respond to N activation are more efficient in producing the ligands. One important aspect of the mechanism described is that the selection of the second pfc is tightly linked to the elimination of the other pfc, as the same signal is required for both. This renders the mechanism of PC pair selection highly efficient. The implication of the N pathway in the protection of cells from apoptosis or in the induction of apoptosis has already been described in several developmental contexts. In the process of PC pair selection, the regulation of DIAP1 expression by Notch possibly provides a direct link that merits further exploration (Vachias, 2010).

The mechanisms by which one cell becomes more efficient in sending the signal remain unknown. Recently, it has been shown that the first anterior and posterior pfc are specified in region IIb of the germarium and that additional pfc are formed later during stages 1 and 2 (Nystul, 2010). One possibility is that this difference in timing is necessary for the first specified pfc to become refractory to N activation and thus more efficient in producing Dl. Moreover, the difficulty in detecting neur mRNA or protein prevents the authors from rejecting the possibility that neur is unequally distributed among the pfc. Similarly, it would also be interesting to determine why one pfc is more efficient at receiving the signal. The simplest explanation is that this depends upon the extent of contact between the sending and the receiving cells (Vachias, 2010).

Three modes of action -- lateral inhibition, lineage decisions and boundary formation -- have been proposed to describe how N activity regulates cell differentiation. Two of the main criteria used to define these modes are the state of the sending and receiving cells (equivalent or not), and the outcomes induced (positive or negative). In lateral inhibition, N signalling occurs between equivalent cells with roughly equivalent developmental properties. The signal-sending activity increases in one cell, which in turn increases the signal-receiving activity in the others, preventing them from adopting the fate of the signal-sending cell (negative outcome). In lineage decisions, the signalling happens between daughter cells and depends on the asymmetrical inheritance of some N regulators, such as Numb or Partner of Numb. Their presence in one cell autonomously antagonises N activation, whereas the activation does occur in the sibling cell, preventing it from taking on the same identity (negative outcome). During boundary formation, N signalling takes place at the interface between two compartments of differentially fated cells. The cells of the first row of each compartment are both the sending and the receiving cells and acquire identical new characteristics (positive outcome). In the mechanism described in this study, PC pair selection commences within a group of equivalent cells, with one cell acquiring specific properties and then signalling to another one to induce an identical fate (positive outcome). Based on the criteria mentioned above, this mechanism cannot be classed with either of the first two modes. First, it differs from the lineage decision mode, as the outcome of this mode is necessarily negative and because the two selected polar cells are not always sibling cells. Second, although the mechanisms used in PC pair selection and in the lateral inhibition mode both act upon a set of equivalent cells and generate Dl-expressing cells and N-expressing cells, the outcomes established are different. The mechanism described in this study is more closely related to the inductive signalling mode, as both generate positive outcomes and promote the acquisition of organizing activities by the Dl-sending and -receiving cells. But the fact that, in the signalling used to select the PC pair, the Dl-sending cell might be in part refractory to N activation because of differentially regulated endocytic trafficking possibly reveals an atypical N mechanism. Support for the existence of such a mechanism comes from recent data about the role of EGFR signalling and phyllopod during Drosophila eye formation. In that case, EGFR signalling suppresses some Nact phenotypes by activating phyllopod, which acts to regulate the residence time of the N components in early endocytic vesicles. In light of these observations, analysis of known trafficking regulators during PC pair formation could help to resolve the mechanisms that allow one pfc to become refractory to N activation. Thus, the data might define a novel aspect of the inductive signalling mode or, alternatively, might be representative of a fourth mode of N action. The discovery of other, similar examples would allow clarification of this point (Vachias, 2010).

Evolution of Notch regulation of the ventral midline in insect embryos

The ventral midline is a source of signals that pattern the nerve cord of insect embryos. In dipterans such as the fruitfly Drosophila melanogaster (D.mel.) and the mosquito Anopheles gambiae (A.gam.), the midline is narrow and spans just 1–2 cells. However, in the honeybee, Apis mellifera (A.mel.), the ventral midline is broad and encompasses 5–6 cells. slit and other midline-patterning genes display a corresponding expansion in expression. Evidence is presented that this difference is due to divergent cis regulation of the single-minded (sim) gene, which encodes a bHLH-PAS transcription factor essential for midline differentiation. sim is regulated by a combination of Notch signaling and a Twist (Twi) activator gradient in D.mel., but it is activated solely by Twi in A.mel. It is suggested that the Twi-only mode of regulation—and the broad ventral midline—represents the ancestral form of CNS patterning in Holometabolous insects (Zinzen, 2006).

Dorsoventral (DV) patterning of the D.mel. embryo is initiated by a nuclear gradient of the Dorsal (Dl) transcription factor, which differentially regulates at least 50 target genes in a concentration-dependent manner. Most of these genes encode sequence-specific transcription factors and components of cell signaling pathways that control gastrulation. Genetic analyses, microarray screens, and DNA-binding assays with defined DV enhancers have elucidated a gene network of functional interconnections among 40 Dl target genes. The goal of this study is to use this information to understand the evolution of DV patterning among divergent insects (Zinzen, 2006).

In D.mel., the Dl gradient leads to localized activation of Notch signaling in single rows of cells straddling the presumptive mesoderm (Bardin, 2006; De Renzis, 2006). This localized Notch signal works together with the bHLH factor Twi to activate sim expression. After invagination of the ventral furrow, the sim-expressing cells converge at the ventral midline, and the bHLH-PAS Sim transcription factor activates target genes required for midline differentiation (Zinzen, 2006).

The ventral midline is a source of localized signals that help pattern the nerve cord. For example, a transmembrane protease encoded by rhomboid (rho) produces a secreted source of the EGF ligand Spitz. Sim also leads to the expression of slit, which encodes a secreted repellant that binds the Roundabout receptor and inhibits the growth of axonal projections across the midline (Zinzen, 2006).

Sim target genes are highly conserved in A.mel., and in situ hybridization assays reveal that they are similarly expressed in the ventral midline of the developing honeybee nerve cord. However, their expression is significantly broader in A.mel. than in D.mel., 5–6 cells versus 1–2 cells, respectively. Evidence is presented that this broader midline is due to divergent regulation of sim expression. In A.mel., sim is regulated solely by Twi and does not depend on Notch signaling, whereas Notch is responsible for restricting sim to single rows of cells in the early D.mel. embryo (e.g., Bardin, 2006; De Renzis, 2006). It is proposed that the acquisition of Notch dependence at the sim locus is sufficient to account for restricted expression of sim and the narrow midline in D.mel (Zinzen, 2006).

The ventral midline in D.mel. embryos encompasses just 1–2 cells that express signaling molecules such as rho and slit. In contrast, orthologous genes are expressed in 5–6 cells in the honeybee embryo. Notably, the initial expression pattern of A.mel. sim is expanded, and the sim-staining pattern remains broad after convergence of the midline following the spreading of neurogenic ectoderm over the mesoderm. In addition to expression in the ventral midline, sim staining is also detected in more lateral clusters of cells exhibiting segmental periodicity in A.mel. embryos; these might be neurons or glial cells migrating away from the midline (Zinzen, 2006).

Previous studies suggest that sim functions as a 'master control gene' to direct differentiation of the ventral midline in D.mel. To determine whether the expanded sim pattern in honeybees can account for the broadening of the midline, whether ectopic sim expression is sufficient to induce transcription of target genes such as slit and rho. The D.mel. sim-coding sequence was placed under the control of the eve stripe 2 enhancer (eve.2) and expressed in transgenic embryos. There is transient sim expression in the stripe 2 domain of early (stages 5–7) embryos in addition to the endogenous pattern (mesectoderm) in the presumptive ventral midline (Zinzen, 2006).

The initial sim expression pattern is established by a distal 5′ enhancer that contains linked Dl-, Twi-, and Suppressor of Hairless [Su(H)]-binding sites. Expression is maintained by a separate autoregulatory enhancer containing Sim/Tango-binding sites; Tango is a ubiquitous bHLH-PAS transcription factor that forms heterodimers with Sim. Though the eve stripe 2 enhancer mediates transient activation, autoregulation maintains expression of the endogenous sim gene in the ventral neurogenic ectoderm of advanced-stage embryos, but not in the mesoderm or dorsal ectoderm (Zinzen, 2006).

Ectopic sim expression leads to the induction of various target genes, including rho, slit, sog, and the transcription factor otd. These results provide evidence that ectopic sim expression is sufficient to expand the ventral midline in D.mel. In principle, the altered midline seen in the honeybee embryo could be explained by a change in sim regulation. The distal 5′ enhancer that establishes sim expression is the most likely site of change, since the autoregulatory enhancer merely maintains expression within the limits of the established pattern (Zinzen, 2006).

To determine the basis for the distinct sim expression patterns in flies and honeybees, it was necessary to isolate the early sim enhancer from A.mel. However, the identification of homologous enhancers is complicated by the rapid turnover of noncoding DNA sequences in insect genomes. For example, the 5′ flanking regions of the sim loci in D.mel. and A.gam. lack simple sequence homology, even though they belong to the same order (Diptera). Nonetheless, it was possible to identify the early sim enhancer in A.gam. based on the clustering of Dl-, Twi-, and Su(H)-binding sites. The D.mel. and A.gam. enhancers are located in similar positions relative to the sim transcription unit (Zinzen, 2006).

A.mel. is a member of the order Hymenoptera and is highly divergent from D.mel. Computational methods used for the in silico identification of the A.gam. sim enhancer were further developed to ensure the accurate identification of the sim enhancer in A.mel. The current method (ClusterDraw2) employs position-weighted matrices (PWMs) to identify binding motif clusters (Zinzen, 2006).

The efficacy of the method was tested by surveying ~50 kb genomic intervals encompassing the sim loci of D.mel. and A.gam.. PWMs of Dl, Twi, Snail, and Su(H) were used in various combinations and individually. The best binding site clusters coincide exactly with the known sim enhancers (Zinzen, 2006).

ClusterDraw2 was used to survey a ~50 kb genomic DNA interval encompassing the sim locus of A.mel.. The best prediction occurs in the 5′ flanking region of the gene, similar to the locations of the fly and mosquito enhancers. However, while the D.mel. and A.gam. sim enhancers contain several optimal Su(H)-binding sites, the A.mel. cluster lacks such sites, but contains several high-scoring Twi sites. This is consistent with the possibility that A.mel. sim is regulated by Twi alone, rather than by the combination of Twi+Notch (Zinzen, 2006).

A 2.2 kb genomic DNA fragment encompassing the predicted A.mel. sim enhancer directs lateral stripes of lacZ expression in transgenic D.mel. embryos. A similar pattern was obtained with a 471 bp fragment containing the predicted Twi-binding sites. This pattern encompasses 3–4 cells on either side of the presumptive mesoderm, similar to the expression of the endogenous A.mel. sim gene, but distinct from the single-row sim patterns in D.mel. and A.gam. (Zinzen, 2006).

The fly, honeybee, and mosquito sim enhancers were crossed into various genetic backgrounds to determine the basis for their distinct expression patterns. The D.mel. m5/8 enhancer was also examined. It is located within the Enhancer of split (E(spl)) complex, where it controls the expression of the m5 and m8 genes within the mesectoderm. The m5/8 enhancer directs lacZ expression in a pattern that is virtually identical to that produced by the D.mel. sim enhance (Zinzen, 2006).

Transgenic D.mel. embryos carrying an eve.2::NICD fusion gene exhibit ectopic Notch signaling in the eve stripe 2 domain. The m5/8-lacZ transgene is strongly induced in the neurogenic ectoderm and dorsal ectoderm, but not in the mesoderm, where the Sna repressor is present. The D.mel. sim-lacZ transgene displays only modest ectopic induction by the eve.2::NICD transgene; this induction appears as a 'pyramid' limited to ventral regions of the neurogenic ectoderm. This pyramid coincides with the intersection of ectopic Notch signaling and the endogenous Twi gradient. The different patterns ('pyramid' versus 'column') seen for the sim and m5/8 enhancers appear to reflect activation by Notch+Twi or regulation by Notch alone, respectively. The m5/8 enhancer contains an SPS (Su(H) Paired Site) motif, and it has been suggested that the endogenous m8 gene is activated solely by Notch signaling (Zinzen, 2006).

The A.mel. sim enhancer is not activated by the eve.2::NICD transgene, consistent with the absence of Su(H) sites in this enhancer. To determine whether it is activated by Twi, the lacZ fusion gene was crossed into embryos carrying an hsp83::twi-bcd-3′UTR transgene that produces high levels of Twi transcripts at the anterior pole. The resulting ectopic anteroposterior Twi protein gradient induces intense expression of the lacZ reporter gene directed by the A.mel. sim enhancer. In contrast, neither the D.mel. sim enhancer nor the m5/8 enhancer is induced by this ectopic gradient. Finally, the D.mel. sim enhancer is inactive in mutant embryos derived from germline clones lacking Su(H) activity, whereas the honeybee sim enhancer is fully active. Thus, unlike the D.mel. sim enhancer, the A.mel. enhancer does not rely on Notch signaling (Zinzen, 2006).

The preceding analysis suggests that the D.mel. sim enhancer is activated by Twi and Notch signaling, whereas the A.mel. sim enhancer is activated solely by Twi. These distinct modes of regulation are reflected by the composition of binding sites in the different enhancers. The A.mel. enhancer contains several optimal Twi sites, but it lacks unambiguous Su(H) sites. In contrast, the D.mel. enhancer contains several optimal Su(H) sites, but just one optimal Twi site. Both enhancers contain binding sites for the Sna repressor, which inhibits expression in the mesoderm (Zinzen, 2006).

sim regulation was examined in the mosquito, A.gam., to determine whether the midline of ancestral dipterans might have been regulated solely by Notch signaling, as seen for the fly m5/8 enhancer. The A.gam. genome contains a clear ortholog of the sim gene, expressed in a single row of cells in the mesectoderm, similar to the pattern seen in D.mel. The A.gam. sim enhancer directs sporadic expression within the mesectoderm of transgenic D.mel. embryos, but it is strongly induced by the eve.2::NICD transgene. This response is similar to that obtained with the D.mel. m5/8 enhancer, but it is distinct from the 'pyramid' pattern seen for the D.mel. sim enhancer (Zinzen, 2006).

To determine whether the sim loci of other drosophilids are regulated by Twi+Notch, as seen in D.mel., or Notch alone, sim enhancers from D. pseudoobscura (D.pse.) and D. virilis (D.vir.) were tested in transgenic eve.2::NICD D.mel. embryos. Surprisingly, these enhancers behave like the A.gam. sim enhancer: they are expressed throughout the neurogenic ectoderm and dorsal ectoderm (“column”) in response to Notch signaling, rather than the “pyramid” pattern indicative of Notch+Twi regulation. These observations suggest that the evolution of sim regulation is highly dynamic, although there is no obvious difference in the number or quality of Su(H) and Twi sites in the different drosophilid enhancers. Perhaps a subtle shift in the organization of binding sites distinguishes regulation by Notch alone versus Notch+Twi (Zinzen, 2006).

