BIOLOGICAL OVERVIEW

Delta is one of the principle molecules in the Notch signaling pathway, and like the other proteins in this pathway is designated a neurogenic gene. As will be seen, Delta plays an especially important role in neurogenesis.

Both Delta, and its receptor Notch, are found expressed throughout the neuroectoderm. As a result of the gastrulation process, this layer of cells is positioned in the ventral part of the embryo (the ventral neuroectoderm). These cells are the precursors of neuroblasts, the stem cells from which the ventral nervous system (CNS) will arise. Cells destined to be neuroblasts delaminate from the neuroectoderm and migrate dorsally into the developing ventral nervous system. Delta plays a key role in the selection of cells that assume the neuroblast fate.

Through a process of selection known as lateral inhibition only a few cells in each segment become neuroblasts. It is a competitive process. A single cell, perhaps the one with the highest levels of Delta, sends a message to neighboring cells via the Notch receptor. This results in the inhibition of neuroblast development. The selected cell then delaminates and migrates dorsally. In this way, neuroblasts are born.

Because the Delta-Notch complex functions in cell adhesion (Fehon, 1990) this complex certainly must function in the delamination and migration process. The process of selection and inhibition is repeated many times, in many different aspects of development. The Delta-Notch pathway is highly conserved: Delta and Notch homologs are found throughout the metazoa. One of the major breakthroughs in developmental biology is an understanding of this mechanism, which has come about only in the last decade.

Expression of Delta has been studied during microchaeta (small bristle) development in Drosophila as well as in the regulatory relationships between the Delta-Notch signaling pathway and the proneural gene, achaete. The adult notum (the dorsal surface of thorax) is derived from the fusion of two heminota found at the anterior ends of the two wing/notal imaginal discs. Fusion takes place between 6 and 8 hours after puparium formation (APF). Within each notum, microchaeta sensory organ precursors (SOPs) arise within stripes of proneural cells arrayed from anterior to posterior. The stripe that develops in the center of the adult notum is designated stripe 1, and the stripe that contains the dorsocentral macrochaetae is designated stripe 5. Microchaeta stripes 1, 3, and 5 develop first, followed by stripes 2 and 4. The majority of microchaeta SOPs arise within proneural stripes between 10 and 12 hours APF. Delta is expressed in all microchaeta proneural cells and microchaeta SOPs, and is expressed dynamically in SOP progeny. Delta expression in microchaeta proneural cells is detected prior to the onset of achaete expression and arises normally in the absence of achaete/scute function, indicating that the initial Delta expression in the notum is not dependent on proneural gene function (Parks, 1997).

The Delta-Notch signaling pathway is required at two steps during proneural cluster formation and SOP specification. (1) Dl prevents specification of supernumerary SOPS. In this function Delta represses achaete expression between microchaeta proneural stripes. Thus Delta function is required to help define microchaeta proneural stripe boundaries. (2) Dl is required later within proneural stripes to repress achaete expression. This second function involves the choice between neural (neuronal and thecogen/glial) and non-neural (tormogen and trichogen support cells) cell fate in the two cells decendent from a single SOP. In the first stage of Delta function, the expression data indicate that Delta is transcribed and Delta protein is localized throughout the entire microchaeta proneural stripe. There are no asymmetries in Delta accumulation within proneural stripes at the transcriptional level; nor is there a decrease in Notch protein levels in nascent SOPs. This is in contrast to the predictions of lateral inhibition models that suggest a Delta-Notch feedback loop might result in higher Delta expression in the cell adopting the SOP fate and higher Notch expression in immediately surrounding cells. Within proneural stripes, therefore the expression data is most consistent with the idea of mutual inhibition, i. e., that microchaeta proneural cells within the entire equivalence group interact via Delta and Notch to inhibit adoption of the SOP fate (Parks, 1997).

After Achaete protein expression has, for the most part, resolved to cells adopting the SOP fate, there do appear to be asymmetries in subcellular Delta protein localization in the vicinity of SOPs. Activation of the Delta-Notch pathway results in loss of Delta protein accumulation, suggesting that Delta expression is post-transcriptionally regulated, in part, by Delta-Notch signaling activity. Thus, Delta signaling is required for correct delineation of early proneural gene expression in developing nota. Later, within microchaeta proneural stripes, Delta-Notch signaling prohibits adoption of the SOP fate by repressing expression of the proneural gene achaete (Parks, 1997).

Not all Delta functions are involved with neurogenesis. During wing margin formation in the wing imaginal disc Delta is required in ventral cells at the dorsal/ventral boundary. Ectopic Delta induces wingless, vestigial and cut and causes adult wing tissue outgrowth in the dorsal compartment. Whereas Delta is required in ventral cells, Serrate, another ligand for Notch is required in dorsal cells, and Notch is required in both. Thus Delta and Serrate function as compartment-specific ligands for Notch in the wing disc, activating Notch, which in turn induces downstream genes required for wing formation (Doherty, 1996).

Neural determination in the Drosophila eye occurs progressively. A diffusible signal, Dpp, causes undetermined cells first to adopt a 'pre-proneural' state in which they are primed to start differentiating. A second signal is required to trigger the activation of the transcription factor Atonal, which causes the cells to initiate overt photoreceptor neurone differentiation. Both Dpp and the second signal are dependent on Hedgehog (Hh) signaling. Previous work has shown that the Notch signaling pathway also has a proneural role in the eye (as well as a later, opposite function when it restricts the number of cells becoming photoreceptors -- a process of lateral inhibition). It is not clear how the early proneural role of Notch integrates with the other signaling pathways involved. Evidence suggests that Notch activation by its ligand Delta is the second Hh-dependent signal required for neural determination. Notch activity normally only triggers Atonal expression in cells that have adopted the pre-proneural state induced by Dpp. Notch drives the transition from pre-proneural to proneural by downregulating two repressors of Atonal: Hairy and Extramacrochaetae (Baonza, 2001).

