Suppressor of Hairless



Su(H) is present in the early syncytial embryo, suggesting it is provided maternally. It reappears in a small number of segmentally reiterated cells in the peripheral nervous system. This expression is specific to the external sensory organs and internal sensory organs (chordotonal organs) (Schwasgath, 1995). Su(H) is necessary to restrict achaete and scute expression to single cells. This function is carried out by the Notch signalling pathway (Lecourtois, 1995).


Loss of Su(H) at this stage causes multiple cells, rather than single cells, to adopt sensory organ precursor fate. Thus Su(H) behaves as a neurogenic gene. Su(H) is expressed in wing, leg, haltere and eye-antennal imaginal discs (Schweisguth, 1995)

Evidence that stem cells reside in the adult Drosophila midgut epithelium: Expression of Su(H)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Effects of Mutation or Deletion

Suppressor of Hairless is lethal on first-day of pupation. In the Su(H) mutant embryo, the number of neuroblasts and sensory organ precursors that segregate from the ventral and dorsolateral neuroectoderm respectively is greatly decreased (Lecourtois, 1995).

Comparison between the phenotypes produced by Notch, Suppressor of Hairless and Enhancer of split mutations in the wing and thorax indicate the Su(H) and Notch requirements are not indistinguishable, but that Enhancer of split activity is only essential for a subset of developmental processes involving Notch function. For example Enhancer of split function is required for the segregation of a single sensory organ precursor in in wing morphogenesis but not for the correct differention of the progeny from each sensory organ precursor, requiring Notch and Su(H). Likewise, the ectopic expression of Enhancer of split proteins does not reproduce all the consequences typical of ectopic Notch activation. For example, no ectopic acitvation of wingless occurs when Enhancer of split proteins are ectopically expressed. It is suggested that the Notch pathway bifurcates after the activation of Su(H) and that Enhancer of split activity is not required when the consequence of Notch function is the transcriptional activation of downstream genes. Transcriptional activation mediated by Su(H) and transcriptional repression mediated by Enhancer of split could provide greater diversity in the response of individual genes to Notch activity (de Celis, 1996)

Suppressor of Hairless mutant alleles exhibit dose-sensitive interactions with Hairless loss-of-function mutations. Genetic interactions with H loss-of-function alleles has led to the definition of two classes of Su(H) mutant alleles: 'loss-of-function' alleles that, like deficiencies, suppress the haplo-insufficient H phenotype, and 'gain-of-function' alleles that, like duplications, enhance it. These gain-of-function alleles were thought to increase N signaling. However, somatic clones of cells mutant for such gain-of-function alleles, produce typical loss-of-function phenotypes. Further genetic analysis shows that gain-of-function alleles are actually partial loss-of-function alleles. It is suggested that the mutant proteins encoded by gain-of-function Su(H) alleles are defective for N-signaling activity but retain their ability to bind H: This binding results in a titration of H, hence in an enhancement of the haplo-insufficient H phenotype. These results provide a simple solution to a paradox that arose from classifying Su(H) mutation alleles using an interaction assay. More importantly, they provide strong genetic evidence that Su(H) is a direct target of H (Schweisguth, 1998).

The activities of Serrate protein were analyzed in wing development. An important outcome of Serrate activity is the induction, through Notch, of the wing margin at the dorsal-ventral interface of the wing imaginal disc. Analysis of the function of Ser indicates that excess Ser can titrate out Notch function in the developing wing, an effect that is suppressed by an increase in the dosage of Notch. Since Serrate has been shown to bind Notch, this effect can be interpreted as an induction of a dominant negative activity of Serrate on Notch. Ubiquitous expression of Ser in the wing pouch throughout the development of the wing curtails wing development and produces wings that lack most of the margin and adjacent tissue (Klein, 1997).

Serrate can activate or inactivate Notch in a concentration-dependent manner as revealed by the expression of two targets of Notch activity: wingless and Enhancer of split. E(spl) is expressed in a stripe that straddles the DV interface at the beginning of the third larval instar. Ectopic expression of Ser product reduces the normal margin and produces two new margins on the ventral side of the developing wing. Ectopic expression of Ser in a Su(H) mutant has no effect on disc development or patterning and results in discs that are identical to those of Su(H) mutants. This demonstrates that the activity of Serrate during wing development requires Su(H). While the inactivation produced by ectopic Ser is likely to be mediated by a dominant negative effect over Notch, the activation is similar to that elicited by Delta and requires the product of the suppressor of Hairless gene (Klein, 1997).