Dynamic filopodia transmit intermittent Delta-Notch signaling to drive pattern refinement during lateral inhibition

The organization of bristles on the Drosophila notum has long served as a popular model of robust tissue patterning. During this process, membrane-tethered Delta activates intracellular Notch signaling in neighboring epithelial cells, which inhibits Delta expression. This induces lateral inhibition, yielding a pattern in which each Delta-expressing mechanosensory organ precursor cell in the epithelium is surrounded on all sides by cells with active Notch signaling. This study shows that conventional models of Delta-Notch signaling cannot account for bristle spacing or the gradual refinement of this pattern. Instead, the pattern refinement observed using live imaging is dependent upon dynamic, basal actin-based filopodia and can be quantitatively reproduced by simulations of lateral inhibition incorporating Delta-Notch signaling by transient filopodial contacts between nonneighboring cells. Significantly, the intermittent signaling induced by these filopodial dynamics generates a type of structured noise that is uniquely suited to the generation of well-ordered, tissue-wide epithelial patterns (Cohen, 2010).

The analysis of lateral inhibition in the notum described in this study reveals an extended period of plasticity during pupal development in which a gradual process of pattern refinement takes place, after which cells take on an epithelial fate or undergo an asymmetric division to initiate the development of the mechanosensory organ. Importantly, the path of this pattern formation process and the final spacing observed can be quantitatively reproduced using a model of Delta-Notch signaling in which cells are able to exchange signals via a population of dynamic, basal filopodia (parameterized based on in vivo observations) that interact over a distance of several cell diameters. As quantitatively predicted by a model of filopodia to filopodia signaling, changes in protrusion length and dynamics lead to corresponding changes in bristle spacing, implying the involvement of filopodia in both signal sending and receiving cells. Moreover, the model suggests that protrusions are likely to determine the spacing of bristle precursors even in instances in which they transmit the minority of the total Delta-Notch signal (Cohen, 2010).

Although previous authors have suggested roles for long, stable protrusions in cell-cell signaling events, this analysis shows that the generation of a robust, well-ordered, and well-spaced pattern of bristle precursors across an entire tissue requires filopodia that are dynamic. In the case of the notum, these protrusions are actin based, and appear to be generated through the action of Rac and the SCAR complex (Georgiou, 2010). Although this is unexpected, given the well-described involvement of the SCAR complex in the generation of lamellipodia, there are precedents for the involvement of the SCAR complex in the generation of filopodia in Drosophila cells. Moreover, filopodia have previously been seen predominating in a three-dimensional (3D) tissue context. It is also noted that the role described here for SCAR-dependent protrusions in determining precursor cell spacing is strongly supported by the recent identification of the full complement of components of the SCAR complex (SCAR/WAVE, Abi, Sra1, Hem/Kette, and HSPC300/Sip1) in a recent genome-wide RNAi screen as genes required to maintain bristle spacing within the notum without affecting subsequent mechanosensory organ development (Mummery-Widmer, 2009). Loss of SCAR function leads to the specific loss of F-actin and dynamic protrusions within the basolateral domain without affecting cell morphology, cell size, cell polarity, or endocytosis (Georgiou, 2008; Georgiou, 2010), making it less likely that SCAR functions by directly altering the Notch signaling pathway. Additionally, it was found that scar mutant cells within the external sensory organ (but not RacN17 expressing cells) differentiate correctly despite having an altered morphology. Conversely, as recently reported by Rajan (2009) and as seen in the Mummery-Widmer screen, the Arp2/3 complex and WASp appear to function in Notch signaling following precursor cell division, leading to a loss of bristles from the nota of adult flies (Cohen, 2010).

Since physical tension has been shown to enhance Notch cleavage and hence Delta-Notch signaling, it is possible that forces generated through the retraction of actin-based protrusions engaged in signaling could function to enhance Notch activation. Testing this hypothesis, however, will likely have to await the future development of tools enabling Notch cleavage to be imaged as it occurs in vivo (Cohen, 2010).

Significantly, theoretical analysis suggests that the dynamics of these actin-based filopodia, as measured in vivo, could induce intermittent Delta-Notch signaling to drive a process defined as 'pattern refinement' -- whereby the self-organizing pattern improves steadily over an extended period of developmental time as cells undergo switches in their gene expression profile. This theoretical prediction fits with the observed shift in bristle-precursor cell patterns observed during development of the notum, and the extended period of time over which the bristle precursor pattern remains labile, as measured using laser ablation and a temperature-sensitive Notch allele. In theory, since intermittent signaling could be generated in the absence of protrusions using cell autonomous oscillations in Notch-Delta signaling, the oscillations seen in some other systems could serve a similar function-aiding gradual pattern refinement. Dynamic filopodia, however, have several features that make them uniquely suited to a role in pattern refinement. A network of filopodia can be quickly established and eliminated, and the length and direction of filopodia tuned in order to define a precise gradient of signaling over a distance of several cell diameters. Thus, dynamic filopodial signaling appears to be an ideal alternative to morphogen diffusion as a mediator of developmental signaling at a distance (Cohen, 2010).

The salvador-warts-hippo pathway is required for epithelial proliferation and axis specification in Drosophila

In Drosophila, the body axes are specified during oogenesis through interactions between the germline and the overlying somatic follicle cells. A Gurken/TGF-alpha signal from the oocyte to the adjacent follicle cells assigns them a posterior identity. These posterior cells then signal back to the oocyte, thereby inducing the repolarization of the microtubule cytoskeleton, the migration of the oocyte nucleus, and the localization of the axis specifying mRNAs. However, little is known about the signaling pathways within or from the follicle cells responsible for these patterning events. It study shows that the Salvador Warts Hippo (SWH) tumor-suppressor pathway is required in the follicle cells in order to induce their Gurken- and Notch-dependent differentiation and to limit their proliferation. The SWH pathway is also required in the follicle cells to induce axis specification in the oocyte, by inducing the migration of the oocyte nucleus, the reorganization of the cytoskeleton, and the localization of the mRNAs that specify the anterior-posterior and dorsal-ventral axes of the embryo. This work highlights a novel connection between cell proliferation, cell growth, and axis specification in egg chambers (Meignin, 2007).

Multicellular organisms develop through an orchestrated temporal and spatial pattern of cell behavior, which is controlled by cell-to-cell signaling. In Drosophila melanogaster, the establishment of the embryonic axes occurs in the oocyte and depends on a sequence of signals between the germline and the somatic cells. First, Gurken (Grk) signals from the oocyte to the adjacent follicle cells (FCs), in which Torpedo (Top, EGFR) is activated, and this signal instructs them to adopt a posterior identity. The posterior FCs (PFCs) then send an unidentified signal back to the oocyte, leading to the movement of the nucleus from the posterior to the dorsoanterior (DA) corner and the repolarization of the microtubule (MT) cytoskeleton, with the minus ends at the anterior and lateral cortex and the plus ends at the posterior. This repolarization results in the localization of the mRNAs that encode key patterning factors. grk mRNA is next to the nucleus at the DA corner of the oocyte. At this corner, Grk instructs the overlying FCs to adopt dorsal fates. In contrast, oskar (osk) and bicoid (bcd) mRNAs are localized at the posterior and anterior pole, respectively, thus defining the anterior posterior (AP) embryonic axis and the germ cells. Although several genes are required in the FCs to control these events, little is known about the signaling pathways within and from the FCs (Meignin, 2007).

One of the genes required for axis formation during oogenesis is the tumor suppressor merlin (mer). However, it is not known whether Mer influences axis specification directly or what signaling pathways lie downstream of Mer. In other tissues, Mer is known to activate the Salvador Warts Hippo (SWH) pathway, which is a tumor-suppressor pathway. Inhibition of the SWH pathway leads to a characteristic overgrowth phenotype in adult organs because of an overproliferation of cells, increased cell growth, and defects in apoptosis. To test whether the SWH pathway is required in the function of Mer in axis formation, the localization of grk, bcd, and osk mRNA was examined in egg chambers with warts (wts) and hippo (hpo) mutant FCs. wts and hpo encode two serine/threonine kinases that are core components of this pathway. In both cases, grk mRNA is mislocalized at the posterior, osk mRNA is mislocalized at the center, and bcd mRNA is mislocalized at the posterior and anterior poles. The mislocalization of these mRNAs could be due to failure of the MTs to repolarize, as has been previously shown in grk/EGFR and mer mutants. In wild-type oocytes, the MTs are organized in an AP gradient. In contrast, in egg chambers with hpo mutant FCs, the MTs are distributed diffusely all over the oocyte cytoplasm. Considering these results, together with previous characterizations of similar phenotypes, it is concluded that the oocyte cytoskeleton in mutant egg chambers for the SWH pathway is disorganized with the MT plus ends at the center and the minus ends at the anterior and posterior poles. These defects resemble those described in oocytes lacking the Grk signal. In wts mutants, however, Grk protein is detected at the posterior pole, where grk mRNA is mislocalized. This demonstrates that the axis-specification defects in wts mutant egg chambers are not a consequence of the absence of Grk protein (Meignin, 2007).

It was shown that mer is required in the FCs for the repolarizing signal back to the germline and consequently for the migration of the oocyte nucleus from the posterior to the DA corner. Similarly, when mutant FC clones were generated for wts, hpo, and expanded (ex), an activator of the SWH pathway, the oocyte nucleus fails to migrate to the anterior. Another protein that is upstream of the SWH pathway is the giant atypical cadherin fat (ft). However, egg chambers with ft mutant FCs show no defects in oocyte polarity, and both the nucleus and Staufen (Stau) [a marker for osk mRNA] are always properly localized. In other epithelia, hpo and wts are required to repress the activity of Yorkie (Yki) and overexpression of yki phenocopies loss-of-function mutations of hpo and wts. Similarly, it was found that overexpression of yki in the FCs also causes the mislocalization of Stau and the oocyte nucleus. These results indicate that the SWH pathway, with the exception of Ft, might be required for the repolarizing signal back from the FCs to the oocyte (Meignin, 2007).

Because this signal is sent by the PFCs, whether the SWH pathway is required only in these cells was analyzed. In egg chambers with wild-type PFCs within an otherwise hpo or wts mutant epithelium, as well as in hpo, wts, and ex germline clones, the oocyte polarity is unaffected. However, in egg chambers with hpo mutant PFCs in an otherwise wild-type epithelium, the oocyte nucleus is mislocalized. It was also observed that when only a few cells at the posterior are mutant, Stau localizes in the region of the oocyte that faces the posterior wild-type cells. The SWH pathway is not required in the polar cells for axis determination because egg chambers with hpo or wts mutant PFCs and wild-type polar cells show oocyte polarity defects. It is concluded that the SWH pathway is required only in the PFCs to induce axis specification in the oocyte (Meignin, 2007).

In contrast to the monolayered wild-type epithelium, anterior and posterior, but not lateral, hpo and wts mutant cells form a bilayered, and occasionally a multilayered, epithelium. Given that the SWH pathway is required to control proliferation in epithelia of imaginal discs, whether the bilayered epithelium is a result of overproliferation was analyzed. At stage 6 of oogenesis, wild-type FCs undergo a Notch-dependent switch from a mitotic cell cycle to an endocycle. For this reason, phosphohistone 3 (PH3), a marker for mitotic cells, is detected only until that stage and never later. In contrast, hpo mutant anterior and posterior FCs are often positive for PH3 at stage 7-10B, indicating that these cells are still dividing. Similar results are obtained in yki overexpressing FCs. Taken together, these findings show that the SWH pathway is required for the control of proliferation at the anterior and posterior FCs (Meignin, 2007).

The formation of a multilayered epithelium was also observed in stage 3-5 mutant FCs, although the number of dividing cells is similar to that of the wild-type. It has been recently shown that the aberrant orientation of the mitotic spindle in the FCs results in the formation of a multilayered epithelium. Therefore the orientation of the mitotic spindle was analyzed in wild-type and hpo mutant cells. It was observed that, contrary to wild-type cells, the mitotic spindle in mutant FCs is often at an angle or perpendicular to the membrane. This aberrant orientation disrupts the remaining daughter cells within the same plane, thereby resulting in a bilayered epithelium (Meignin, 2007).

Often, tumor suppressors are important for the polarity of the epithelia. To determine whether this is the case for the SWH pathway, the atypical (novel) Protein Kinase C (nPKC), an apical marker, and Disc large (Dlg), a lateral marker, were examined in the FCs. In wild-type cells, as well as in hpo mutant FCs that maintain a monolayer epithelium, nPKC and Dlg localize at the apical and lateral membrane, respectively. However, when the mutant epithelium forms several layers of cells, nPKC and Dlg are often mislocalized, with a reduction of the nPKC staining and an expansion of the Dlg-positive membrane. Nevertheless, a certain degree of the polarity in these cells is maintained because nPKC is always apical in the cells that are in contact with the oocyte (Meignin, 2007).

Because SWH pathway mutant cells do not exit mitosis and keep dividing, it is possible that their differentiation is impaired. To address this question, the expression of Fasciclin III (FasIII) and eyes absent (eya) were analyzed in wild-type and wts and hpo mutant FCs. FasIII and Eya are downregulated in a Notch-dependent manner in the main-body FCs after stage 6 of oogenesis. However, the levels of FasIII in hpo mutant PFCs and Eya in wts mutant PFCs remain high after stage 6. To further assess the effect of the SWH pathway on the Notch-dependent maturation of the FCs, the expression of Hindsight (Hnt), a transcription factor that is upregulated by Notch signaling in all FCs was examined.. In hpo posterior FC clones, this Hnt upregulation is blocked. Contrary to notch clones, however, hpo lateral and anterior clones do not show defects in FasIII, Eya, or Hnt expression. Furthermore, border, centripetal, and stretched cells that are mutant for hpo migrate normally. Considering all these results together, it is concluded that the SWH pathway is essential for the PFCs to fully differentiate (Meignin, 2007).

The findings described above, together with the proliferation defects in hpo and wts mutant cells, suggest that the SWH pathway is required for Notch signaling. To test whether this is the case, the expression of universal Notch transcriptional reporters was analyzed in wild-type and hpo mutant FCs. In wild-type egg chambers, the Notch reporter E(spl)mß7-lacZ is expressed in all FCs upon Notch activation at stage 6 of oogenesis. In contrast, it was found that in hpo mutant cells, the levels of E(spl)mß7-lacZ are weakly reduced in 53% of the clones and normally expressed in the rest. It has been shown that in wing imaginal discs, mer and ex are required to control Notch localization in the cell and consequently its activity. Similarly, the subcellular distribution of Notch is affected in hpo mutant FCs. Contrary to the wild-type, in which Notch accumulates in the apical membrane, Notch expands to other membranes and is often detected in clusters in hpo clones. The results point out that hpo is essential in the PFCs for the Notch-dependent expression of several differentiation markers, such as FasIII, Eya, and Hnt, and for Notch subcellular localization. These observations and the weak defects on the Notch reporters support a function of the SWH pathway in modulating Notch signaling (Meignin, 2007).

Because the SWH pathway is required for the polarization of the oocyte, as well as for the differentiation of the PFCs, whether the mutant cells are competent to respond to Grk and indeed adopt a posterior fate was analyzed. Dystroglycan (DG) is expressed in all FCs at early stages of oogenesis, but upon Grk signaling, DG forms an AP gradient with lower levels at the PFCs. The fact that this Grk-dependent gradient of DG is also observed in the hpo mutant epithelia suggests that the mutant cells are responsive to the Grk signaling. Similarly, when hpo clones affect only a portion of the PFCs, the posterior fate marker pointed is expressed as in the wild-type in 40% of the cases. However, in 60% of the egg chambers with partial hpo posterior clones, and in all cases when all the PFCs are mutant, the expression of pointed is abolished. These results illustrate that hpo is required to fully process the Grk/EGFR signal in the PFCs. Conversely, in grk mutant egg chambers, the Hpo-dependent expression of Hnt is not affected, suggesting that the EGFR pathway is not required for the activation of the SWH pathway in the PFCs (Meignin, 2007).

