Notch
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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 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).
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).
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 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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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