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).
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).
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
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