Considering all these results together, it is concluded that the SWH pathway is involved in the Notch- and Gurken-dependent maturation of the PFCs. Whether the SWH pathway modulates this maturation directly or indirectly, for example by affecting membrane properties, needs to be further investigated (Meignin, 2007).

To study whether the oocyte polarity defects in egg chambers with FCs mutants for the SWH pathway are a consequence of the FCs proliferation and differentiation defects, egg chambers with ex and ft mutant PFCs were analyzed. Egg chambers with ft PFCs occasionally form a bilayer, although they never have defects in oocyte polarity, suggesting that the morphological disruption of the epithelia in itself does not block the repolarizing signal. Egg chambers with ex PFCs show weak defects in the epithelium, with a bilayer rarely formed and restricted to only a few mutant cells, but Stau is never properly localized. However, Hnt is not properly expressed in stage 7 ex mutant FCs, suggesting that the mislocalization of Stau is a consequence of the ex mutant cells being undifferentiated at the stage when the repolarizing signal is sent to the oocyte. These results suggest that the defects in oocyte polarity are probably due to a lack of proper differentiation of FCs in SWH mutant egg chambers (Meignin, 2007).

This study has analyzed the requirement of the SWH pathway during oogenesis. Several of the components of this pathway, but not ft, are required in the PFCs to induce the axis specification in the germline. The defects in oocyte polarity, however, are probably due to a lack of proper differentiation of the PFCs in SWH mutant egg chambers. In addition, the pathway is required in the terminal cells to control their proliferation. It has already been shown that terminal follicle cells are different from lateral follicle cells. The distinct spatial requirement of the SWH pathway for differentiation and proliferation is another feature that distinguishes the terminal from the lateral FCs, and the posterior from the anterior FCs. These results point out that this dual function of the SWH pathway might be achieved by modulation of the Notch and EGFR signals. In conclusion, the SWH pathway lies at the intersection of two signaling pathways and is permissive for the signal that is sent from the follicle cells to repolarize the oocyte.

Salvador-warts-hippo signaling promotes Drosophila posterior follicle cell maturation downstream of notch

The Salvador Warts Hippo (SWH) network limits tissue size in Drosophila and vertebrates. Decreased SWH pathway activity gives rise to excess proliferation and reduced apoptosis. The core of the SWH network is composed of two serine/threonine kinases Hippo (Hpo) and Warts (Wts), the scaffold proteins Salvador (Sav) and Mats, and the transcriptional coactivator Yorkie (Yki). Two band 4.1 related proteins, Merlin (Mer) and Expanded (Ex), have been proposed to act upstream of Hpo, which in turn activates Wts. Wts phosphorylates and inhibits Yki, repressing the expression of Yki target genes. Recently, several planar cell polarity (PCP) genes have been implicated in the SWH network in growth control. This study shows that, during oogenesis, the core components of the SWH network are required in posterior follicle cells (PFCs) competent to receive the Gurken (Grk)/TGFβ signal emitted by the oocyte to control body axis formation. These results suggest that the SWH network controls the expression of Hindsight, the downstream effector of Notch, required for follicle cell mitotic cycle-endocycle switch. The PCP members of the SWH network are not involved in this process, indicating that signaling upstream of Hpo varies according to developmental context (Polesello, 2007).

Body axis formation is a critical stage of development in most multicellular organisms. In Drosophila melanogaster, the anteroposterior (AP) body axis is determined by the polarization of the developing oocyte. The egg chamber is composed of 16 germ cells (15 nurse cells plus the oocyte) and the follicular epithelium. Specification of the AP axis requires active transport of several mRNAs along the microtubule network, thereby resulting in asymmetric mRNA and protein localization inside the oocyte. For example, bicoid (bcd) and oskar (osk) mRNAs localize to and control the formation of the anterior and posterior poles, respectively. This process is initiated through bidirectional signaling between the oocyte and the adjacent follicle cells. In midoogenesis egg chambers, grk mRNA is localized between the oocyte nucleus and the plasma membrane at the presumptive posterior pole and targets the Grk signal to the posterior follicle cells (PFCs) only. Grk is believed to be the ligand for the Torpedo/DER (EGFR) signaling pathway, which controls PFC identity. Once they are specified, the PFCs send an unknown signal back to the oocyte; this signal is required to establish oocyte posterior polarity (Polesello, 2007).

Mer, which has recently been proposed to be part of the SWH network in tissue-size control, has been suggested to play a role in signal back. Therefore whether other members of this network could play a role in body axis formation was addressed. It was tested whether hpo, like mer, is required in PFCs to control oocyte polarity by generating FLP/FRT mitotic clones of mutant cells in the egg chamber was tested with either a kinase-dead (hpoJM1) or a truncating (hpoBF33) allele of hpo. These two alleles behave similarly in all subsequent experiments (Polesello, 2007).

In wild-type egg chambers, the RNA-binding proteins Staufen (Stau) and Osk are localized in a crescent at the posterior pole of the oocyte. When the PFCs were mutant for hpo (visualized by the lack of GFP), both Osk and Stau are mislocalized. If all PFCs were mutant, both Stau and Osk were found in the middle of the oocyte or were absent in some cases for Osk. When hpo clones affected only a portion of the PFCs, Stau was mislocalized almost exclusively in the mutant part, showing the importance of the crosstalk between PFCs and the oocyte (Polesello, 2007).

In hpo germline clones, Stau localization is unaffected if the PFCs are wild-type, suggesting that Hpo is not required for secretion of the Grk signal by the oocyte. Similarly, hpo activity in polar cells is not sufficient to rescue hpo PFC phenotypes because chambers with mutant PFCs and wild-type (GFP-positive) polar cells show disrupted Stau localization. Together, these data suggest that hpo is required in the PFCs to control oocyte polarity (Polesello, 2007).

By using Stau localization as a readout, it was found that like mer and hpo, ex, sav, mats, wts, and yki are playing a role in PFCs to control oocyte polarity, suggesting that 'canonical' Hpo signaling is responsible for the observed phenotype. In contrast, fat (ft) and discs overgrown (dco) are not required in PFCs to control oocyte polarity. This suggests that the core components of the SWH network but not the SWH-associated PCP genes are required for anteroposterior axis formation (Polesello, 2007).

The microtubule cytoskeleton plays an active role in the correct localization of posterior determinants such as Osk mRNA and Stau. Therefore, whether the microtubules are normally organized when the PFCs were mutant for hpo was tested. The oocyte nucleus is initially positioned at the posterior pole (up to stage 6) and migrates to an anterodorsal localization in a microtubule-dependent manner after the signal back from the PFCs (stages 7-14). The oocyte nucleus fails to migrate to an anterodorsal position in 50% of egg chambers with PFC hpo clones. The expression of a tubulin-GFP fusion protein was drived in the germline to visualize the microtubule network. In control oocytes, tubulin-GFP forms a regular network of filaments with a stronger accumulation at the anterior pole corresponding to the nucleation site. Egg chambers with hpo mutant PFCs present ectopic Tubulin-GFP accumulation at the posterior pole of the oocyte. Apart from this defect, the general aspect of the microtubule network is normal in egg chambers with hpo PFC clones, even when the oocyte nucleus has failed to migrate to the anterior end. Finally, microtubule polarity was examined by using both Nod-βGalactosidase (Nod-βGal, minus end marker-anterior) and Kinesin-βGalactosidase (Kin-βGal, plus end marker-posterior) fusion proteins. When the PFCs were mutants for hpo, Nod-βGal was present at both poles or only at the posterior of the oocyte when the nucleus failed to migrate. When all PFCs were hpo mutant, Kin-βGal localization was in a diffuse cloud in the middle of the ooplasm. As for Stau, only half of the Kin-βGal was normally localized when only part of the PFCs were hpo mutant. Together these data support the idea that core components of the SWH pathway are required in the PFCs to build oocyte polarity, controlling microtubule-network orientation (Polesello, 2007).

Because the SWH network is known to control cell number, a phosphorylated Histone 3 (PH3) antibody was used to follow cell division in the follicle cells. During egg-chamber development, follicle cells undergo normal mitotic divisions up to stage 6, thereby giving rise to ~650 follicle cells surrounding the germ cells. Follicle cells then switch from mitotic cycles to three rounds of endoreplication cycles (endocycles) during stages 7-10A. Thus, follicle cells normally stop proliferating after stage 6, as assayed by the absence of PH3-positive cells. hpo PFC clones still contained PH3-positive cells until stage 10B. This excess proliferation observed in hpo mutant cells gives rise to both a reduction of the size of follicle cell nuclei (reduced endocycling) and formation of double layers of cells at the posterior of the egg chamber. Formation of extra layers in the follicular epithelium has been reported to result from misorientation of the mitotic spindle. Normally, the mitotic spindle is parallel to the surface of the germline cells but appears randomly oriented in hpo mutant PFCs because both parallel and perpendicularly oriented spindles were observed. This defect in the mitotic-spindle orientation is probably responsible for the double-layer formation. The proliferation defect specifically affects PFCs because reduced nuclei, ectopic PH3 foci or double layers were not obvious elsewhere. Finally, it was found that loss of the core components of the SWH network, but not of ex for which the proliferation defect is weaker, produced a double cell layer (Polesello, 2007).

In imaginal discs, loss of SWH pathway genes leads to increased expression of Yki target genes. Whether this is also the case in PFCs was tested. As expected, disruption of SWH activity in PFCs gave rise to an increase in ex expression, although no changes were detected in DIAP1 or cycE expression. ex upregulation was restricted to the PFCs in both wts mutant cells and yki gain-of-function experiments. These results suggest that core components of the SWH network specifically control proliferation of a particular subset of follicle cells required for body axis establishment (Polesello, 2007).

Because hpo mutant PFCs were still dividing after stage 6, whether hpo loss of function could affect PFC polarity was assessed. Armadillo (Arm) and Discs large (Dlg) normally label the adherens junctions and the lateral region of the cell, respectively. In hpo mutant PFCs, these were found all around the cells. In addition, the level of Arm, atypical Protein Kinase C (aPKC), and phosphorylated Moesin (P-Moe) were increased. Nevertheless, some aspects of the polarity in these cells were preserved because aPKC was still localized in the apical domain facing the oocyte (Polesello, 2007).

Grk signals via the EGF receptor Torpedo (Top) and activates the Ras signaling pathway, specifying the PFC identity. The PFC fate can be followed by the expression of the Ras target pointed (pnt-LacZ). In the absence of hpo, pnt-LacZ expression was disrupted in most but not all PFC clones. Nevertheless, hpo mutant PFCs were still able to activate the Jak/STAT pathway in response to a signal emerging from the polar cells, (monitored with a STAT reporter suggesting that the polarity defect observed in hpo mutant PFCs does not affect their ability to receive secreted signals in general. wts mutant PFCs were negative for the dpp-LacZ reporter, a specific marker of the anterior follicle cell fate (stretch and centripetal cells), suggesting that when the SWH pathway is compromised, the PFCs are not merely transformed into anterior cells. In addition, it was found that hpo mutant PFCs present characteristics of immature cells such as maintenance of Fasciclin III (FASIII) and eyes absent (eya) expression. Normally, the level of these two genes is downregulated when the follicle cells switch from mitotic cycles to endocycles. It is noted that, when hpo mutant PFCs were FASIII positive, they did not express pnt-LacZ and vice versa. In addition, it was found that pnt-LacZ-positive hpo mutant PFCs have normal Stau localization. This suggests that the primary defect in hpo mutant cells is the failure to mature. In the rare cases where hpo mutant PFCs mature properly, they are competent to transduce the Grk signal, and oocyte polarity is normal (Polesello, 2007).

Notch (N) is required in the follicle cells for the mitotic-endocycle switch that occurs at stage 6 and for controlling follicle cell identity. N mutant follicle cells, like hpo mutant PFCs, keep proliferating because they are stuck in an immature state and continue to express undifferentiated markers such as FASIII. Recently, members of the SWH network were reported to modulate N activity by affecting its subcellular localization. N protein, which localizes to the apical part of the follicle cells, is downregulated at midoogenesis. This downregulation is delayed in wts and hpo mutant PFCs, possibly causing a defect in N signaling. Hindsight (Hnt), a target of N, which starts to be expressed in all follicle cells at stage 7 after N activation, was examined. Expression of Hnt in hpo mutant PFCs is compromised. In addition, it was found that the expression of Cut, which is normally inhibited by Hnt at stage 7, was maintained in hpo and wts clones up to stage 10. Finally, whether the modulation of N activity by the SWH network was direct was tested by looking at the expression of direct N reporters. no obvious reduction of the m7-LacZ reporter was found in hpo PFC clones. However, because of the perdurance of the β-galactosidase protein, this type of reporter is more suitable to follow increases rather than decreases in signaling. It therefore cannot be entirely rule out that the SWH network might directly affect Notch activity. Nevertheless, together these data show that inactivation of the SWH network compromises the regulation of downstream targets of Notch such as Hnt and Cut. As is the case for FASIII, misregulation of these genes is restricted to the PFCs in a SWH mutant background (Polesello, 2007).

Because of this spatial restriction of SWH activity to PFCs, whether the SWH network could be part of the Torpedo/Ras pathway acting downstream of the Grk signal was tested. ras, wts double loss-of-function clones were examined. ras, wts clones present characteristics of both ras and wts single-mutant clones, namely upregulation of Dystroglycan (DG), as observed in ras clones, and maintenance of FASIII protein, as observed in wts clones. In addition, grk mutant egg chambers present only DG upregulation but no FASIII modification and no substantial change in ex expression. It is therefore concludes that the SWH network and EGFR/Ras signaling are likely to act in parallel to control respectively PFC maturation and identity and that Grk is not the ligand that controls the SWH network activation (Polesello, 2007).

A last concern was to test whether the SWH network is involved in the PFC signal back that controls oocyte polarity. To tackle this point, attempts were made to uncouple the possible signal back to the oocyte from the PFC maturation phenotypes. ex loss of function, which affects Stau localization but presents a very reduced proliferation rate and double-layer formation compared to other SWH members, was examined. Unfortunately, ex loss of function still affected Arm, FASIII, and Cut protein levels in the PFCs, in particular at midoogenesis, when both the N and Grk signals act. Therefore mer, cut double mutants were generated. In theory, this should force the cells to differentiate (lack of cut) and still affect SWH activity (lack of mer). As expected, whereas mer loss of function alone elicited both Cut upregulation and Stau mislocalization, mer/cut PFC clones were able to induce normal oocyte polarity, manifested by correct Stau localization. It is concluded that the activity of the SWH network is required to control PFC maturation, but this pathway is probably not involved in the signal-back process (Polesello, 2007).

In conclusion, this study has shown that the core components of the SWH network are required specifically to allow the maturation of the PFCs receiving the Grk signal, thus controlling AP body axis formation. The PFC defect is due to a lack of Hnt expression in response to Notch signaling. Because the function of the SWH network is restricted to the PFCs, one interesting speculation is that it is an added layer of Notch regulation specific to PFCs, which, given their crucial role in initiating body axis formation, need robust control of signaling. Placing this regulatory element in complement and in parallel to the signal that initiates PFC specification (Grk) would ensure, in cooperation with the Unpaired signal (Jak/STAT pathway) from the polar cells, a tight and robust boundary between the PFCs and the rest of the follicle cells (Polesello, 2007).