Loss of Notch signaling leads to a loss of neural differentiation. Cells within clones of a null allele of Notch fail to upregulate Atonal expression from its initial low, uniform level. This implies that Notch signaling is required for the initiation of neural development but not for the first low level expression of Atonal. To examine in detail the role of Notch signaling in promoting neural differentiation, clones of cells expressing the Notch ligand Delta were made and their ability to induce neural differentiation was examined. In the wing disc, similar ectopic expression of Delta in clones induces the activation of Notch signaling within the clone as well as non-autonomously in cells surrounding it (Baonza, 2001).

Clones were generated using the Gal4/UAS system combined with the Flip-out technique and third instar larval eye discs were labelled with different markers to assess neural development. The phenotype of Delta-expressing clones depends on their position with respect to the morphogenetic furrow. Clones in the anterior part of the disc have no effect unless they are within 12-15 cell diameters of the furrow. Within this zone close to the furrow, Delta induces the ectopic expression of Atonal, both autonomously within the clone and non-autonomously, in cells surrounding the clone. In some of these clones there are also cells ectopically expressing the neural antigen Elav. This indicates that once Atonal expression is activated, the full neural program is initiated. Thus, the primary proneural function of Notch signaling is the activation of Atonal (Baonza, 2001).

Consistent with the neural-promoting properties of Delta, clones that span the furrow from posterior to anterior cause the anterior displacement of Atonal and Elav expression. This displacement implies that the furrow accelerates as it moves through the clone. In the region of these clones that lies posterior to the furrow, the domain of Atonal expression is expanded and the Atonal-expressing cells are disorganized and more numerous. In this region repression of neural differentiation, visualized with the expression of Elav, is also observed. This later phenotype reflects the function of Notch signaling pathway in preventing neural differentiation posterior to the morphogenetic furrow (Baonza, 2001).

Similar clones were also produced expressing the alternative Notch ligand, Serrate, and unlike Delta-expressing cells, these clones cause no neural induction ahead of the furrow. Conversely, when posterior to the furrow, Ser-expressing clones behave like those expressing Delta and prevent neural differentiation. This implies that anterior to the furrow, the two Notch ligands are not equivalent in their ability to activate the receptor. The reason for this has not been explored, but it is noted that the Notch glycosyltransferase Fringe, which makes Notch resistant to Serrate, is strongly expressed anterior to the furrow. The inability of Serrate to induce proneural Notch signaling is consistent with previous reports, which show that loss of Serrate caused no effects on eye development (Baonza, 2001).

These results imply that there is a zone of about 12-15 cell diameters ahead of the morphogenetic furrow, where the activation of Notch signaling by Delta, but not by Serrate, is sufficient to trigger neural fate (Baonza, 2001).

The simplest explanation of these results is that some signal or signals emanating from the cells posterior to, or within, the morphogenetic furrow are necessary for the specification of a neural competence zone ahead of the furrow. Within this zone, cells can respond to Delta-induced Notch activation by upregulating Atonal expression. A candidate for such a signal is the secreted protein Dpp. Dpp is expressed within the furrow in response to Hh signaling and has been proposed to define a 'pre-proneural' state in a zone anterior to the furrow. In order to analyse whether the function of Dpp is sufficient to generate the condition necessary for the neural activation by Notch signaling, clones that simultaneously express ectopic dpp and Delta were produced (Baonza, 2001).

Clones of cells that express dpp alone only induce neural differentiation along the margin of the eye discs; internal clones have no effect on neural induction. By contrast, clones that co-express Dl and dpp trigger neural differentiation everywhere ahead of the furrow. In all the clones studied, ectopic expression of Atonal and Elav was observed. The induction of neural differentiation occurs in all the cells surrounding the clone and not, as in Delta-expressing clones, only in the cells within the competence zone. In most of the clones analyzed, Atonal expression was found to be associated with an ectopic morphogenetic furrow induced by the clones. Thus, it is possible to observe clones with ectopic Atonal expression several cells away from the border of the clone and with Atonal expression restricted to isolated cells within the clone, reproducing the pattern of Atonal expression of the endogenous furrow. One interpretation of this result is that once Atonal is activated within and in the cells surrounding the clone, the normal cascade of ommatidial development is triggered, inducing an ectopic furrow that begins to move away from the clone (Baonza, 2001).

These observations lead to the conclusion that the expression of dpp is sufficient to enable all cells anterior to the furrow to activate neural differentiation in response to Notch. It is postulated that during normal development, Dpp primes the cells to become competent to differentiate neurally in response to Notch signaling, at a range of 12-15 cells anterior to the furrow (Baonza, 2001).

Loss of Dpp signaling during eye development causes furrow progression to slow down but not to stop: partial redundancy allows Hh signaling to induce neural differentiation in cells in which the Dpp signaling is blocked. Furthermore, clones of ectopic expression of Hh always induce neural differentiation and an ectopic furrow, even beyond the zone of Dpp-influenced cells, indicating that Hh is sufficient to trigger neural differentiation. The current model is that Dpp is important for furrow progression to occur efficiently and at a normal rate, but that it is not essential for neural differentiation to occur. This study shows that Dpp signaling has an important role in promoting the proneural function of Notch signaling by generating the 'pre-proneural' state ahead of the furrow. This does not, however, rule out the possibility that Hh signaling could also produce a similar effect. If the function of Dpp signaling can be rescued by Hh signaling, then it would be expected that the effects of ectopic activation of Notch signaling would be identical in a background where Dpp signaling is blocked (because in this case, Hh would replace Dpp function) (Baonza, 2001).

An examination was made of the effect of the ectopic expression of Delta when Dpp signaling is blocked, by inducing clones that co-express Delta and the negative Dpp signal regulator brinker. The use of brinker expression was evaluated as a way of inhibiting Dpp function in the eye by examining the phenotype of clones of brinker-expressing cells. brinker-expressing clones indeed mimic mad null clones in their ability to prevent the initiation of the morphogenetic furrow when they occur at the posterior margin of the disc (Baonza, 2001).

Double clones of brinker- and Delta-expressing cells only activate Atonal expression when they lie within four to five cells of the morphogenetic furrow. In addition, the position of the endogenous morphogenetic furrow is only slightly altered compared with control clones expressing Dl alone. Thus, the proneural action of ectopic Notch signaling anterior to the morphogenetic furrow is substantially reduced in cells in which Dpp signaling is inhibited. These results suggest that despite some partially rescuing short-range signal near the furrow (which is presumed to be Hh), Dpp signaling is required for the longer range ability of cells to initiate neural differentiation in response to Notch activation (Baonza, 2001).