Expression of Ser leads to smaller wings with thick veins. When wingless and Serrate are coexpressed, the resulting flies bear large wings that are covered with bristles. These wings have a different shape from those in wild-type: they lack a defined margin and are more round rather than elongated. This experiment shows that increased functional Wingless not only can suppress the dominant negative effect of Serrate expression, but can cooperate with Serrate to promote wing development. These wings are very similar to those that result from the expression of Delta and indicate that wingless enables Serrate to activate Notch. Coexpression of Ser and Notch generates very large wings, bigger than those that result from expression of Delta or coexpression of Serrate and wingless. These large wings have a clearly define margin with an antineurogenic phenotype. These results indicate that regulation of the concentration of Serrate during development must be an important way of regulating its activity.

Two different models are proposed. In one view the role of Serrate is to bind Notch at the DV interface to free Notch-bound Delta, which then would trigger events at the DV boundary that lead to wing outgrowth. This view is consistent with the observation of a dominant negative activity associated with Serrate and with the ability of Serrate to mimic Delta by activating Notch, leading to signaling through Su(H) and the consequent outgrowth of the wing. A different view sees Serrate as the active component of the signaling system, either alone or in combination with Wingless (Klein, 1997).

A single copy of Hairless, is able to suppress the wing defects of heterozygous strawberry notch, suggesting that Hairless and sno exhibit related antagonistic activities downstream of the Notch pathway. In a similar fashion, a single copy of Suppressor of Hairless and a single copy of sno show enhanced defects, indicating that Su(H) and Sno cooperate closely in patterning the wing. As Su(H) and Sno have not been shown to physically interact, this may mean that the two proteins work in parallel or that the interaction is too weak to be detected (Majumdar, 1997).

The Notch signaling pathway is involved in many processes where cell fate is decided. Previous work has shown that Notch is required at successive steps during R8 specification in the Drosophila eye. Initially, Notch enhances atonal expression and promotes atonal function. After atonal autoregulation has been established, Notch signaling represses atonal expression during lateral specification. Once ato autoregulation is established, lateral specification starts to limit ato expression to R8 precursor cells. Thus Notch signaling is required at successive steps during R8 specification, initially to promote neural potential and later to suppress it through lateral specification. Consequently the phenotype of loss of Notch gene function varies with time. If Notch function is removed conditionally once ato expression has been enhanced, supernumerary R8 cells differentiate because lateral specification is affected. If N function is absent from the outset, such as in a clone of cells lacking N, little R8 specification can occur. For this reason clones of N null mutant cells in the eye disc almost completely lack neural differentiation, contrasting with the neurogenic phenotype of null mutant embryos. Using clonal analysis it is shown that Delta, a ligand of Notch, is required along with Notch for both proneural enhancement and lateral specification (Ligoxygakis, 1998).

Recent studies have identified Su(H) as a common component in the Notch signal transduction pathway. Ligand binding (Delta or Serrate) to Notch activates Su(H), which can shuttle between the cytoplasm and the nucleus and act as a transcription factor. Activated Su(H) turns on a number of downstream target genes mediating Notch signaling in lateral specification or inductive processes. In order to investigate the role of Su(H), clones of cells homozygous for an apparent null allele of Su(H) were generated by FLP-mediated recombination. In the eye imaginal disc Su(H)- mutant cells are associated cell autonomously with neural hypertrophy. Many of the ectopic neural cells are R8 photoreceptors, based on expression of the R8- specific protein Boss. It appears that, like the E(spl)-C, Su(H) is required for lateral specification but not for R8 differentiation. To confirm this conclusion ato expression was examined. In wild type, initial broad expression of Ato protein is replaced by R8-specific expression that persists for 6-8 hours (3-4 columns of ommatidia) and then fades. Whereas ato expression begins normally in Su(H) mutant cells, ato expression is maintained in many more R8 cells than in wild type, indicating failure of lateral specification. Expression of ato then fades from Su(H) mutant R8 cells at the same time as from wild-type cells. Thus, like the E(spl)-C, Su(H) is required for lateral specification but not for the proneural function of Notch in the retina. Interestingly, although many extra R8 precursors form in Su(H) mutant clones, not all Su(H) mutant cells maintain ato expression or subsequently express the R8-specific Boss protein. Instead clusters of R8-like cells often seem interspersed with non-R8 neurons. ato expression in wild type first becomes patterned into regular ‘intermediate groups’ of about ten ato-expressing cells before resolving to individual R8 precursors. These results support the conclusions that initial spacing of intermediate groups is not part of the N-dependent lateral specification process, and so does not depend on E(spl) or Su(H) (Ligoxygakis, 1998).