Finally the results make a clear distinction between the core components of the SWH network (hpo, sav, wts, mats, and yki) and mer, ex on one hand and the PCP genes (ft and dco) on the other. It is speculated that the core components are used in a variety of contexts during development, whereas the PCP genes are restricted to organ-size specification (Polesello, 2007).

Notch signal organizes the Drosophila olfactory circuitry by diversifying the sensory neuronal lineages

An essential feature of the organization and function of the vertebrate and insect olfactory systems is the generation of a variety of olfactory receptor neurons (ORNs) that have different specificities in regard to both odorant receptor expression and axonal targeting. Yet the underlying mechanisms that generate this neuronal diversity remain elusive. This study demonstrates that the Notch signal is involved in the diversification of ORNs in Drosophila. A systematic clonal analysis showed that a cluster of ORNs housed in each sensillum were differentiated into two classes, depending on the level of Notch activity in their sibling precursors. Notably, ORNs of different classes segregated their axonal projections into distinct domains in the antennal lobes. In addition, both the odorant receptor expression and the axonal targeting of ORNs were specified according to their Notch-mediated identities. Thus, Notch signaling contributes to the diversification of ORNs, thereby regulating multiple developmental events that establish the olfactory map in Drosophila (Endo, 2007).

In the Drosophila olfactory system there are about 50 different types of ORNs, each of which is characterized by the expression of a specific odorant receptor. Yet little is known about the molecular mechanisms underlying the generation of this diverse array of ORNs. By analyzing the projection patterns of ORNs and the cell lineage of the olfactory organ in mastermind (mam) and numb (nb) clones, it was shown that differential Notch activity in the two sibling ORN precursors leads to ORN diversification and thereby regulates multiple developmental events in the organization of the olfactory system (Endo, 2007).

There are several types of sensory organs in Drosophila, and the component cells in each organ are generally derived from a single SOP. All of the neuronal and non-neuronal cells that constitute the olfactory sensilla are derived from three precursors that arise before early pupal stages. MARCM-clone analyses was used to examine developmental events in the olfactory sensory lineage that precede these stages, and it was found that all the cells in the olfactory sensillum are generated from a single SOP that arises as early as 30 h BPF. Therefore, the olfactory sensilla seem to adopt developmental mechanisms similar to those seen in other sensory organs. However, although only one or two neurons are produced in the canonical, or ancestral, peripheral nervous system lineage, 1 to 4 ORNs are generated in the lineages of the olfactory sensilla. These findings reveal that this difference in the number of generated neurons is a consequence of the generation of more inner-cell precursors in the olfactory sensilla lineage. Interestingly, the gustatory sensillum is also innervated by 2 to 4 gustatory receptor neurons. It is proposed that the gustatory and olfactory systems share similar developmental mechanisms for producing a variety of neurons (Endo, 2007).

Although Notch signaling asymmetrically differentiates the ORN precursors, lineage analysis of nb clones in the antenna suggests that Notch does not diversify the Notch-ON-class ORN siblings (referring to a class with activate Notch signaling) on their generation from a common precursor. This notion is consistent with the results of systematic mam- and nb-clone analyses in the antennal lobe, where most ORNs are categorized into either Notch-ON or Notch-OFF classes. Therefore, the Notch-ON-class ORN siblings seem to be diversified by Notch-independent mechanisms. The asymmetric distribution of other proteins may mediate this process in ORN siblings. Thus, the olfactory sensory lineages use both Notch-dependent and Notch-independent mechanisms to generate a variety of neurons as well as non-neuronal cells (Endo, 2007).

The data in this study indicate that the genetic information that specifies the characteristics of the olfactory organ, such as sensilla and ORN type, is derived from SOPs in the developing antennal disc. Part of the information in SOPs is possibly encoded by the proneural genes atonal and amos, because they are essential for the differentiation of SOPs that produce different types of sensilla. It is proposed that the identities of SOPs arise, in part, from positional information allocated along the axes of the disc. This hypothesis is supported by the observation that ORNs expressing different types of odorant receptors are roughly segregated into distinct domains that are distributed along the proximo-distal axis. Positional information along other axes is also likely to refine genetic information in SOPs, as evidenced by a correlation between the regional localization of sensilla types on the antenna and the patterned projections of the corresponding ORNs in the antennal lobe. The data of lineage analysis indicate that Notch signaling further diversifies the genetic information of SOPs to produce the outer and inner cell lineages, and subsequently differentiates ORN precursors to generate two Notch-mediated classes of ORNs. Importantly, Notch signaling diversifies not only the genetic information specific to each SOP, but also the information shared by a subset of SOPs, as evidenced by the observation that a subset of ORNs of a given Notch-mediated class share their axonal pathways, target domains or both in the antennal lobe. Thus, Notch signaling controls multiple aspects of ORN development that contribute to the organization and function of the olfactory circuit (Endo, 2007).

In the adult olfactory system, odor information is first represented as a spatial map of activated glomeruli in the antennal lobes. Recent optical recording studies in vertebrates have shown that distinct chemical classes activate distinct regions of glomeruli, forming a chemotopic map. In Drosophila, although the larval olfactory system has a glomerular organization, in which two distinct domains represent specific classes of chemicals, the chemotopic organization of glomeruli in the adult brain is less obvious than that in the fly larvae and vertebrates. The glomerular domains identified in this study may be correlated with the chemical properties of some odorants, but no specific chemical classes have been assigned to these domains, and there are no significant sequence similarities among odorant receptors expressed in ORNs that project to the same glomerular domains compared with those expressed in ORNs that project to distinct domains. One possible implication of this domain organization is that glomeruli are physically segregated, as is typically found in the posterior group, and the sensory information from ORNs is independently processed in distinct domains. Alternatively, these domains may evoke different behavioral responses. In the Drosophila gustatory system, the axonal targets of the gustatory receptor neurons are segregated by taste category: neurons that recognize sugars project to a different region from those that recognize noxious substances. Currently, little is known about the relationship between the olfactory circuitry and behavior. Further functional studies would be necessary to assess whether Notch-mediated glomerular domains function in olfactory coding and behavior (Endo, 2007).

Notch is required in adult Drosophila sensory neurons for morphological and functional plasticity of the olfactory circuit

Olfactory receptor neurons (ORNs) convey odor information to the central brain, but like other sensory neurons were thought to play a passive role in memory formation and storage. This study show that Notch, part of an evolutionarily conserved intercellular signaling pathway, is required in adult Drosophila ORNs for the structural and functional plasticity of olfactory glomeruli that is induced by chronic odor exposure. Specifically, it was shown that Notch activity in ORNs is necessary for the odor specific increase in the volume of glomeruli that occur as a consequence of prolonged odor exposure. Calcium imaging experiments indicated that Notch in ORNs is also required for the chronic odor induced changes in the physiology of ORNs and the ensuing changes in the physiological response of their second order projection neurons (PNs). It was further shown that Notch in ORNs acts by both canonical cleavage-dependent and non-canonical cleavage-independent pathways. The Notch ligand Delta (Dl) in PNs switches the balance between the pathways. These data define a circuit whereby, in conjunction with odor, N activity in the periphery regulates the activity of neurons in the central brain and Dl in the central brain regulates N activity in the periphery. This study highlights the importance of experience dependent plasticity at the first olfactory synapse (Kidd, 2015).

Drosophila follicle cells are patterned by multiple levels of Notch signaling and antagonism between the Notch and JAK/STAT pathways

The specification of polar, main-body and stalk follicle cells in the germarium of the Drosophila ovary plays a key role in the formation of the egg chamber and polarisation of its anterior-posterior axis. High levels of Notch pathway activation, resulting from a germline Delta ligand signal, induce polar cells. This study shows that low Notch activation levels, originating from Delta expressed in the polar follicle cells, are required for stalk formation. The metalloprotease Kuzbanian-like, which cleaves and inactivates Delta, reduces the level of Delta signaling between follicle cells, thereby limiting the size of the stalk. Notch activation is required in a continuous fashion to maintain the polar and stalk cell fates. Mutual antagonism between the Notch and JAK/STAT signaling pathways provides a crucial facet of follicle cell patterning. Notch signaling in polar and main-body follicle cells inhibits JAK/STAT signaling by preventing STAT nuclear translocation, thereby restricting the influence of this pathway to stalk cells. Conversely, signaling by JAK/STAT reduces Notch signaling in the stalk. Thus, variations in the levels of Notch pathway activation, coupled with a continuous balance between the Notch and JAK/STAT pathways, specify the identity of the different follicle cell types and help establish the polarity of the egg chamber (Assa-Kunik, 2007).

Stalk formation between adjacent egg chambers is induced by directional signaling from the anterior polar cells of the older (posterior) egg chamber. Signaling via the JAK/STAT pathway provides an essential component of this process, but various indications have suggested a role for the Notch pathway as well. To verify the requirement for Notch signaling in the induction of stalk cells, follicle cell clones were generated that are mutant for Dl, the primary Notch ligand during oogenesis. Despite the proper specification of polar cells, egg chambers containing Dl follicle cell clones often failed to form a stalk on their anterior side, and as a result fused to the neighboring egg chamber. Such clones always encompassed follicle cells at the anterior portion of the egg chamber, indicating that Dl produced by anterior follicle cells is necessary to form an anterior stalk. However, the stalk positioned on the posterior side of these egg chambers was normal, even when the Dl clone surrounded the entire germline cyst. This is in keeping with the observation that posterior follicle cells do not contribute to stalk formation (Assa-Kunik, 2007).

In order to determine which cells of the anterior follicle cell population provide the signal for stalk formation, small anterior Dl-mutant follicle cell clones were analyzed. In all cases where Dl-mutant clones led to loss of the stalk, the anterior polar cells were included in the mutant clone, suggesting that these cells are the source of Dl signaling. A few instances were observed in which an anterior stalk formed even though both polar cells were mutant for Dl. Since the polar cell population defined by expression of Fng is initially larger, and is reduced to two cells by programmed cell death, this most probably resulted from the presence of wild-type Dl-expressing polar cells that provided the signal prior to their apoptosis. No phenotype was observed when the stalk cells themselves were mutant for Dl, indicating that Dl production by the stalk cells is not required for stalk specification (Assa-Kunik, 2007).

These results indicate that Notch signaling is required for at least two processes of follicle cell patterning during early oogenesis: specification of polar cells induced by Dl from the germ line and induction of stalk by Dl provided by anterior polar cells. How are these two signals distinguished, and what is the temporal relationship between them (Assa-Kunik, 2007)?

The universal Notch transcriptional reporter Gbe+Su(H)m8-lacZ was to follow the activation profile of Notch signaling throughout oogenesis. During stages 2-3 of oogenesis, variations were observed in the strength of Notch pathway activation within different anterior follicle cell types. Activation of Notch was observed in the polar cells, but no activation could be detected at this resolution in the stalk cells. These observations indicate that the level of Notch activation in the stalk cells is significantly lower than in the polar cells. Utilization of a second Notch reporter (m7-lacZ) identified essentially the same pattern. However, as this reporter appears to be more sensitive than Gbe+Su(H)m8-lacZ, low levels of Notch activation in the stalk cells at early stages could also be observed (Assa-Kunik, 2007).

Expression of Fng specifically in the future polar cells, provides a possible basis for the enhanced magnitude of Notch signaling in these cells. Polar cells are also part of the follicle cell population adjacent to the germline nurse cell complex, in which overall levels of Dl protein appear relatively high. However, the fraction of Dl localized to the nurse-cell membranes is difficult to quantify, preventing attribution with confidence the differences in signaling levels during early oogenesis to this parameter (Assa-Kunik, 2007).

To define the temporal sequence of polar and stalk cell induction, the expression of specific markers was followed for each cell type. Polar and stalk cell markers are first detected in stage 1 egg chambers (region 3 of the germarium). Markers of both cell types could be detected simultaneously in some egg chambers, where they were aligned as broad adjacent bands, with the polar cell marker always positioned towards the posterior. All other egg chambers at this stage displayed expression of the polar cell marker alone. These observations imply that polar cells are induced first, and, in agreement with the genetic evidence, are properly positioned to signal and induce stalk cell formation at the anterior end of the egg chamber (Assa-Kunik, 2007).

Taken together, these data suggest that distinctions in both the strength of signaling via the Notch pathway and the temporal sequence of pathway activation contribute to distinct cell-fate outcomes within the population of anterior follicle cells during early Drosophila oogenesis (Assa-Kunik, 2007).

It has been shown that the metalloprotease Kuzbanian-like (Kul) cleaves Dl in a cell-autonomous manner, leading to its downregulation. Modulation of Kul levels therefore provides a sensitive tool for manipulating Dl signaling activity in vivo. Attempts were made to determine whether Kul functions within follicle cells during early oogenesis. The expression pattern of Kul during oogenesis was monitored by fluorescent RNA in situ hybridization. Whereas Kul RNA was not detected in the germ line, prominent expression was observed in follicle cells, up to stage 3 (Assa-Kunik, 2007).

Kul levels can be effectively reduced by expression of a specific UAS-dsRNA construct. Since expression of Kul dsRNA by various GAL4 drivers resulted in lethality, expression of this construct was restricted to adult stages through the use of a temperature-sensitive GAL80 inhibitor system. This approach was used throughout the study to enable expression of various UAS-based transgenes during oogenesis. The GAL80ts system was used in conjunction with the neur-GAL4 driver (A101-GAL4) to specifically express Kul dsRNA in polar cells, and assess the effect of Kul on Notch signaling in early follicle cells. Notch transcriptional reporter activity was examined in these egg chambers, and the position and intensity of staining compared with wild-type egg chambers that were processed under identical conditions. Following expression of dskul in polar cells, Notch reporter levels were significantly elevated, both in the germarium and in stage 1-3 egg chambers. These observations indicate that Kul acts as an attenuator of Dl signaling in early-stage follicle cells. Interference with Kul function in this fashion thus provides a means to address the significance of follicle cell Dl levels for proper stalk cell induction. Indeed, expression of dskul in the polar cells led to a significant increase in stalk-cell number, from an average of 7.0 to 10.3 cells per stalk (Assa-Kunik, 2007).

These results indicate that the size of the stalk is highly sensitive to the amount of Dl signaling between follicle cells. This is in agreement with previous experiments, in which the size of the stalk was dramatically increased following a mild hyperactivation of Notch. Consistent with these data, ovaries from heterozygous Dl females have a reduced number of stalk cells, underscoring the sensitivity of the system to levels of Dl signaling (Assa-Kunik, 2007).

To determine whether stalk cells remain sensitive to Notch pathway signaling following their differentiation, dskul was expressed in the stalk cells themselves, using the 24B-GAL4 stalk cell-specific driver, and an increase was observed in the number of stalk cells to an average of 9.0. Kul thus attenuates Dl levels even after the stalk is formed, implying that stalk-cell number is regulated by Dl signaling from both polar cells and the stalk cells themselves. In a converse experiment, Notch signaling was reduced or eliminated from the stalk cells. Expression of dsNotch, or of a dominant-negative Notch construct, by the 24B-GAL4 stalk cell-specific driver led to the disappearance of the stalk marker Big brain (Bib). Thus, persistent, low level activation of Notch is required to maintain stalk cell fate. The low levels of Dl employed for this purpose are presented initially at the polar cell-stalk cell boundary, but as the stalk becomes elongated they might be displayed by neighboring stalk cells (Assa-Kunik, 2007).