The fact that the ectopic expression of Dpp does not reproduce the effects cause by the overexpression of Hh, indicates that additional Hh-dependent signals are needed to promote neural differentiation. The results suggest that Notch signaling could be one of these. According to this model blocking Notch and Dpp signaling would be sufficient to prevent neural differentiation, since it would block both Hh-induced intermediate signals. To analyze this possibility, double mutant clones of the strong Delta allele Dl^rev10 and the medea allele med⁸ were induced. Medea is the Drosophila homologue of the mammalian MAD-related protein Smad4, and is required for transduction of the Dpp signal. Clones of med⁸ along the posterior eye margin cause similar phenotypes to mad minus clones, preventing the initiation of the morphogenetic furrow. Internal clones of med⁸ can reduce the expression of Atonal, especially the initial uniform expression. Occasionally (1/17), the expression of Atonal is totally removed in part of the clone. These phenotypes are similar to those described when Dpp signaling is blocked in mutant clones of the Dpp receptor thick vein (tkv). One phenotype of med⁸ clones could be found not accounted for by phenotypes caused by loss of other members of the pathway: in some clones (6/11) posterior to the morphogenetic furrow, Atonal is ectopically expressed, always in isolated cells. The basis for this phenotype is not understood, but it does not affect the region anterior to the furrow, which is under consideration here (Baonza, 2001).

Double Dl^rev10;med⁸ mutant clones show a combination of the phenotypes observed in independent mutant clones of Delta and med. Thus, all internal clones analysed show Delta-like reduction of Atonal expression. In some of these clones there are regions where Atonal expression is totally lost, a phenotype observed in med clones. Also as in medea clones, posterior Dl^rev10;Med⁸ clones are found that express Atonal ectopically. However, in this case, the Atonal expression is in clusters of cells, reflecting the fact that lateral inhibition is blocked in the absence of Delta (Baonza, 2001).

These results indicate that the initial expression of Atonal can be induced in the absence of Notch and Dpp signaling, implying that Hh signaling can, directly or via yet another intermediate, overcome the loss of function of both pathways (Baonza, 2001).

The progression of the morphogenetic furrow correlates with the modulated expression of the negative regulators of Atonal expression, Emc and Hairy. Hairy is expressed in a broad stripe anterior to the furrow and rapidly switched off in the furrow. Emc protein is present in all cells but the highest levels are present in a dorsoventral stripe of cells anterior to the domain of Hairy expression, whereas the lowest levels are observed in the furrow. Thus, the increase of Atonal expression in the proneural groups within the furrow is associated with the downregulation of both Emc and Hairy. Whether this downregulation of Emc and Hairy is mediated by Notch was tested by analyzing the expression of Emc and Hairy when Notch signaling is blocked and when it is ectopically activated (Baonza, 2001).

In mitotic clones of the Notch null allele N^54/9, the expression of Hairy is displaced posteriorly extending behind the morphogenetic furrow. The consequent ectopic expression of Hairy within the furrow is accompanied by a reduction in Atonal expression: Atonal levels remain at the low level normally observed anterior to the furrow. Similar results were obtained with Delta clones. Reciprocally, when Notch signaling is ectopically activated in clones of Delta-expressing cells, Hairy is downregulated, both within the clone and in the cells immediately surrounding it. In these clones Emc is also downregulated within the clone, although for reasons that are not understood, Emc levels are unusually high in the wild-type cells that border the clone. The downregulation of Emc and Hairy caused by the ectopic expression of Delta correlates with increased expression of Atonal ahead of the furrow. It is concluded from these results that Delta/Notch signaling promotes Atonal activation and neural differentiation by downregulating the repressors Hairy and Emc (Baonza, 2001).

The most well characterized role of Notch signaling in R8 photoreceptor determination is mediating the process of lateral inhibition, which refines Atonal expression from a small group of cells to a single cell. However, an earlier and opposite role for Notch, this time promoting neural determination, has also been recognized, although how this 'proneural' function integrates with other pathways necessary for neural differentiation has been unclear. In this work, it has been shown that in normal eye development the proneural function of Notch signaling depends on prior Dpp signaling. Emc and Hairy, two negative regulators of Atonal expression, mediate the proneural function of Notch signaling in the eye. Thus, a model is proposed that links the upregulation of Atonal in the proneural groups with the downregulation of Hairy and Emc through the activation of Delta/Notch signaling (Baonza, 2001).

Thus a model is proposed specifically to integrate proneural Notch signaling into the concept of a progression of cell states, from undetermined to pre-proneural to proneural. Hh in the cells posterior to the morphogenetic furrow activates the expression of Dpp in the furrow. The data support the idea that as Dpp acts at a longer range than Hh, this relays a signal to a zone extending about 15 cells anterior to the furrow, priming these cells for differentiation. This makes cells competent to receive a later signal that upregulates Atonal expression, thereby initiating overt neural differentiation. This second signal is also dependent on Hh, but operates only much closer to the furrow: the evidence implies that it consists of Delta activating Notch signaling. The initial 'pre-proneural' state is molecularly defined by the accumulation of the repressors of atonal transcription Hairy and Emc, as well as by the positive regulator of Atonal, the HLH transcription factor Daughterless. Therefore, although Atonal and Daughterless are both expressed in this pre-proneural zone, neural differentiation is not initiated, as Hairy and Emc ensure that Atonal activity remains below a threshold. The Hh-dependent activation of Delta/Notch signaling triggers the transition from this pre-proneural state to the proneural state by downregulating both Hairy and Emc. This negative regulation of the Atonal repressors is sufficient to allow the accumulation of active Atonal in the proneural groups to a level where R8 determination is initiated.

Notch can only trigger Atonal upregulation in a zone extending 12-15 cells anterior to the furrow, and this zone is defined as the cells that receive the diffusible factor Dpp, whose source is in the furrow. Dpp acts to define a ‘pre-proneural’ state that prepares cells for the imminent initiation of neural determination. This pre-proneural state is defined as the zone of cells that initiate Hairy and Atonal expression in response to Dpp signaling. A functional definition to this state can be added: all these cells are primed for neural differentiation because all can respond to Notch activation by upregulating Atonal levels (Baonza, 2001).