Dl is required for establishment and maintenance of the stalk cell fate. The sensitivity of stalk size to the levels of Dl provided by the stalk cells themselves suggests that Dl also affects stalk cell proliferation or survival. To examine this possibility, the anti-apoptotic protein p35 was expressed in both polar and stalk cells using the 109-53-GAL4 driver. A greater abundance was observed of cells not properly arranged into a one-cell-wide stalk. This suggests that excess stalk cells are normally eliminated by apoptosis, and would support a model in which Dl is required for stalk cell survival, as well as stalk differentiation (Assa-Kunik, 2007).

The above observations suggest that different levels of Notch signaling determine the final fate of cells from within the polar/stalk precursor population - a strong germline signal induces the polar cell fate, whereas a weaker follicle cell signal induces the stalk. As an additional test of this model, the effects were examined of strongly elevating the Notch follicle cell signal, by overexpression of Dl specifically in polar cells. Overexpression of Dl using polar cell-specific GAL4 drivers had dramatic effects on anterior follicle cell fate and tissue morphology. Significantly, this alteration in Notch signaling resulted in an excess of polar cells. Supernumerary polar cells formed primarily at the expense of stalk cells, as evidenced by their expression of both polar and stalk cell markers, and as fusions between adjacent egg chambers. Some of the excess polar cells expressed the main-body follicle cell marker Eya, suggesting that the elevated Dl signal was capable of recruiting polar cells from this neighboring population as well. Furthermore, overexpression of Dl within the stalk cells themselves, using the 24B-GAL4 driver, induced the expression of a polar cell marker within the stalk (Assa-Kunik, 2007).

The JAK/STAT ligand Upd is expressed in polar cells, and like Dl is required for induction of the stalk. The binding of Upd to its receptor, Domeless, activates the JAK kinase Hopscotch, which then phosphorylates STAT (Stat92E) to induce its translocation into the nucleus, where it regulates transcription. The observed shift from stalk to polar cell fate upon overexpression of Dl implies that Notch activation has the capacity to antagonize JAK/STAT signaling. To explore this issue further, the Notch m7-lacZ and the STAT92E-GFP transcriptional reporters were used to simultaneously monitor Notch and JAK/STAT signaling in the ovary. Two distinct distributions of transcriptional activation wee observed. During early stages of oogenesis, Upd signaling from the polar cells is capable of inducing strong STAT activation in stalk cells, but fails to elicit activation in either the polar cells themselves, or in the neighboring main-body follicle cells. At later stages, however, follicle cell populations, including main-body and border cells, exhibited concomitant Notch and STAT activation. This analysis highlights a continuous requirement for both the Notch and JAK/STAT signaling pathways during follicle cell differentiation, throughout oogenesis. As predicted, Notch signaling can antagonize STAT activation in follicle cells, but this capacity is spatially and temporally restricted (Assa-Kunik, 2007).

The antagonistic effect of Notch signaling in early egg chambers was further pursued by following nuclear localization of STAT as an assay for JAK/STAT pathway activity. Nuclear STAT staining was pronounced throughout the stalk separating the germarium from the polar cells of the adjacent, posterior egg chamber in wild-type ovaries. Consistent with the STAT92E-GFP reporter pattern, the anterior polar cells did not exhibit nuclear localization of STAT, indicating that although they produce the Upd ligand, they themselves are refractory to this signal. STAT also remained cytoplasmic in the main-body follicle cells adjacent to the polar cells (Assa-Kunik, 2007).

When Upd was overexpressed using a polar cell-specific driver, the anterior range of nuclear STAT localization was significantly increased. Consistent with this enhanced activation of JAK/STAT signaling, longer stalk-like structures were observed. In spite of the higher levels of Upd, nuclear STAT was still only seen in cells anterior to the source, including the future stalk and posterior polar cells of the adjacent younger egg chamber. By contrast, JAK/STAT signaling in the anterior polar cells themselves, and in the neighboring main-body follicle cells, was not activated (Assa-Kunik, 2007).

In light of the suggestion of an antagonistic relationship between Notch and JAK/STAT signaling, one possible explanation for failure of the polar and main-body follicle cells to respond to Upd is the higher level of Notch activation in these cells. To test this hypothesis, Notch-mutant clones were generated in the main-body follicle cells, and the nuclear localization of STAT was monitored. Elimination of Notch in these cells led to nuclear accumulation of STAT in mutant cells situated within four cell-diameters of the polar cells. No nuclear localization was detected in Notch-mutant cells situated further away, presumably owing to restricted diffusion of Upd from the polar cells (Assa-Kunik, 2007).

These results indicate that moderate to high levels of Notch activation inhibit JAK/STAT signaling, and that this inhibition acts before the nuclear translocation of activated STAT. Furthermore, the results demonstrate that correct specification of the polar, main-body and stalk follicle cells depends on crosstalk between distinct levels of Notch activity and the JAK/STAT pathway. High Notch activation induces polar cell fate, including expression of Upd, and antagonizes JAK/STAT signaling. Intermediate levels of Notch activation in the main-body follicle cells antagonize JAK/STAT signaling, without inducing expression of Upd. Finally, low levels of Notch activation synergize with Upd signaling to induce stalk cell fate and to regulate the size of the stalk (Assa-Kunik, 2007).

Maintaining the moderate level of Notch signaling that is induced by Dl expressed in the follicle cells, is essential for producing a stalk with the correct cell number, and this is achieved at least in part by the activity of Kul in the signal-sending cells. The possibility that Notch signaling is also attenuated in the signal-receiving cells by the activity of JAK/STAT was examined by monitoring oogenesis in hopscotch (hop) hypomorphs, in which JAK/STAT signaling is compromised. Stalks formed at early stages of oogenesis in hopmv1/GA32 females, and the oocyte moved to the posterior of the egg chamber as in wild type. However, stalk cells failed to intercalate, and the stalk consisted of two rows of cells linked by adherens junctions. At later stages, the stalk collapsed and, as was observed for strong hop alleles, the stalk cells reverted to the polar cell fate. These cells now clustered at the anterior corners of the older cyst, whilst remaining in contact with the oocyte of the younger egg chamber (Assa-Kunik, 2007).

The conversion of stalk cells to polar cells when the level of JAK/STAT signaling was compromised suggests that Notch signaling in the stalk cells is normally attenuated by the JAK/STAT pathway. When this inhibition is relieved in hop hypomorphs, the increase in the level of Notch signaling leads to their conversion to polar cells. Since the entire polar/stalk precursor cell population expresses Fng, even activation by the lower levels of Dl produced by these cells may be sufficient to give rise to polar cells, in the absence of repression by JAK/STAT (Assa-Kunik, 2007).

Notch signaling controls germline stem cell niche formation in the Drosophila ovary

Stem cells, which can self-renew and generate differentiated cells, have been shown to be controlled by surrounding microenvironments or niches in several adult tissues. However, it remains largely unknown what constitutes a functional niche and how niche formation is controlled. In the Drosophila ovary, germline stem cells (GSCs), which are adjacent to cap cells and two other cell types, have been shown to be maintained in the niche. In this study, Notch signaling is shown to control formation and maintenance of the GSC niche, and cap cells help determine the niche size in the Drosophila ovary. Expanded Notch activation causes the formation of more cap cells and bigger niches, which support more GSCs, whereas compromising Notch signaling during niche formation decreases the cap cell number and niche size and consequently the GSC number. Furthermore, the niches located away from their normal location can still sufficiently sustain GSC self-renewal by maintaining high local BMP signaling and repressing bam as in normal GSCs. Finally, loss of Notch function in adults results in rapid loss of the GSC niche, including cap cells and thus GSCs. These results indicate that Notch signaling is important for formation and maintenance of the GSC niche, and that cap cells help determine niche size and function (Song, 2007).

At the onset of the larval-pupal transition, all of the 16 to 20 terminal filament (TF) stacks have formed and initiate ovariole formation, while another group of somatic cells, cap cells, start to occupy a position between the TFs and the germ cells. The primordial germ cells (PGCs) in direct contact with cap cells are further anchored through E-cadherin and are further expanded through symmetric division and develop into permanent GSCs in the adult ovary. Actin-filament regulator, Cofilin/ADF, and ecdysone signaling, are required for TF formation. However, no studies have been carried out to investigate the formation of cap cells, which are a key component of the GSC niche. In this study, the role of N signaling in controlling cap cell formation was investigated (Song, 2007).

In late third-instar larval female gonads, Dl is expressed in newly formed TFs, while the N receptor is expressed in all the somatic cells, including TFs and cap cells. Consequently, N signaling is active in newly formed TFs and cap cells and its activation is sufficient to induce cap cell formation, suggesting that TF-expressed Dl activates N signaling to induce cap cell formation. To further support the idea that N signaling specifies cap cell fate, reduction of N signaling results in a reduced number of cap cells. Induction of cap cells by N signaling can take place only during the late third instar and early pupal stages, suggesting that active N signaling promotes only cap cell formation along with other factors provided at particular stages. Cap cells can still form without germ cells. This suggests that Dl is unlikely to be required in germ cells for cap cell formation. Therefore, it is concluded that N signaling, activated probably by Dl from newly formed cap cells, specifies cap cell fate through direct induction. In this study, it was also shown that N signaling is required for maintaining the GSC niche in the adult ovary, since loss of N function results in rapid loss of cap cells and GSCs. Taken together, the results of this study demonstrate that N signaling is important for controlling niche formation as well as niche maintenance (Song, 2007).

Although niches have been defined for GSCs in the Drosophila ovary and testis, as well as in several tissue types of the mammalian systems, it remains unclear whether they still function properly for controlling stem cell self-renewal after their location and size are changed. In this study, two pieces of experimental evidence have been provided supporting the idea that expanded niches are functional for controlling GSC self-renewal. First, increased cap cells in the normal location express all known cap cell markers, such as hh-lacZ, wg-lacZ, Lamin C and E-cadherin, and behave like normal cap cells. Second, these expanded cap cells can support self-renewal of extra GSCs, which behave similarly to normal ones based on Dad-lacZ and bam-GFP expression, and their ability to self-renew and generate differentiated germ cells. Even when cap cells cover the anterior half of the germarium, the GSCs associated with the cap cells also appear to be capable of self-renewing and generating differentiated germ cells. These findings show that GSC niche size can be expanded by adding more niche cells (Song, 2007).

This study has also demonstrated that the GSC niche could function in ectopic locations. Ectopic cap cells, which are surrounded by IGS cells or follicle cells, also express known cap cell markers and sufficiently support functional GSCs, supporting the idea that TFs and IGS cells are not essential components of the GSC niche. This is consistent with early published studies showing that the numbers of cap cells and GSCs are closely correlated and that TFs and cap cells express the genes important for GSC self-renewal such as piwi, Yb, hh and dpp/gbb. In light of the recent evidence that ESCs in direct contact with cap cells and GSCs are required for maintaining GSCs, it remains formally possible that some unidentified escort stem cells cells associated with expanded or ectopic cap cells contribute to niche function. In any case, this study demonstrates that the size and location of the GSC niche can be genetically manipulated while it maintains its functions. The ability to manipulate niche location and size will further increase the capacity to investigate how niche formation is controlled and how the niche controls stem cell function in general (Song, 2007).

One of the major unsolved issues for the GSC niche in the Drosophila ovary is how BMPs function as a short-range signal to control GSC self-renewal and allow the GSC daughter adjacent to the GSC to differentiate at the same time. Several previous studies have shown that BMP signaling activity is primarily restricted to the GSC based on Mad phosphorylation and Dad expression, two indicators of BMP signaling. Early work has also shown that dpp is primarily expressed in TF and cap cells, while gbb is expressed in TF and cap cells as well as in IGS cells. In this study BMP signaling activity has been shown to spread two or more cell diameters based on expression of Dad-lacZ and bam-GFP when more cap cells exist. Furthermore, when more cap cells accumulate in ectopic sites, the GSCs associated with the cap cells as well as the germ cells lying two or three cells away are capable of activating BMP signaling and repressing bam expression. One of the explanations for these observations is that cap cells are the source of BMP and more cap cells would produce more BMP to diffuse further away to repress differentiation of germ cells lying two or more cell diameters away. Another explanation is that the ratio of cap cells to ESCs or escort cells increases so that BMP inhibitors, such as Sog, produced by ESCs or escort cells, are diluted or deterred by more cap cells, and consequently more active BMP is available for reaching and activating cells lying more than two cell diameters away. In Xenopus gastrula-stage embryos, an effective BMP-4 activity gradient is established, not by diffusion of BMP-4 protein but by the long-range effects of two BMP-4 inhibitors, Noggin and Chordin. Finally, it is also possible that a combination of both mechanisms contributes to restriction of BMP signaling activity to one cell diameter in the GSC niche. Observations from this study have suggested that a limited amount of active BMP produced by cap cells is probably responsible for its short-range effect on GSC self-renewal in the GSC niche (Song, 2007).

Notch and Mef2 synergize to promote proliferation and metastasis through JNK signal activation in Drosophila

Genetic analyses in Drosophila revealed a synergy between Notch and the pleiotropic transcription factor Mef2 (myocyte enhancer factor 2), which profoundly influences proliferation and metastasis. This study shows that these hyperproliferative and invasive Drosophila phenotypes are attributed to upregulation of eiger, a member of the tumour necrosis factor superfamily of ligands, and the consequent activation of Jun N-terminal kinase signalling, which in turn triggers the expression of the invasive marker MMP1. Expression studies in human breast tumour samples demonstrate correlation between Notch and Mef2 paralogues and support the notion that Notch-MEF2 synergy may be significant for modulating human mammary oncogenesis (Pallavi, 2012).

A genetic modifier screen was undertaken in Drosophila and a number of genetic modifiers of Notch signals were identified that affect proliferation. Further examination of one of these modifiers, Mef2, established that its synergy with Notch signals directly triggers expression of the Drosophila JNK pathway ligand eiger, consequently activating JNK signalling that profoundly influences proliferation and metastatic behaviour. It might perhaps be worth noting that metastatic behaviour in Drosophila may not be completely equivalent to mammalian metastasis, notwithstanding the fact that they share molecular signatures, for example, MMP activation (Pallavi, 2012).

Cancer is characterized by the deregulation of the balance between differentiation, proliferation and apoptosis; thus, it is not surprising that the Notch signalling pathway, which plays a central role in all these developmental events, is increasingly implicated in oncogenic events. The rationale of this study is based on the fact that synergy between Notch and other genes is key in understanding how Notch signals contribute to oncogenesis. It remains a remarkable fact that while activating mutations in the Notch receptor have been associated with >50% of T-cell lymphoblastic leukemias (T-ALLs), a search for mutations in other cancers, despite a few suggestive reports, remains essentially unfruitful. Yet, correlative studies have linked Notch activity with a broad spectrum of human cancers and work in mice suggests that while Notch activation promotes proliferation, it is the synergy between Notch and other factors that eventually leads to cancer. Similar synergies have been identified before but the extraordinary complexity of the gene circuitry that modulates the Notch pathway suggests that more such relationships will be uncovered as exemplified by the discovery of Mef2 as a Notch synergistic partner affecting proliferation (Pallavi, 2012).

The transcription factor Mef2 plays an essential role in myogenic differentiation, but several studies have also shown a broad pleiotropic role of Mef2. Mef2 can integrate signals from several signalling cascades through chromatin remodelling factors and other transcriptional regulators to control differentiation events. This study extends the functionality of Mef2 by uncovering the profound effect it can have on proliferation and metastatic cell migration in synergy with Notch signals (Pallavi, 2012).

This is not the first study to link Mef2 with Notch. They have been linked before in the context of myogenesis both in Drosophila and in vertebrates. A ChIP-on-chip analysis of Mef2 target regions identified several Notch pathway components as potential Mef2 targets during Drosophila myogenesis. In human myoblasts, Mef2C was suggested to bind directly to the intracellular domain of Notch via the ankyrin repeat region, suppressing Mef2C-induced myogenic differentiation. Mef2 has also been reported to interact with the Notch coactivator MAML1 and suppress differentiation (Pallavi, 2012).

While upregulation of Mef2 alone does not show overt proliferation effects, these analyses demonstrate that in vivo it can activate MMP1. Even though Mef2 was ectopically expressed in the whole wing pouch, MMP1 expression was confined around the D/V boundary, where endogenous Notch signals are active. This effect of Mef2 overexpression depends on Notch signals, a notion corroborated by the fact that inhibiting Notch activity by RNAi reverses the effects of Mef2 on MMP1 (Pallavi, 2012).

The polarity gene scribble cooperates with Ras signalling to upregulate the JNK pathway, promoting invasiveness and hyperplasticity. However, the synergy seen in this study appears to be scribble independent. The fact that both the scribbled/Ras and the Notch/Mef2 metastatic pathways converge at the level of JNK signal activation suggests that JNK is a crucial regulator of oncogenic behaviour, which is controlled by inputs from multiple signals. Even though there is little evidence that twist activates JNK signalling, it is a crucial regulator of epithelial-to-mesenchymal transition and metastasis and has also been independently linked to both Mef2 and Notch in myogenesis. However, it is noted that the Notch-Mef2 synergy seems to be independent of twist, as Twist cannot replace Mef2 in the synergistic relationship (Pallavi, 2012).

Numerous reports link JNK signalling to normal developmental events requiring cell movement and to metastatic phenomena both in Drosophila and in vertebrates. JNK signals seem to be crucial for controlling gene activities involved in epithelial integrity and the observations from Drosophila suggest that the Nact and Mef2 synergy may be important in JNK-linked carcinogenesis. A role for Notch in controlling JNK signals has been reported previously. While studies carried out using breast cancer samples can only be correlative now, the observations suggest that metastatic breast tumours harbour higher levels of Notch and Mef2 paralogue pairs, consistent with observations in Drosophila (Pallavi, 2012).

Although the majority of studies on Mef2 are focused on muscle development/differentiation, some intriguing links between Mef2C and leukaemias are noteworthy. MEF2C and Sox4 synergize to cause myeloid leukaemia in mice. Analysis of T-ALL patient samples revealed increased levels of Mef2C; however, Mef2C alone could not cause cellular transformation of NIH3T3 cells, but it could do so in the presence of RAS or myc. Given the role of Notch in T-ALL it will be important to examine how activated Notch mutations, often the causative oncogenic mutation, correlate with Mef2 family members. The functional differences between the different Mef2 homologues in humans are not well understood and the specific role each may play in the Notch synergy remains to be elucidated (Pallavi, 2012).

This analysis clearly indicates that, in Drosophila, the underlying molecular mechanism of the Notch/Mef2 synergy relies on the direct upregulation of expression of the prototypical TNF ligand egr through the binding of Mef2 and Su(H), the effector of Notch signals, to regulatory sequences on the egr promoter. In Drosophila, egr is the only JNK ligand while in humans, the superfamily is large and includes the cytokines TNFα (TNF), TRAIL and RANKL which have been associated with tumour progression in numerous human cancers including breast. RANKL plays a key role in bone metastasis of breast cancer, and is the target of a therapeutically effective monoclonal antibody. In breast cancer cells, TNFα, which can signal through several pathways, including JNK and NF-κB, affects proliferation and promotes invasion and metastasis (Pallavi, 2012).

In human breast cancer, clinical relapse after initial treatment is almost always accompanied by metastatic spread and it is almost invariably lethal. ER- tumours tend to respond well to first-line chemotherapy, but a significant subset of these tumours recur. Recurrent ER- tumours are typically resistant to chemotherapy and radiation, and are highly lethal. The current data suggest that ER- tumours that recur but not ER- tumours that do not recur show significant positive correlation between NOTCH1 and all four MEF2 paralogues. Further, the data show that even within the recurrent subset, NOTCH1 expression predicts poor survival but MEF2 expression does not. While these observations do not establish causality, they are consistent with the hypothesis that NOTCH1/MEF2 coexpression identifies a set of breast cancers that are more likely to relapse, and that MEF2 genes act as NOTCH cofactors rather than independently of NOTCH (Pallavi, 2012).

In conclusion, this study in Drosophila uncovers a new functional role for Mef2, which in synergy with Notch affects proliferation and metastasis. Mechanistically, this synergy relies on the direct upregulation of the JNK pathway ligand eiger. The correlation analysis and tumour staining of human cancer samples suggests that the observations in Drosophila may well be valid in humans, defining Notch-Mef2 synergy as a critical oncogenic parameter, one that may be associated with metastatic behaviour, emphasizing the value of model systems in gaining insight into human pathobiology (Pallavi, 2012).

Lola regulates cell fate by antagonizing Notch induction in the Drosophila eye

Lola is a transcription repressor that regulates axon guidance in the developing embryonic nervous system of Drosophila. Lola regulates two binary cell fate decisions guided by Notch inductive signaling in the developing eye: the R3-R4 and the R7-cone cell fate choices. Lola is required cell-autonomously in R3 for its specification, and Lola transforms R4 into R3 if overexpressed. Lola also promotes R7 fate at the expense of cone cell fate. Lola antagonizes Notch-dependent gene expression and Notch-dependent fate transformation. Expression analysis shows that Lola is constitutively present in all photoreceptors and cone cells. It is proposed that when a precursor cell receives a weak Notch inductive signal, it is not sufficiently strong to overcome the constitutive repression of target gene transcription by Lola. A precursor that receives a strong Notch inductive signal expresses target genes despite a constitutive repression by Lola. The predicted consequence of this mechanism is to sharpen a cell’s responsiveness to Notch signaling by creating a threshold (Zheng, 2008).

Lola regulates two cell-fate decisions in the eye: the R3 vs. R4 decision and the R7 vs. cone cell decision. Lola promotes R3 fate at the expense of the R4 fate, and it promotes R7 fate at the expense of the cone cell fate. Lola appears to influence these binary fate decisions by acting on responsiveness to Dl-N signaling. Lola regulates N-dependent gene expression, and Lola is able to suppress the effects of constitutively-active N on cell fate decisions. This latter result argues that Lola either acts downstream of N in the N signaling pathway or acts in a pathway that is convergent downstream with the N signaling pathway (Zheng, 2008).

Lola antagonizes 'strong N' signaling, which is evident by overexpressed Lola antagonizing strong N-dependent gene expression in R4 and transforming it into an R3-type. This is interpreted to mean that Lola normally makes the R3 precursor more resistant against induction into a R4-type, an interpretation that is consistent with mosaic analysis indicating that Lola is required in the R3 cell. Within the R3 precursor cell, Lola might repress transcription of genes that are normally activated by strong N signaling, such as the E(spl)m∂ gene. Doing so, Lola then would ensure the precursor would not inappropriately adopt an R4 cell fate. However, this notion raises a paradox: since Lola is expressed somewhat equivalently in all photoreceptors including R4, why does it not repress genes such as E(spl)m∂ in the R4 cell? One explanation is that it represses these genes in the R4 precursor but this repression is not strong enough to block their expression when induced by strong N signaling. Two lines of evidence support this explanation. Overexpression of Lola in the R4 precursor is sufficient to block expression of the E(spl)m∂ gene. This argues that Lola activity is a limiting component in N-dependent gene expression. Second, analysis of mutant lola clones reveals that R4 cells in mosaic ommatidia are more likely to be mutant for the lola gene. This result implies that Lola antagonizes N-activated programming of R4 fate in the R4 cell itself (Zheng, 2008).

Based on these observations, a model is proposed in which Lola acts as a transcription repressor of certain N-dependent genes that program the R4 cell fate. It acts on these genes in all cells as they are induced by EGFR to differentiate. In cells that receive no Dl-N signal or a weak Dl-N signal, repression is sufficiently strong to block N-dependent gene expression. However, in cells such as the R4 precursor that receive a strong Dl-N signal, repression is not sufficient to block gene expression, and the cells follow an R4 fate. Thus, Lola functions to make cells respond with a sharper threshold to Dl-N signaling. Since Lola functions in this manner only in cells that have already been induced by EGFR signaling, Lola seems to aid in crosstalk between the two signal transduction pathways. Moreover, the lola gene is repressed by Dl-N signaling; loss of N activity results in ectopic lola expression in many precursor cells. This maybe ensures that only cells induced to differentiate will respond with a sharp threshold to later Dl-N signals they might receive (Zheng, 2008).

Lola protein has sequence-specific transcriptional repressor activity and it genetically interacts with histone remodeling proteins. Thus, Lola could antagonize N signaling by virtue of it specifically binding and repressing transcription of genes that are activated by N signaling. Alternatively, Lola might render genes less responsive to N by specifically interacting with effectors of N signaling. In this regard, strong genetic interactions have been observed between Lola and Hairless, a co-repressor of Su(H) in Drosophila (Muller, 2005). It is not yet possible to distinguish between these two models of interaction (Zheng, 2008).

The Dl-N pathway is also required to specify both R7 and cone cell fates; strong Dl-N signaling induces cone fate and weaker Dl-N signaling induces R7 fate. Precursors with high levels of constitutively-active Notch become cone cells. This transformation is suppressed by Lola overexpression and leads to formation of more R7 cells. Conversely, loss of Lola leads to fewer precursors adopting an R7 fate and more adopting a cone fate, Again, these data fit with a model in which Lola sharpens the threshold response to levels of Dl-N signaling. It helps block strong N-dependent expression in the R7 precursor receiving a weak Dl-N signal, while it is insufficient to block the expression of the same genes in the cone precursors that receive a stronger Dl-N signal. However, in contrast to Lola's role in the R3/R4 fate switch, Lola has less effect on the R7/cone fate switch. Loss of lola causes infrequent R7 cell loss, and there is no significant correlation between the lola genotype of R7 cells in mosaic ommatidia and their normal development. Moreover, flies overexpressing Lola in cone cells only occasionally exhibit ectopic R7 formation. The reason why Lola has less impact on the R7/cone cell fate choice is unclear. Perhaps other factors play a redundant role with Lola in this fate switch (Zheng, 2008).

Erect wing regulates synaptic growth in Drosophila by integration of multiple signaling pathways

Formation of synaptic connections is a dynamic and highly regulated process. Little is known about the gene networks that regulate synaptic growth and how they balance stimulatory and restrictive signals. This study shows that the neuronally expressed transcription factor gene erect wing (ewg) is a major target of the RNA binding protein ELAV and that EWG restricts synaptic growth at neuromuscular junctions. Using a functional genomics approach it was demonstrated that EWG acts primarily through increasing mRNA levels of genes involved in transcriptional and post-transcriptional regulation of gene expression, while genes at the end of the regulatory expression hierarchy (effector genes) represent only a minor portion, indicating an extensive regulatory network. Among EWG-regulated genes are components of Wingless and Notch signaling pathways. In a clonal analysis it was demonstrated that EWG genetically interacts with Wingless and Notch, and also with TGF-β and AP-1 pathways in the regulation of synaptic growth. These results show that EWG restricts synaptic growth by integrating multiple cellular signaling pathways into an extensive regulatory gene expression network (Haussmann, 2008).

Several pathways have been identified that stimulate synaptic growth at NMJs of Drosophila larvae (Wnt/Wingless, TGF-β/BMP and jun kinase). Overexpression of AP-1 and mutants in regulatory genes involved in Wnt/Wingless and TGF-β/BMP pathways (spinster, highwire, shaggy and the proteasome) can increase bouton numbers, suggesting that synaptic growth is regulated through the balance of stimulatory and restrictive signals. This study has identified such a restrictive role for the transcription factor EWG and, through the analysis of EWG-regulated genes, for the N pathway in the regulation of synaptic growth. Using genetic mosaics, it was further demonstrated that EWG's role in synaptic growth regulation is cell-autonomous, suggesting that the transcriptional regulator EWG mediates this restrictive effect through the alteration of transcription pre-synaptically (Haussmann, 2008).

Analysis of genes differentially expressed in ewgl1mutants revealed a rather unexpected set of genes involved in synaptic growth regulation, besides an expected number of metabolic genes due to homology of EWG to human NRF-1. Most genes that could account for the phenotype of ewgl1mutants, and that are thus expressed in the nervous system, are involved in transcriptional and post-transcriptional regulation of gene expression. Although changes of transcript levels in ewgl1mutants were mostly moderate, their significance was validated through mRNA profiling with rescued ewgl1mutants under the same conditions of RNA preparation and microarray hybridization. In addition, differences in gene expression in ewgl1mutants were validated using quantitative RT-PCR and biochemical assays with regard to predicted changes in glycogen levels based on differential regulation of genes involved in gluconeogenesis. Furthermore, genetic interaction experiments in double mutants with increased bouton numbers support that these co-regulated genes are functionally connected in regulating synaptic growth (Haussmann, 2008).

The group of neuronal genes among those differentially regulated in ewgl1mutants that have been demonstrated to have roles in synaptic growth or could account for it, is remarkably small. In particular, from the large number of cell adhesion molecules and cytoskeletal proteins present in the Drosophila genome only a handful is differentially regulated. Similar results have also been obtained in response to JNK and AP-1 signaling. These results are in contrast to changes in gene expression induced by acute or chronically enhanced neuronal activity in Drosophila seizure mutants, which also result in synaptic overgrowth. Here, the vast majority of differentially regulated genes are for cell adhesion molecules and cytoskeletal proteins or their regulators, and genes involved in synaptic transmission and neuronal excitability; transcriptional or post-transcriptional regulators comprise only a minor portion. These differences could be explained by separate pathways regulating growth independent of neuronal activity (Haussmann, 2008).

Particularly striking is the large number of genes involved in RNA processing among genes differentially expressed in ewgl1mutants. Although local regulation of gene expression is required in growth cones of navigating axons, a prominent role for pre-synaptic regulation of gene expression at the RNA level is only just emerging, but is a hallmark of post-synaptic plasticity. Several RNA binding proteins have been implicated in memory storage . osk and CPSF (cleavage and polyadenylation specificity factor) are among the genes differentially regulated in ewgl1mutants. Other genes involved in RNA processing differentially regulated in ewgl1mutants comprise the whole spectrum of regulation at the post-transcriptional level, from nuclear organization (otefin), alternative pre-mRNA processing (Pinin, CPSF, Rox8) and export/import (Segregetion distorter, Nxf2, CG11092, Karyopherin, Transportin) to transport, localization and translation (oskar, swallow, ribosomal protein genes S5 and Rpl24), and likely also include the regulation of mRNA stability (Rox8) (Haussmann, 2008).