Simultaneous loss of Hairy and Emc activity leads to the precocious differentiation of photoreceptors in a competent region ahead of the morphogenetic furrow, a phenotype that resembles that caused by ectopic expression of Delta. In addition, ectopic Notch signaling downregulates Hairy and Emc ahead of the morphogenetic furrow, causing the accumulation of Atonal at high levels; conversely, loss of function of Notch signaling increased the levels of Hairy. It is concluded that Delta/Notch signaling regulates the expression of these negative regulators in the eye. Consistent with this proposal, Emc is also regulated by Notch in the developing wing disc (Baonza, 2001).

Although Notch signaling negatively regulates both Hairy and Emc, the ectopic expression of Delta does not affect both genes identically. Thus, whereas Hairy is removed both within the clone and in the neighboring cells, Emc is only downregulated autonomously within the clone. This distinction could be an artifact caused by the perdurance of ß-galactosidase. Alternatively, these differences may reflect a different requirement for Notch signaling in the regulation of both genes. Furthermore, the expression pattern of Hairy and Emc is different during the normal progression of the morphogenetic furrow. Hairy is precisely regulated, being expressed only in the cells anterior to the furrow, and is rapidly downregulated in the furrow. This precise regulation is crucial as shown by the ectopic expression of hairy. Emc has a much broader expression pattern in the eye disc, although it shows a similar upregulation followed by downregulation in the zone immediately anterior to the furrow (Baonza, 2001).

It is also worth pointing out that not only does the expression pattern of Emc and Hairy differ, but their exact mechanism of repression is also distinct. Hairy regulates bHLH proteins by a mechanism of direct DNA binding and transcriptional repression. Emc, however, forms complexes with bHLH proteins, preventing their DNA binding. Thus, Emc can antagonize the proneural function of Atonal by two distinct mechanisms: (1) Emc presumably binds to Atonal, rendering it incapable of activating its targets; (2) Emc controls the levels of Atonal. By analogy to its regulation of two other bHLH transcriptional regulators, Achaete and Scute, it is expected that Emc interferes with the autoregulatory upregulation of atonal expression. This positive autoregulation is an essential component of its accumulation in cells within the morphogenetic furrow. In conclusion, the proneural action of Notch signaling increases Atonal activity by two mechanisms: atonal is transcriptionally upregulated, and at the same time a repressive co-factor is removed. These concerted actions lead to the accumulation of active Atonal and thereby the initiation of neural differentiation (Baonza, 2001).

Delta and Hairy establish a periodic prepattern that positions sensory bristles in Drosophila legs

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

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

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

This study identified Dl as a second prepattern gene that functions together with hairy to establish ac expression in the leg microchaete proneural fields. Several lines of evidence are are presented that support this conclusion. First, it was found that, beginning at 4 h APF, Dl expression is up-regulated in domains overlapping the microchaete proneural fields. This distribution of Dl is similar to that, in the notum, where Dl has been shown to regulate proneural ac expression. Second, it is shown that ac expression is expanded in legs with reduced Dl function. Third, it was found that elevated N signaling throughout the tarsus results in severely reduced ac expression. Finally, activation of N signaling was observed within the hairy-OFF interstripes (ac interstripes that do not express hairy), in agreement with the genetic requirement for Dl/N signaling in these domains. Based on these results, it is proposed that ac expression is activated broadly during mid-prepupal leg development but is confined to the microchaete proneural fields by a previously generated prepattern of repression, established by Hairy and Dl/N signaling. This hypothesis is supported by analysis of cis-regulatory elements that direct ac expression in the leg microchaete proneural fields (Joshi and Orenic, unpublished cited in Joshi, 2006). By generating rescue and reporter constructs, an enhancer has been identified that specifically controls expression of ac in the microchaete proneural fields. Unlike the hairy leg enhancers, no modular organization of the cis-regulatory elements that control expression of ac stripes in different regions of the leg is observed. Rather, preliminary analyses suggest that there is one enhancer consisting of an activation element that directs broad expression of ac along the leg circumference and two repression elements, which are N- or Hairy-responsive. This finding is consistent with genetic studies and the model for regulation of ac expression in the leg microchaete proneural fields (Joshi, 2006).

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

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

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

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

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

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

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

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

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

Functional analysis of the NHR2 domain indicates that oligomerization of Neuralized regulates ubiquitination and endocytosis of Delta during Notch signaling

The Notch pathway plays an integral role in development by regulating cell fate in a wide variety of multicellular organisms. A critical step in the activation of Notch signaling is the endocytosis of the Notch ligands Delta and Serrate. Ligand endocytosis is regulated by one of two E3 ubiquitin ligases, Neuralized (Neur) or Mind bomb. Neur is comprised of a C-terminal RING domain, which is required for Delta ubiquitination, and two Neur homology repeat (NHR) domains. Previous studies have shown that the NHR1 domain is required for Delta trafficking. This study shows that the NHR1 domain also affects the binding and internalization of Serrate. Furthermore, it was shown that the NHR2 domain is required for Neur function and that a point mutation in the NHR2 domain (Gly430) abolishes Neur ubiquitination activity and affects ligand internalization. Finally, evidence is provided that Neur can form oligomers in both cultured cells and fly tissues, which regulate Neur activity and, by extension, ligand internalization (Liu, 2012).

Neur is an E3-ubiquitin ligase that plays an essential role in Notch signaling by regulating the endocytosis of Notch ligands. It contains NHR domains, which are rare and conserved between vertebrates and invertebrates but not present in viruses, bacteria, fungi, or plants. In the Drosophila proteome, besides Neuralized, there are two other NHR-containing proteins, CG3894 and Bluestreak. In mammals, proteins containing NHR domains (also known as NEUZ) include the β-catenin regulator OzzE3 and lung-inducible Neuralized-related C3HC4 RING protein (LINCR). Recent studies reported that the human homologue of Bluestreak serves to localize to the centrosome. Although the general role of the NHR domains is unclear, these domains tend to cluster, and most proteins contain two to six NHR domains. The significance of having more than one NHR domain in one protein is to yet be determined (Liu, 2012).