An intriguing connection between ewg and signaling pathways involved in regulating synaptic growth is indicated by differentially regulated components of the Wg and N pathways (gro and Hairless) in ewgl1mutants. Consistent with a role of the co-repressor gro in Wg and N mediated transcriptional regulation of synaptic growth, Wg and N signaling pathways do not operate independently of ewg in genetic interaction experiments. The transcriptional regulatory networks of EWG, Wg and N seem to be highly interwoven. Overexpression of pan, the transcriptional mediator of canonical Wg signaling, which is repressed by gro, does not lead to a further expansion of synaptic growth in ewg mutants, suggesting a requirement for ewg-regulated genes. This effect could be mediated by deregulated N signaling, which is also repressed by gro, but antagonistic to Wg in synaptic growth. Thus, removal of gro, as in ewg, will relieve the repressive effect of N and antagonize the stimulatory effect of pan. In the complementary situation, removal of N increased bouton numbers further in the absence of EWG, which is consistent with an increase in Wg signaling as a result of down-regulated gro in ewg mutants. Antagonism between N and Wg pathways has also been found in wing discs, where N inhibits armadillo (arm), the transcriptional co-activator of canonical Wg signaling. Intriguingly, gro has also been found to be a target of receptor tyrosine kinase signaling and, thus, can combine additional pathways with N and Wg signaling. In addition to transcriptional hierarchies, chromatin remodeling has also been implicated in synaptic plasticity. Strikingly, CG6297, a Drosophila homologue of the histone deacetylase RPD3, is differentially expressed in ewgl1mutants and physically interacts with gro (Haussmann, 2008).

How ewg exerts its effect on TGF-β signaling is less clear. A prominent regulatory step in this pathway is the regulated degradation of the SMAD co-factor Medea by Highwire. Several genes involved in regulating protein stability are differentially down-regulated in ewg mutants (CG6759, CG3431, CG4973, CG7288, CG3455, CG9327 and CG9556). Lower expression levels of these genes might interfere with stabilization of Medea and explain why the effect of activated TGF-β signaling is not additive in the absence of EWG (tkvA GOF ewg LOF). Bouton numbers in wit null mutants are marginally increased in the absence of EWG, suggesting further that genes regulated by SMADs are involved in mediating synaptic overgrowth in ewgl1mutants. Potentially, ewg could also regulate TGF-β signaling through the endosomal pathway involving spinster and/or spichthyin (Haussmann, 2008).

Functionally related genes have been shown to be co-regulated, suggesting additional ELAV targets in EWG-regulated gene networks. Indeed, ELAV negatively regulates alternative splicing of the penultimate exon in armadillo (arm). Exclusion of this exon, which truncates the carboxyl terminus of arm, reduces Wg signal transduction, which is in agreement with ewg's antagonistic role relative to Wg signaling. Another known ELAV target gene is neuroglian (nrg), where a role in synapse formation has recently been demonstrated in the giant fiber system. Taken together, the establishment of a gene network regulated by EWG will now serve as valuable tool to identify further ELAV regulated modules that shape the synapse (Haussmann, 2008).

The transcription factor EWG is a major target of the RNA binding protein ELAV, which regulates EWG protein expression via a splicing mechanism. EWG is required pre-synaptically and cell-autonomously at third instar neuromuscular junctions to restrict synaptic growth, demonstrating that restrictive activities at gene expression levels are also required for synaptic growth regulation. EWG mediates regulation of synaptic growth primarily by increasing transcript levels of genes involved in transcriptional and post-transcriptional regulation of gene expression. Genes at the end of the gene expression hierarchy (effector genes) represent only a minor portion of EWG-regulated genes. Since analysis of mutants in genes differentially regulated in ewgl1mutants revealed that these genes are involved in both stimulatory and restrictive pathways of synaptic growth, and since ewg genetically interacts with a number of signaling pathways (Wingless, Notch, TGF-β and AP-1), the results suggest that synaptic growth in Drosophila is regulated by the interplay of multiple signaling pathways rather than through independent pathways (Haussmann, 2008).

Chromatin modification of Notch targets in olfactory receptor neuron diversification

Neuronal-class diversification is central during neurogenesis. This requirement is exemplified in the olfactory system, which utilizes a large array of olfactory receptor neuron (ORN) classes. An epigenetic mechanism was discovered in which neuron diversity is maximized via locus-specific chromatin modifications that generate context-dependent responses from a single, generally used intracellular signal. Each ORN in Drosophila acquires one of three basic identities defined by the compound outcome of three iterated Notch signaling events during neurogenesis. Hamlet, the Drosophila Evi1 and Prdm16 proto-oncogene homolog, modifies cellular responses to these iteratively used Notch signals in a context-dependent manner, and controls odorant receptor gene choice and ORN axon targeting specificity. In nascent ORNs, Hamlet erases the Notch state inherited from the parental cell, enabling a modified response in a subsequent round of Notch signaling. Hamlet directs locus-specific modifications of histone methylation and histone density and controls accessibility of the DNA-binding protein Suppressor of Hairless at the Notch target promoter (Endo, 2011).

This study analyzed the ORN lineage history that gives rise to three primary ORN identities (Naa, Nab and Nba). These three identities arise in a sensillum via iterated rounds of Notch-mediated binary cell-fate decisions. Together with previous findings, these results suggest that diversification of Drosophila ORN classes is the result of the combined output of two predominantly hardwired mechanisms; spatially localized factors determine at least 21 types of sensilla, and Notch and Ham then act in each sensillum to maximize ORN class variety (Endo, 2011).

Biochemical and molecular analyses of Ham function indicate that it can repress Notch target enhancers. In the ORN lineage, Notch signaling is used in consecutive cell fate decisions, and it was found that Ham acts to turn off Notch targets before a subsequent a round of selective reactivation. Ham is expressed specifically in pNa, the neuronal-intermediate precursor with high Notch activity, and inherited by both of the pNa daughter cells. In addition to the current findings, studies in other contexts have observed that some Notch targets require a Notch signal for their transcriptional induction, but not for maintaining their expression. These Notch targets could aberrantly persist in both pNa progeny without the intervention of a mechanism to erase the effects of the preceding Notch signal (Endo, 2011).

ham mutants showed an unusual ORN fate switch. They not only transformed ORN fate with respect to Notch state, but also altered sublineage-specific identity (low-Notch Nab to high-Notch Nba identity). This phenotype suggests that, in addition to suppressing the previous round of Notch activation, Ham may delineate the selection of the next round of targets. As Ham activity resulted in altered chromatin modifications at Notch targets, this suggests that Ham could set an epigenetic context in which the terminal round of Notch signaling occurs (Endo, 2011).

Although this was demonstrated with respect to Ham, it is suggested that this approach to modifying the transcriptional outputs of a signaling pathway may have widespread importance in other lineages that utilize iterative signals. Notch signaling iteration is a widespread phenomenon. One important example is in the maintenance of neural and other stem cells, and it is now known that some chromatin-modifying factors promote stem cell self-renewal. Notably, several Prdm factors have regionalized expression in neural precursor domains of the embryonic mouse spinal cord and could modify and diversify stem cell identity during mammalian CNS development (Endo, 2011).

In Drosophila, Notch signaling and Ham expression are transient in nascent ORNs. Thus, Notch- and Ham-mediated fate choices must be perpetuated during the later selection of alternative axon guidance factors and odorant receptors. It is possible that chromatin methylation not only sets the context of immediate Notch signaling outcomes, but also maintains initial fate choice by priming or silencing promoters for readout during differentiation. The existence of such mechanisms in neural development is now beginning to emerge. It was recently shown that, in mouse cortical precursor cells, the trithorax factor Mixed-lineage leukemia1 (Mll1) prevents epigenetic silencing of the neural differentiation gene Distal-less homeobox 2 (Dlx2), enabling it to be properly upregulated during differentiation stages. In contrast, in the mammalian olfactory system, epigenetic repression is used during the transition from multipotent precursor to immature ORN to silence all ~2,800 odorant receptor genes before subsequent de-repression of a single odorant receptor per neuron (Endo, 2011).

To determine how chromatin modifications create a context-dependent outcome from signaling and how resultant cell-fate choices are perpetuated during Drosophila ORN differentiation, it will be necessary to elucidate the components and action of the chromatin-modification complex targeted by Ham. Furthermore, genome-wide identification of the promoters targeted by Su(H) and Ham will reveal the genes regulated by these factors to confer specific ORN fates (Endo, 2011).

The insulin receptor is required for the development of the Drosophila peripheral nervous system

The Insulin Receptor (InR) in Drosophila presents features conserved in its mammalian counterparts. InR is required for growth; it is expressed in the central and embryonic nervous system and modulates the time of differentiation of the eye photoreceptor without altering cell fate. This study shows that the InR is required for the formation of the peripheral nervous system during larval development and more particularly for the formation of sensory organ precursors (SOPs) on the fly notum and scutellum. SOPs arise in the proneural cluster that expresses high levels of the proneural proteins Achaete (Ac) and Scute (Sc). The other cells will become epidermis due to lateral inhibition induced by the Notch (N) receptor signal that prevents its neighbors from adopting a neural fate. In addition, misexpression of the InR or of other components of the pathway (PTEN, Akt, FOXO) induces the development of an abnormal number of macrochaetes, which are Drosophila mechanoreceptors. These data suggest that InR regulates the neural genes ac, sc and sens. The FOXO transcription factor, which becomes localized in the cytoplasm upon insulin uptake, displays strong genetic interaction with the InR and is involved in Ac regulation. The genetic interactions between the epidermal growth factor receptor (EGFR), Ras and InR/FOXO suggest that these proteins cooperate to induce neural gene expression. Moreover, InR/FOXO is probably involved in the lateral inhibition process, since genetic interactions with N are highly significant. These results show that the InR can alter cell fate, independently of its function in cell growth and proliferation (Dutrieux, 2013).

A model is proposed in which the InR receptor plays a role in the development of the peripheral nervous system mainly through FOXO cell localization independently of its role in proliferation and apoptosis. The role of the InR/FOXO pathway appears early in PNS development before SOP formation. The use of different mutants involved in growth indicates that the TOR pathway does not play a major role in the phenotypes observed. The results using genetic and molecular methods strongly suggest that InR/FOXO controls the level of proneuronal genes such as ac, sc and Sens early in PNS development. This explains the interaction observed with N55e11 (Dutrieux, 2013).

Several arguments indicate that the phenotypes observed when InR is overexpressed are not due, at least for the most part, to proliferation, growth or lack of apoptosis. First using anti-PH3 staining that allows to visualize mitotic cells, no extra mitoses are observed in the clusterOverexpression of genes such as dE2F1, or dacapo did not lead to a significant increase or decrease in the number of macrochaetes. In addition co-expression of these genes with InR indicates no interaction. Moreover, the effects of InR and FOXO when overexpressed on respectively the increase and the decrease in cell number, could be estimated by the number of Ac-positive cells in the DC and SC clusters. No significant differences were observed between the control and the overexpressed strain (either InR or FOXO) in the number of cells positive for Ac. If the possibility that proliferation is somehow involved in cluster size cannot be discarded, it does not account for the effects observed since the ratio of Sens-positive cells when InR is overexpressed over the control strain is much higher than the ratio of Ac-positive cells. A similar role for FOXO in apoptosis could also be discarded on the same basis. No clear interactions were observed between FOXO and genes involved in inhibition of apoptosis like diap1 (Dutrieux, 2013).

Along the same line it has been shown that the InR/TOR pathway plays a role in controlling the time of neural differentiation. This has been observed in photoreceptor formation but also in the chordotonal organs of the leg that develop on the same basis as thoracic bristles. The dynamic formation of the SOPs, particularly after a block of InR signaling was undertaken. No differences were observed before the end third larval instar in the test and in the overexpressed strain. Only an increase in the number of positive Sens stained cells are observed in the sca>InR strain (Dutrieux, 2013).

Using Pros staining that marks pIIb cells, this study shows that staining appears in the late third instar larvae at the level of DC SOPs in sca>InR; this is not observed in the control strain. In addition in sca>FOXO RNAi wing discs it also leads to Pros staining. This indicates that the time of differentiation is advanced in the InR strain through the absence of nuclear FOXO. However it was verified that in very early third instar larvae the first scutellar SOP appears at the same time in the control and in the overexpressed strains and that no differences were observed in mid third instar (Dutrieux, 2013).

In addition the observations show that the increase in the number of macrochaetes in sca>InR is independent of the TOR pathway since none of the members induces a similar phenotype as does InR or interacts either with InR or FOXO in this process. However, some interactions were observed with raptor and Rheb that could be the consequence for the latter of its role in PIIa and PIIb formation regulating N (Dutrieux, 2013).

Are InR and FOXO acting on the same target in SOP formation? Several arguments are in favor of this possibility. First underexpression experiments (InR clones, InR RNAi or FOXO RNAi overexpression and FOXO homozygotes and even heterozygotes,) induce exactly opposite phenotypes. This is also true for overexpression experiments with InR and hFOXO3a-TM. Moreover overexpression of both transgenes leads to an intermediate phenotype, very different from the control phenotype. Finally, overexpression of InR in a heterozygote FOXO mutant background leads to an increase in the number of macrochaetes compared to InR alone. FOXO null flies are fully viable and do not usually display any phenotype. However an increase in the number of pDC and aSC macrochaetes is observed in some FOXO homozygotes and even heterozygotes that are nor observed in the control strain. This could indicate that FOXO function is in part dispensable. Even if the InR/FOXO double heterozygote is completely normal, the double null mutant InR/FOXO shows either an excess or a lack of macrochaetes, that is in favor of the hypothesis that InR acts through FOXO. FOXO null clones do not display any phenotype comparable to FOXO RNAi overexpression. However overexpression of InR in a FOXO null clone leads to stronger phenotypes than overexpression of InR alone in a clone. Yet, it cannot be excluded that part of the InR overexpression phenotype is not due to the absence of FOXO or its cytoplasmic retention (Dutrieux, 2013).

The absence of FOXO, using FOXO RNAi, or its retention in the cytoplasm by InR or Akt overexpression produces the same neurogenic phenotypes that are exactly the opposite when nuclear hFOXO3a-TM is overexpressed. In addition overexpression of both hFOXO3a-TM and InR leads to a decrease in the number of highly positive Ac and Sens expressing cells compared to overexpression of InR alone. Finally, overexpression of FOXO RNAi in dpp regulatory sequences, induces Ac expression. All these results should be explained by the same molecular process. One possibility would be that InR/FOXO regulates one or several neural genes involved in cluster formation and maintenance. The results are in favor of the hypothesis that genes of the Ac/Sc complex could be regulated by InR. Either InR via nuclear FOXO represses the Ac/Sc pathway, or FOXO activates a repressor of the pathway (Dutrieux, 2013).

Since it has been well established that InR induces cell proliferation, it remains possible that these functions could affect the size of the proneural clusters when the genes are overexpressed. However, when the number of the Ac-positive cells in the DC and SC clusters in the different genotypes was estimated, it was not significantly different (Dutrieux, 2013).