This study has investigated the role of the highly conserved NHR domains in Neur function. The NHR1 domain alone mediates the interaction between Neur and both Delta and Serrate. It was also shown that the NHR2 domain is required for Neur function, and while it is not required for the interaction with Notch ligands, it is involved in Dl internalization, a critical step in Notch activation. Moreover, the NHR domains play a role in Neur oligomerization, which in turn could contribute to Neur ubiquitination activity and ligand endocytosis (Liu, 2012).

The NHR1 domain mediates the interaction between Neur and the Notch ligands Delta and Serrate. Previous studies have shown that the NHR1 domain of Neur is both necessary and sufficient for the interaction with the Notch ligand Dl. Specifically, it was found that a point mutation in a highly conserved glycine residue within the NHR1 domain (G167E) abolishes the ability of Neur to bind Delta. Whether the NHR1 domain was also required to bind to Serrate, however, was unknown. In fact, in vitro studies in vertebrates suggested that the NHR2 domain in mouse Neuralized- like 1 (Neurl1) is sufficient to bind to Jagged1, the mouse orthologue of Serrate. In contrast, the current study found that the NHR2 domain is not required for Neur to bind Serrate in Drosophila and that the interaction is mediated entirely by the NHR1 domain. Other studies reported previously that the motifs on Dl and Ser that mediate the interaction with Neur are conserved. In comparisons of protein sequences, Jagged1 and Serrate share 40.7% similarity overall, while the overall similarity between Jagged1 and Dl is 33.8%. The NHR1 and NHR2 domains from Drosophila Neuralized have the same degree of amino acid similarity with the mouse Neurl1 NHR2 domain (33%). Since there is no clear correlation between protein sequence similarity and the ability of either the NHR1 or the NHR2 domain to interact with Notch ligands, whether NHR1 or NHR2 is important for interacting with ligands is likely to be species dependent. The Neur-Ser interaction was found to be abrogated by the G167E mutation in the NHR1 domain. Given that the NeurG167E mutant still retains ubiquitination activity, it is unlikely to affect overall protein folding. A previously reported structural analysis of the Drosophila NHR1 domain suggested that Gly167 resides in a hydrophobic core and that the Gly167 mutation presumably destabilizes the surrounding microenvironment. Therefore, the Gly167 mutation may result in spatial changes in the neighboring residues of the core, thus abolishing binding to ligands (Liu, 2012).

The G430E mutation reveals a distinct role for the NHR2 domain in the regulation of Neur activity and Delta trafficking. The data demonstrate that the NHR2 domain is required for Neur function in vivo. NeurG430E fails to rescue neur mutant embryos, while NeurNHR2 has some residual activity, which suggests that they affect different aspects of Neur function. The expression of NeurG430 in a heterozygous background, which does not have a neurogenic phenotype on its own, resulted in a significant increase in the percentage of neurogenic embryos, suggesting that NeurG430E has a negative effect on Neur function. In contrast, NeurNHR2 overexpression did not have any effect on heterozygous embryos, suggesting that it behaves as a loss-of-function allele. Despite the fact that the two NHR2 mutant proteins NeurG430E and NeurNHR2 behave differently, they both localize to the plasma membrane in the presence of Delta both in vitro and in vivo, and they are both capable of binding to the Notch ligands Delta and Serrate. However, both mutant proteins affect the extent of Dl internalization to various degrees. NeurG430E exhibits severely compromised ubiquitination activity and is no longer capable of inducing Delta internalization. NeurNHR2, on the other hand, retains ubiquitination activity but is much less efficient than WT Neur at directing Dl internalization in vivo or in Kc cells. The precise mechanism by which NeurNHR2 affects Delta endocytosis is unclear (Liu, 2012).

One possibility is that the NHR2 domain is required for Neur oligomerization. Neur was shown to form NHR domain-mediated oligomers by coimmunoprecipitation experiments. Therefore, the deletion of the NHR2 domain (NeurNHR2) may simply reduce the oligomerization potential of Neur, leading to a decrease in ligand endocytosis. In contrast, the point mutation (NeurG430) might disrupt the overall structure of the NHR2 domain, preventing oligomerization and resulting in a protein that has no ubiquitination activity and therefore can no longer internalize ligands. However, this model cannot fully explain the data, which show that NHR2 does not prevent Neur oligomerization and that Neur oligomerization still occurs in the absence of any NHR domains, suggesting that while the NHR domains may play a role in oligomerization, they are not necessary for this process. The data also show that although the G430E mutant loses ubiquitination activity, the double mutant containing the NHR1 deletion and the G430E mutation retains ubiquitination activity, which argues that G430E does not affect the overall folding of the NHR2 domain (Liu, 2012).

Another possibility is that the NHR1 and NHR2 domains initially form an intramolecular structure that is inactive and must be resolved for ubiquitination to promote ligand internalization. The G430E mutation may lock Neur into an intramolecular conformation through an NHR1-NHR2 interaction, such that this inactive form can still bind to Dl and Ser but cannot form oligomers and has no ubiquitination activity as a consequence of dysfunctional oligomerization. This model, in contrast to the former one, is supported by the data that demonstrate that the G430E mutant no longer forms oligomers and has no ubiquitination activity. Furthermore, when the NHR1 domain was removed from the G430E mutant (NeurNHR1G430E), the ubiquitination activity was restored, suggesting that the intramolecular loop can no longer form, whereas intermolecular interactions between NHR1 and NHR2 domains can occur. It may also explain the negative effect of NeurG430E on NeurWT: although NeurG430E has a reduced ability to bind WT Neur, it is still be able to sequester some portion of NeurWT into a nonfunctional intermolecular oligomer that can no longer ubiquitinate targets. In contrast, the deletion of the NHR2 domain would prevent intramolecular interactions but would be expected to have a reduced ability to form productive oligomers, leading to a defect in ligand internalization. Whether the NHR2 domain has additional roles in recruiting a protein(s) that promotes Notch ligand ubiquitination and endocytosis remains to be determined. Like Neuralized, other RING domain E3 ligases often function as oligomers, and they multimerize in different ways: some form heterodimers, such as Mdm2-MdmX, and some can form homo-oligomers, such as TRAF. The functional significance of RING E3 oligomerization is poorly defined. One previously proposed model is that the oligomerization of E3 ligases may functionally resemble the dimerization of receptor tyrosine kinases in such a way that autoubiquitination yields a mark that serves as a platform to assemble a signaling complex. Consistent with this idea, ubiquitination was seen in anti-Neur IPs when Dl was not present, consistent with the idea that Neur may be autoubiquitinated. Whether this autoubiquitination can initiate a cascade of further downstream ubiquitination events remains to be determined. It is possible that oligomerization is mediated via autoubiquitination and the interaction of ubiquitinated Neur with itself through a ubiquitin-binding motif (UIM). If so, then the NHR1-NHR2 interaction could also function to keep Neur in an inactive state by occluding the putative UIM. Such a model of Ubi-UIM complex formation was previously proposed for other endocytic proteins (Liu, 2012).

In summary, this study has shown that NHR domains are protein-protein interaction modules that are required for many aspects of Neur function. The NHR1 domain mediates the interaction between Neur and its targets Dl and Ser. Both NHR domains appear to regulate Neur activity by affecting its ability to form oligomers and/or interact with proteins required for the endocytosis of Notch ligands. Interestingly, NHR domains have been identified in several other proteins that are conserved between flies and humans. Whether Neur can form heterodimers with these other NHR-containing proteins and whether these heterodimers play a role in Notch signaling and other developmental processes remain to be determined (Liu, 2012).

Sensitized genetic backgrounds reveal differential roles for EGF repeat xylosyltransferases in Drosophila Notch signaling

In multicellular organisms, glycosylation regulates various developmental signaling pathways including the Notch pathway. One of the O-linked glycans added to epidermal growth factor-like (EGF) repeats in animal proteins including the Notch receptors is the xylose-xylose-glucose-O oligosaccharide. Drosophila glucoside xylosyltransferase (Gxylt) Shams negatively regulates Notch signaling in specific contexts. Since Shams adds the first xylose residue to O-glucose, its loss-of-function phenotype could be due to the loss of the first xylose, the second xylose or both. To examine the contribution of the second xylose residues to Drosophila Notch signaling, biochemical and genetic analysis was performed on CG11388, which is the Drosophila homolog of human xyloside xylosyltransferase 1 (XXYLT1). Experiments in S2 cells indicated that similar to human XXYLT1, CG11388 can add the second xylose to xylose-glucose-O glycans. Flies lacking both copies of CG11388 (Xxylt) are viable and fertile and do not show gross phenotypes indicative of altered Notch signaling. However, genetic interaction experiments show that in sensitized genetic backgrounds with decreased or increased Notch pathway components, loss of Xxylt promotes Delta-mediated activation of Notch. Unexpectedly, it was found that in such sensitized backgrounds, even loss of one copy of the fly Gxylt shams enhances Delta-mediated Notch activation. Taken together, these data indicate that while the first xylose plays a key role in tuning the Delta-mediated Notch signaling in Drosophila, the second xylose has a fine-tuning role only revealed in sensitized genetic backgrounds (Pandey, 2018).

Notch signaling regulates neural stem cell quiescence entry and exit in Drosophila

Stem cells enter and exit quiescence as part of normal developmental programs and to maintain tissue homeostasis in adulthood. Although it is clear that stem cell intrinsic and extrinsic cues, local and systemic, regulate quiescence, it remains unclear whether intrinsic and extrinsic cues coordinate to control quiescence and how cue coordination is achieved. This study reports that Notch signaling coordinates neuroblast intrinsic temporal programs with extrinsic nutrient cues to regulate quiescence in Drosophila. When Notch activity is reduced, quiescence is delayed or altogether bypassed, with some neuroblasts dividing continuously during the embryonic-to-larval transition. During embryogenesis before quiescence, neuroblasts express Notch and the Notch ligand Delta. After division, Delta is partitioned to adjacent GMC daughters where it transactivates Notch in neuroblasts. Over time, in response to intrinsic temporal cues and increasing numbers of Delta-expressing daughters, neuroblast Notch activity increases, leading to cell cycle exit and consequently, attenuation of Notch pathway activity. Quiescent neuroblasts have low to no active Notch, which is required for exit from quiescence in response to nutrient cues. Thus, Notch signaling coordinates proliferation versus quiescence decisions (Sood, 2022).

This study reports that Notch signaling regulates quiescence, entry and exit in Drosophila central brain (CB) NBs. Increasing Notch pathway activity induces CB NBs to exit cell cycle via a Dap-dependent mechanism. Dap, a cyclin-dependent kinase inhibitor and CIP/KIP family member, is a known Notch target gene as is Cyclin E, String (Cdc25) and E2F. Whether Notch regulates other cell cycle genes required for CB NB exit remains unknown. Once CB NBs stop dividing, Notch pathway activity becomes attenuated. Low to no Notch activity is required for CB NBs to exit quiescence in response to dietary nutrient cues. Thus, levels of CB NB Notch activity regulate both the entry and exit from quiescence. High Notch is required for entry, whereas low Notch is required for exit. Intestinal stem cells (ISCs) also experience a period of low to no Notch activity during mid-pupal stages and, if Notch is ectopically induced at this time, ISCs terminally differentiate into secretory enteroendocrine cells. Whether any of the quiescent CB NBs with ectopic Notch terminally differentiate remains unknown (Sood, 2022).

Although Notch is required for quiescence, most CB NBs do stop dividing, albeit late. This suggests that other genes or signaling pathways are required or that residual Notch activity is sufficient to induce CB NB quiescence. Unfortunately, Notch null mutants are embryonic lethal. In addition, this study has shown that Notch is sufficient to induce quiescence. Although quiescence occurs prematurely, it is still restricted to late embryonic stages. Restriction of Notch function (cell cycle exit) is likely due to CB NB intrinsic temporal programs. Temporal programs that likely vary across CB NB lineages, as is the case in the ventral nerve cord. Whether temporal programs regulate levels of Notch pathway activity or provide additional factors needed for CB NB cell cycle exit remains unanswered (Sood, 2022).