Several relevant arguments exist suggesting that InR is necessary for SOP formation and regulation of neural gene expression. (1) The phenotype of the overexpression experiments either with InR or with InR RNAi suggests that InR perturbs the normal pattern of singling out a cell in the proneural cluster that will become an SOP. The fact that the sensitive period occurs in the late second/beginning third instar is in accordance with this hypothesis. The phenotype of the InR null clones comfort this hypothesis. (2) When InR is overexpressed the level of Ac is significantly higher. This is confirmed by the IMARIS technique that estimated that in this genotype, the number of cells with the highest scores (106 and 107 units) is larger than in the control strain. These 'highly Ac-positive cells' seem to also be Sens positive cells indicating a correlation between the two events. (3) In sca>InR the level of Sens, measured by the IMARIS technique is higher than in the test raising the possibility that InR regulates several neural genes independently. However another possibility would be that this high Sens expression level would be indirectly due to the induction by InR of a Sens-positive regulator such as sc. (4) Several sc enhancers are regulated by InR, the sc promoter, and the SRV and DC enhancers. As sc is auto-regulated through its different enhancers, it is difficult to evaluate if a specific enhancer is involved although the effect on the 3.8 kb sc promoter is the most striking. For FOXO the absence of FOXO using the FOXO RNAi strain shows that Ac is induced. The double expression of InR and hFOXO3a-TM produces an intermediate phenotype and decreases the effects of InR, on Ac and Sens expression. The results using the sc enhancers when hFOXO3a-TM is overexpressed showed that only a decrease in the expression of the SRV enhancer is observed. However, the phenotypes observed in sca>hFOXO3a-TM agree with the hypothesis of repression of ac and sc by hFOXO3a-TM. As expected, overexpression of FOXO RNAi induces sc-lacZ enhancer. (5) Overexpression of both InR and sc leads to a significant increase in the effect of a single transgene. This indicates that both transgenes have a common target; one of them could be sc itself. An opposite effect is observed with constitutively active hFOXO3a-TM. This favors the model whereby InR and FOXO act in opposite ways on the sc target in SOP formation. (6) Highly significant genetic interactions are observed between sc and InR, and sc and FOXO. (7) Another gene charlatan (chn) which is both upstream and downstream of sc, strongly interacts genetically with InR (Dutrieux, 2013).

Lateral inhibition is determined by the activity of the N receptor. When N is mutated, cell fate changes and extra macrochaete singling appear. Using the N deletion (N55e11) to test possible genetic interaction with InR and with FOXO in heterozygote females, interaction was observed with the InR RNAi strain. Moreover strong interaction is observed with InR overexpression. This indicates that InR impairs lateral inhibition and cooperates with N in this process. In parallel, as for Inr overexpression, the absence of nuclear FOXO either using FOXO25 homozygotes (or even heterozygotes) or FOXO RNAi overexpression induces an increase in the neurogenic phenotype. With this latter strain, tufted microchaetes were observed, indicating that FOXO could also act later in development. Overexpression of hFOXO3a-TM displays highly significant interaction with N55e11 as the neurogenic phenotype is increased compared to overexpression in a wild type background. However, overexpression of InR RNAi in a N55e11 heterozygote background leads to a significant increase but only at the level of aSC, raising the possibility of a local interaction or appearing at a specific time for the different clusters (Dutrieux, 2013).

Moreover the fact that there is no differences when Suppressor of Hairless (Su(H)) which transduces the N pathway, is expressed with or without the InR, indicates that lateral inhibition is not affected. In addition in the InR strain, Sens stained cells were clearly individualized and separated from one another. These results clearly indicate that InR and FOXO act with N on the choice of the cell that will become an SOP (Dutrieux, 2013).

EGFR has also been implicated in macrochaete development. Indeed EGFR mutants and EGFR null clones display macrochaete phenotypes. This could be explained since in EGFR hypomorphic mutants the level of Sc is reduced in some clusters and increased in others suggesting a different requirement of EGFR for the different SOPs. If RasV12 was overexpressed with an ubiquitous driver, sc was ectopically expressed. Thus, Ac/Sc induction by Ras overrules lateral inhibition due to N. Moreover N downregulation enhances EGFR signaling. A model has been established of antagonist interaction between EGFR and N in which Ac/Sc activates both pathways that in turn act on the same SOP specific enhancers (Dutrieux, 2013).

Moreover, the InR/TOR pathway regulates the expression of some of the components of the EGFR signaling pathway such as argos, rhomboid and pointed. The results suggest that both the InR and the EGFR/Ras pathways induce sc in a synergic manner and this further overrules the lateral inhibition mechanism induced by N. The fact that overexpression of RasV12 in an InR null heterozygote background significantly lowers the phenotype observed with RasV12 only, is in agreement with this hypothesis. The interactions observed with the EGFR RNAi strain seem to be FOXO independent (Dutrieux, 2013).

Taken together these results show that InR and several components of the pathway such as PTEN, Akt and FOXO are involved in PNS development independently of their role in growth, proliferation and delay in the time of neural differentiation. The function of InR in PNS development seems to be independent of TOR/4E-BP. FOXO cytoplasmic retention either by InR activation or by the use of FOXO RNAi produces opposite phenotypes suggesting that nuclear FOXO could be a repressor of PNS development. These results using antibody staining and reporters of sc enhancers indicate that InR targets are the neural genes ac, sc and sens. However, as most of these neural genes display a complex co-regulation, it is difficult to demonstrate whether or not sc is the primary target of the pathway. A strong interaction is observed between the EGFR/Ras pathways and InR suggesting that both could act together to induce neural gene expression and this would explain the strong interaction observed between InR/FOXO and N (Dutrieux, 2013).

Roles of PINK1, mTORC2, and mitochondria in preserving brain tumor-forming stem cells in a noncanonical Notch signaling pathway

The self-renewal versus differentiation choice of Drosophila and mammalian neural stem cells (NSCs) requires Notch (N) signaling. How N regulates NSC behavior is not well understood. This study shows that canonical N signaling cooperates with a noncanonical N signaling pathway to mediate N-directed NSC regulation. In the noncanonical pathway, N interacts with PTEN-induced kinase 1 (PINK1) to influence mitochondrial function, activating mechanistic target of rapamycin complex 2 (mTORC2)/AKT signaling. Importantly, attenuating noncanonical N signaling preferentially impairs the maintenance of Drosophila and human cancer stem cell-like tumor-forming cells. These results emphasize the importance of mitochondria to N and NSC biology, with important implications for diseases associated with aberrant N signaling (Lee, 2013).

These results uncover a novel mechanism of N in regulating NSC self-renewal and maintenance through a noncanonical signaling pathway involving PINK1, mTORC2, and AKT. A central feature of this noncanonical N signaling pathway is specific mitochondrial roles of N in regulating respiratory chain complex (RCC) function through direct interactions with PINK1 and select RCC subunits and in activating mTORC2. N could act through a number of possible mechanisms, such as facilitating the import or assembly of RCC components, as suggested by its interaction with complex I subunits, or directing the quality control of mitochondria, as has been implicated for PINK1. Future studies will determine the exact domains of N involved in PINK1 interaction and whether noncanonical N signaling is ligand-dependent. Although the exact mechanism remains to be determined, the results will help in understanding earlier observations in Drosophila that mutations in N affected mitochondrial respiration and data from mammalian systems implicating N in mitochondrial and metabolic regulation. The physiological significance of this newly defined noncanonical N pathway is also underscored by the phenotypes of various diseases associated with N dysregulation. For example, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a disease caused by mutations in NOTCH3, is associated with mitochondrial impairment. On the other hand, GOF mutations of Notch1 are implicated in over half of human T-cell acute lymphoblastic leukemia (T-ALL), in which a pathogenic role of mTORC2 has been proposed, although how N impinges on mTORC2 in this setting is unknown. Mammalian N has been shown to act through AKT and mitochondria to promote T-cell survival, although the mechanism is distinct from the one uncovered in this study. Finally, mTORC2 was shown to be required for the self-renewal and maintenance of cancer stem cells but dispensable in normal stem cells. The findings that cancer stem cell-like brain tumor-forming cells are particularly dependent on the noncanonical N pathway in flies and humans identify the newly discovered noncanonical N signaling pathway as a potential target for disease intervention (Lee, 2013).

Notch-dependent epithelial fold determines boundary formation between developmental fields in the Drosophila antenna

Compartment boundary formation plays an important role in development by separating adjacent developmental fields. Drosophila imaginal discs have proven valuable for studying the mechanisms of boundary formation. This study examined the boundary separating the proximal A1 segment and the distal segments, defined respectively by Lim1 and Dll expression in the eye-antenna disc. Sharp segregation of the Lim1 and Dll expression domains precedes activation of Notch at the Dll/Lim1 interface. By repressing bantam miRNA and elevating the actin regulator Enabled, Notch signaling then induces actomyosin-dependent apical constriction and epithelial fold. Disruption of Notch signaling or the actomyosin network reduces apical constriction and epithelial fold, so that Dll and Lim1 cells become intermingled. These results demonstrate a new mechanism of boundary formation by actomyosin-dependent tissue folding, which provides a physical barrier to prevent mixing of cells from adjacent developmental fields (Ku, 2017).

This study attempted to unravel the molecular and cellular mechanisms of boundary formation in the Drosophila head. Focus was placed on the antennal A1 fold that separates the A1 and A2-Ar segments. The results showed that the expression of the selector genes Lim1 and Dll, which are expressed in A1 and A2-Ar, respectively, was sharply segregated. This step was followed by differential expression of Dl, Ser and Fng, as well as activation of N signaling at the interface between A1 and A2. N signaling then induced apical constriction and epithelial fold, possibly through repression of bantam to allow levels of the bantam target Ena to become elevated, with this latter inducing the actomyosin network. The actomyosin-dependent epithelial fold then provided a mechanical force to prevent cell mixing. When N signaling or actomyosin was disrupted, or when bantam was overexpressed, the epithelial fold was disrupted and Dll and Lim1 cells become mixed. Thus this study describes a clear temporal and causal sequence of events leading from selector gene expression to the establishment of a lineage-restricting boundary (Ku, 2017).

Sharp segregation of Dll/Lim1 expressions began before formation of the A1 fold, suggesting that fold formation is not the driving force for segregation of Dll/Lim1 expression. Instead, the fold functions to safeguard the segregated lineages from mixing. Whether Dll/Lim1 segregated expression is due to direct or indirect antagonism between the two proteins is not known (Ku, 2017).

Actomyosin-dependent apical constriction is an important mechanism for tissue morphogenesis in diverse developmental processes, e.g., gastrulation in vertebrates, neural closure and Drosophila gastrulation, as well as dorsal closure and formation of the ventral furrow and segmental groove in embryos. This study describes a new function of actomyosin, i.e., the formation of lineage-restricting boundaries via apical constriction during development (Ku, 2017).

This actomyosin-dependent epithelial fold provides a mechanism distinctly different from other known types of boundary formation. The cells at the A1 fold still undergo mitosis, suggesting that mitotic quiescence is not involved. Perhaps epithelial fold as a lineage barrier is needed in situations in which mitotic quiescence does not happen. Mechanically and physically, epithelial folds could serve as stronger barriers than intercellular cables when mitotic activity is not suppressed. The drastic and sustained morphological changes, including reduced apical area and cell volume, may be accompanied by increased cortical tension of cells along the A1 fold, with such high interfacial tension then preventing cell intermingling and ensuring Dll and Lim1 cell segregation. Although similar to actomyosin boundaries, the epithelial fold in the A1 boundary is distinctly different from the supracellular actomyosin cable structure in fly parasegmental borders, the wing D/V border, and the interrhombomeric boundaries of vertebrates. The adherens junction protein Echinoid, which is known to promote the formation of supracellular actomyosin cables, is not involved in A1 fold formation. Although actomyosin is enriched in a ring of cells in the A1 fold, it does not exert a centripetal force to close the ring, unlike the circumferential cable described in dorsal closure and wound healing (see review. In the A1 fold, the constricting cells become smaller in both their apical and basolateral domains, thus differing from ventral furrow cells where cell volume remains constant (Ku, 2017).

A tissue fold probably provides a strong physical or mechanical barrier to prevent cell mixing. In addition, whereas in a flat tissue where the boundary involves only one to two rows of cells, the tissue fold involves more cells engaging in cell-cell communication. The close apposition of cells within the fold may allow efficient signaling within a small volume. This may be an evolutionarily conserved mechanism for boundary formation that corresponds to stable morphological constrictions such as the joints in the antennae and leg segments (Ku, 2017).

Although N signaling has been reported to be involved in many developmental processes, a role in inducing actomyosin-dependent apical constriction and epithelial fold is a novel described function for N. For the A1 boundary, N activity is possibly mediated through repression of bantam and consequent upregulation of Ena. In the wing D/V boundary, N signaling is also mediated through bantam and Ena, but the outcome is formation of actomyosin cables, i.e., without apical constriction and epithelial fold [19]. Thus, the N/bantam/Ena pathway for tissue morphological changes is apparently context-dependent (Ku, 2017).

Tissue constriction also occurs later in joint formation of the legs and antennae. N activation also occurs in the joints of the leg disc and is required for joint formation. This role is conserved from holometabolous insects like the fruitfly Drosophila melanogaster and the red flour beetle Tribolium castaneum to the hemimetabolous cricket Gryllus bimaculatus. It is possible that for segmented structures that telescope out in the P/D axis, like the antennae, legs, proboscis and genitalia, N signaling is used to demarcate the boundaries between segments, which are characterized by tissue constriction. N-dependent epithelial fold morphogenesis has also been reported in mice cilia body development without affecting cell fate, suggesting that such N-dependent regulation in morphogenesis is evolutionarily-conserved (Ku, 2017).

It is proposed that N signaling is important in all boundaries that involve stable tissue morphogenesis. For those boundaries corresponding to stable morphological constrictions, e.g., the joints in insect appendages, N acts via actomyosin-mediated epithelial fold. The wing D/V boundary represents a different type of stable tissue morphogenesis. It becomes bent into the wing margin and involves N signaling via actomyosin cables, rather than apical constriction. In contrast, actomyosin-dependent apical constrictions do not involved N signaling and are involved in transient tissue morphogenesis, such as gastrulation in vertebrates, neural closure, Drosophila gastrulation, dorsal closure, as well as formation of the ventral furrow, eye disc morphogenetic furrow, and segmental groove in embryos (Ku, 2017).

N signaling is also involved in the boundary between new bud and the parent body of Hydra, where it is required for sharpening of the gene expression boundary and tissue constriction at the base of the bud [78]. Whether the role of N in these tissue constrictions is due to actomyosin-dependent apical constriction and epithelial fold is not known (Ku, 2017).

Boundaries may be established early in development. As the tissue grows in size through cell divisions and growth, boundary maintenance become essential. This study found that N activity is maintained by actomyosin, suggesting feedback regulation to stably maintain the boundary. Mechanical tension generated by actomyosin networks has been suggested to enhance actomyosin assembly in a feedback manner. Interestingly, the N-mediated wing A/P and D/V boundaries, which form actomyosin cables rather than tissue folds, did not exhibit such positive feedback regulation. Instead, the stability of the Drosophila wing D/V boundary is maintained by a complex gene regulatory network involving N, Wg, N ligands and Cut. Perhaps this is necessary for a boundary not involving tissue morphogenesis (Ku, 2017).

The segmented appendages of arthropods (antennae, legs, mouth parts) are homologous structures of common evolutionary origin. It has been proposed that the generalized arthropod appendage is composed of a proximal segment called the coxopodite and a distal segment called the telopodite, either of which can further develop into more segments. The coxopodite is believed to be an extension of the body wall, whereas the telopodite represents the true limb, and thus represents an evolutionary addition. Dll mutants lack all distal segments except for the coxa in legs and the A1 segment in antennae. Lineage tracing studies have shown that Dll-expressing cells contributed to all parts of the legs except the coxa. These results indicate that the leg coxa and antenna A1 segment correspond to the Dll-independent coxopodite, and that Dll is the selector gene for the telopodite. Therefore, the antennal A1 fold is the boundary between the coxopodite and telopodite. It is postulated that the same N-mediated epithelial fold mechanism also operates in the coxopodite/telopodite boundary of legs and other appendages (Ku, 2017).

Notch continued: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Post-transcriptional regulation of Notch mRNA | Effects of Mutation | References

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