In mammals, Notch signaling is more complicated because of gene duplication. Yet, recently, Notch signaling has been shown to regulate neural stem cell quiescence. In Notch2 conditional knockout mice, more neural stem cells are actively dividing in the hippocampus and subventricular zone brain regions in adult animals compared with controls. This results in premature depletion of the neural stem pool and reduced neurogenesis in older mice. This is similar to what is reported in this study and, in the future, it will be interesting to determine which Notch ligands are required and whether neural stem cells in mammals use their newborn daughters for pathway activation (Sood, 2022).

The Notch pathway regulates the Second Mitotic Wave cell cycle independently of bHLH proteins

Notch regulates both neurogenesis and cell cycle activity to coordinate precursor cell generation in the differentiating Drosophila eye. Mosaic analysis with mitotic clones mutant for Notch components was used to identify the pathway of Notch signaling that regulates the cell cycle in the Second Mitotic Wave. Although S phase entry depends on Notch signaling and on the transcription factor Su(H), the transcriptional co-activator Mam and the bHLH repressor genes of the E(spl)-Complex were not essential, although these are Su(H) coactivators and targets during the regulation of neurogenesis. The Second Mitotic Wave showed little dependence on ubiquitin ligases neuralized or mindbomb, and although the ligand Delta is required non-autonomously, partial cell cycle activity occurred in the absence of known Notch ligands. This study found that myc was not essential for the Second Mitotic Wave. The Second Mitotic Wave did not require the HLH protein Extra macrochaetae, and the bHLH protein Daughterless was required only cell-nonautonomously. Similar cell cycle phenotypes for Daughterless and Atonal were consistent with requirement for neuronal differentiation to stimulate Delta expression, affecting Notch activity in the Second Mitotic Wave indirectly. Therefore Notch signaling acts to regulate the Second Mitotic Wave without activating bHLH gene targets (Bhattacharya, 2017).

Differences were observed between how Notch signaling regulates the SMW compared to how Notch regulates photoreceptor differentiation (see Model of the cell cycle and cell fate specification in the morphogenetic furrow). The Notch pathway suppresses the specification of photoreceptor cells in a manner similar to regulation of neurogenesis by Notch in many other tissues. That is, activation of the transmembrane ligand Delta by the ubiquitin ligase Neuralized leads allows Delta to activate Notch, leading to release of the Notch intracellular domain, which then acts in a nuclear complex with Su(H) and Mastermind to induce the transcription of the E(spl)-C family of transcriptional repressors and prevent neural fate specification and differentiation. By contrast to the pathway regulating neural differentiation, cell cycle entry in the SMW occurred in the absence of the E(spl)-C. This suggested that a distinct transcriptional target of Su(H) is involved, but unusually, this Su(H) function did not seem to require the co-activator Mam. Although most studies find that Mam is required for transcriptional activation by Su(H), mutations in mam often have weaker phenotypes than other neurogenic genes. It is possible that Mam protein might exhibit exceptionally strong perdurance, but there is also in vitro evidence for mam-independent transcriptional activation by Su(H) (Bhattacharya, 2017).

SMW entry also occurred in the absence of neur. In the case of neurogenesis, cells lacking neur show a weaker neurogenic phenotype than cells lacking other components of the pathway, both in the embryonic CNS and in the eye, which is thought to reflect the activities of two other ubiquitin ligases, Mindbomb and Mindbomb2. No effect was found on the SMW of mutated mib1, but neither mib2 nor cells lacking two or more of these possibly redundant ubiquitin ligases were examined (Bhattacharya, 2017).

Eoles of ligands were examined in more detail. It has been reported that SMW entry depended on Delta, which is also the ligand necessary for regulation of neurogenesis in the eye as in other parts of the nervous system, but although their figures show an obvious reduction in cell cycle entry in the clones of cells lacking Dl, some cell cycle entry still seems to occur. Those experiments were repeated using cells lacking both Dl and the other known ligand Ser, with similar results, ie, cell cycle entry was obviously disrupted in clones lacking both Notch ligands, nevertheless some cells still entered the cell cycle. A potential complicating factor is that reducing Notch function typically results in excessive neurogenesis, raising the question of whether some Dl Ser mutant cells were prevented from entering the cell cycle by differentiation as photoreceptors. The excess neurogenesis of such clones was partially suppressed by expression of Argos or Ras-DN. Although there is clearly a considerable disruption of the SMW in the absence of N ligands, there is also clear evidence of cell cycle entry, occurring at approximately the same time as the normal SMW (Bhattacharya, 2017).

There are at least three possible explanations for these findings. First, it possible that another ligand besides Dl or Ser activates N in the SMW. Secondly, it has been reported that Dl can signal across several cell diameters using filopodia. During neurogenesis, rescue of Dl mutant clones appears to extend beyond cells immediately adjacent to wild type cells. Signaling across many cell diameters would be required to account for the cell cycle progression seen in Dl Ser mutant clones. Thirdly, Notch can be activated independently of ligands if delivered to certain cellular compartments. Recently it has been suggested that such ligand-independent activation is suppressed by cis-inactivation, in which ligands inhibit N signaling in the same cells, so that ligand-independent activation can be revealed in cells lacking Dl and Ser. Cis-inactivation is potentially an important contributor to the patterning of Notch-mediated lateral inhibition during neurogenesis, but direct evidence of such a role has not yet been obtained. If ligand-independent signaling is occurring in Dl Ser clones, some other pathway must be active near the SMW to explain why ligand-independent signaling cell cycle entry was limited to cells at the same stage posterior to the furrow as those that undergo the normal, ligand-dependent SMW (Bhattacharya, 2017).

The cell cycle targets of Notch are of some interest. Many Drosophila cell cycles depend on regulated transcription of Cyclin E. Cyclin E is clearly required for the SMW suggested that Cyclin E might not be limiting for SMW cell cycle entry, however, because Cyclin E was seen to accumulate more in the differentiating photoreceptor precursors that do not enter the SMW. Alternatively, the lack of Cyclin E accumulation in SMW cells might reflect Cyclin E protein instability in S phase, with accumulation only in differentiating cells that can't enter the cell cycle. A Cyclin E transcriptional reporter appears to accumulate in the same cells as the Cyclin E protein, however, which is not indicative of post-translational regulation (Bhattacharya, 2017).

It has been suggested that Cyclin A was part of the Notch dependent machinery promoting cell cycle entry in the SMW, although it was concluded that it could not be the only important Notch target. What was observed, however, was dependence of Cyclin A protein levels on Notch signaling that exactly paralleled the dependence of S-phase entry on Notch signaling in various genotypes. Like Cyclin B, Cyclin A protein is degraded by APC/Cyclosome activity during G1, so that Cyclin A protein cannot accumulate until the G1/S transition. Therefore these results are also expected if Cyclin A simply accumulates whenever the G1/S transition has occurred, and is not a direct target of Notch signaling (Bhattacharya, 2017).

In mammalian cells, c-Myc appears to be an important Notch target. Indeed, human Notch was first identified as a proto-oncogene that causes leukemia through c-Myc. c-Myc is an activator of the nucleolar protein fibrillarin, which transiently increases during the SMW. Although Dmyc is important for growth in Drosophila, so that clones of myc null mutant cells in the eye disc are small, they nevertheless enter the SMW at the normal time, indicating that myc is unlikely to be the critical Notch target in the SMW (Bhattacharya, 2017).

Emc is a transcriptional target of N signaling in the eye as in other tissues. Even though Emc protein levels seem to be less affected by N signaling than is emc RNA, it has been suggested that N regulates the eye cell cycle by upregulating emc, thereby inhibiting Da, which is proposed to inhibit cell cycle entry. Contrary to this model, previous studies have reported that Da is in fact required for the SMW. We investigated this potential role for emc directly, finding no evidence that emc was required for the SMW. These studies confirmed previous findings that da is required for S phase entry into the SMW, although not apparently for cell cycle activity anterior to the morphogenetic furrow (Bhattacharya, 2017).

The cell-autonomy of da requirement in the cell cycle had not been determined previously. da mutant cells sometimes enter the SMW at the posterior boundaries of da clones, where they were adjacent to wild type cells. This suggested that da was required for the production of a short-range non-autonomous signal that is required for SMW entry, and could be provided to da mutant cells that were near to wild type cells (Bhattacharya, 2017).

It is instructive to compare the effect of da on the cell cycle to that of ato. Ato is the heterodimer partner of Da that is required for R8 specification. Da is also required independently of ato to specify R2,R3,R4 and R5. Interestingly, this requirement in R2-5 seems to be incomplete, since some da mutant R2-5 cells do differentiate at reduced frequencies. Because of this requirement for da outside R8, R1-7 photoreceptor cells can differentiate near borders of ato mutant clones, but fewer photoreceptors are expected near the borders of da mutant clones, and mostly of the R1, R6 and R7 cell types. Proneural genes often promote Notch ligand expression, and accordingly ato is required for normal Dl expression during these stages of eye development. Reduced Dl expression is a plausible explanation for the absence of the SMW within ato clones, and the more extensive requirement for da in photoreceptor differentiation and Dl expression may explain why non-autonomous rescue of the SMW is more pronounced near the borders of ato clones than near the borders of da clones (Bhattacharya, 2017).

A model is present for SMW. Unpatterned proliferation of the eye imaginal disc is terminated ahead of the morphogenetic furrow by Hh and Dpp signaling. Hh and Dpp affect many aspects of eye development including expression of master regulators of retinal determination such as Eyeless, Teashirt, Dachshund, and Sine Oculis, as well as Homothorax. Cells posterior to the furrow can still respond to growth, because extra cell cycles are driven in unspecified cells by overexpression of CycD/cdk4, Inr, or myc, as well as by Cyclin E. It is possible that Notch regulates cellular growth in the SMW, since there appears to be a small but discernible increase in nucleolar size at this stage, and progenitor cells are not noticeably smaller after dividing, indicating that growth accompanies the SMW cell division. The SMW, however, does not depend on myc. It appears to depend on a transcriptional target of Su(H) outside of the E(spl)-C of bHLH genes and that can be transcribed in the absence of Mam. The possibility of a function of Su(H)/Nicd other than transcription cannot be ruled out. The SMW clearly depends on CycE, but CycE may not be the N-dependent gene required for SMW entry, because it is not obviously elevated in SMW cells. Contrary to a recent proposal, the SMW does not depend on the HLH gene emc and is not inhibited by the bHLH protein Da. Instead da is required for entry into the SMW. The positive requirement for da is cell-nonautonomous, as is the requirement for its heterodimer partner ato, and consistent with the role of ato and da in promoting Dl expression during retinal differentiation, which is likely to account for the cell cycle defect. Surprisingly, the requirement for Dl in the SMW cell cycle was not absolute, either because an unidentified ligand exists, long-range cell-nonautonomy occurs, or Dl (and Ser) may contribute to cis-inactivation of N, so that some ligand-independent Notch signaling could occur in the unphysiological circumstance that Notch ligands are completely absent (Bhattacharya, 2017).

GENE STRUCTURE

There are two major transcripts and three minor ones. The 4.5 transcript is maternal (Kopczynski, 1989). The transcripts differ in the length of their 3'UTR. Minor transcripts consisting of introns of the Delta gene accumulate in the nucleus (Haenlin, 1990).

genomic DNA length - 25kb

cDNA clone length - 5.4 and 4.5 kb

Bases in 5' UTR -136

Exons - six

Bases in 3' UTR - 224

PROTEIN STRUCTURE

Amino Acids - 832

Structural Domains

The protein sequence deduced from the cDNA sequence contains the following four elements: an N-terminal signal peptide required for transport across the membrane, an extracellular domain to residue 596, a transmembrane domain to residue 617, and an intracellular domain of 214 amino acids. There are five consensus asparagine-linked potential glycosylation sites. From residue 217 to 566 there are nine repeats of a 44 amino acid domain homologous to the vertebrate epidermal growth factor receptor. EGF repeats are found in Notch and in many other proteins including integrin and laminin (Kopczynski, 1988). The EGF domain has a major role in differentiation in many characterized developmental systems.

date revised: 18 February 2024

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