Immunohistochemical detection of Mastermind on polytene chromosomes reveals binding at >100 sites. Chromosome colocalization studies with RNA polymerase and the Groucho corepressor protein implicate Mam in transcriptional regulation (Bettler, 1996).

Database comparisons to Mam do not reveal string similarities in nonrepetitive domains, however, limited similarities between Mam and some leucine zipper proteins have been noted. The basic DNA binding domain that flanks the leucine zipper of the proteins encoded by cap 'n' collar, junD, fos and ATF-3 exhibits some features in common with Mam, although Mam does not contain a leucine zipper. The similarity exends to Skn-1, a C. elegans protein that likewise does not contain a leucine zipper, but shows more significant similarity to the zipper class of proteins. Skn-1 binds DNA as a monomer, in a sequence-specific fashion. Two leucine zipper class proteins, ATF-2/ATF-1 and ACR1, contain an additional small block of sequence similarity to Mam. Thus, it is conceivable that Mam represents a DNA-binding protein that is related to, but highly diverged from the leucine zipper class (Bettler, 1996).

If Mam functions late in the neurogenic pathway as a nuclear regulatory protein, there are two principal roles to consider: activation of products of the E(spl) complex and/or repression of the proneural loci. The genetic interaction between mam and Suppressor of Hairless points to the former possibility. Based on its similarity to CBF1, it has been suggested that Su(H) protein may need to recruit a coactivator for E(spl) induction; it is conceivable that Mam performs this function (Bettler, 1996).

The Notch pathway plays a key role in the formation of many tissues and cell types in Metazoans. Notch acts in two pathways to determine muscle precursor fates. The first is the 'standard' Notch pathway, in which Delta activates the Notch receptor, which then translocates into the nucleus in conjunction with Su(H) to reprogram transcription patterns and bring about changes in cell fates. The second pathway is poorly defined, but known to be independent of the ligands and downstream effectors of the standard pathway. The standard pathway is required in many different developmental contexts; it was of interest to determine if there is a general requirement for the novel pathway. The novel Notch pathway is required for the development of each of five examined cell types. Holonull Notch mutants (mutants null for maternal and zygotic Notch) have a more extreme phenotype than null mutants for Su(H), Delta, neuralized or mastermind. In Notch holonull embryos, clusters of 10 or 15 eve expressing RP2-like cells are found in place of a normal single RP2. The phenotype for the other neurogenic genes is far less severe. Notch and other neurogenic genes are involved in the determination of the mesectoderm and the visceral mesoderm. The Notch holonull phenotype is more severe in both cases than that of other holonull embryos. These results indicate that the novel pathway is a widespread and fundamental component of Notch function. Both Notch pathways operate in the differentiation of the same cell types. In such cases, the novel pathway acts first and appears to set up or limit the size of equivalence groups. The standard pathway then acts within the equivalence groups to limit individual cell fates (Rusconi, 1999).

Su(H)-independent activity of Hairless during mechano-sensory organ formation in Drosophila

Formation of mechano-sensory organs in Drosophila involves the selection of neural precursor cells (SOPs) mediated by the classical Notch pathway in the process of lateral inhibition. The subsequent cell type specifications rely on distinct subsets of Notch signaling components. Whereas E(spl) bHLH genes implement SOP selection, they are not required for later decisions. Most remarkably, the Notch signal transducer Su(H) is essential to determine outer but not inner cell fates. In contrast, the Notch antagonist Hairless, thought to act upon Su(H), influences strongly the entire cell lineage, demonstrating that it functions through targets other than Su(H) within the inner lineage. Thereby, Hairless and Numb may have partly redundant activities. This suggests that Notch-dependent binary cell fate specifications involve different sets of mediators depending on the cell type considered (Nagel, 2000).

The decision between the tormogen (socket) versus trichogen (shaft) fate of the pIIa progeny seems to depend strictly on the balanced doses of H and Su(H). Changes in the dose of either one pushes the equilibrium completely towards the opposite fate. Accordingly, Su(H) protein accumulates to very high levels in the future tormogen, and can thus override the elevated levels of H protein within this cell. The epistasis of H over dx regarding outer bristle cell fates can be easily explained by the dominating activity of Su(H) within the pIIa progeny. The choice between neuron and thecogen (sheath) fate is based on a quite different mechanism, because unlike H, Su(H) is not necessary for the emergence of the two opposing cell types. The default state of pIIIb descendants is neuronal. The Notch signal redirects one of these cells into thecogen fate. Although both Su(H) and dx, positively influence Notch signaling in the presumptive thecogen, none of the two is required for the generation of this cell type. Thus, the Notch signal in the thecogen might be transduced by a molecular mechanism independent of Su(H) or dx involving as yet unknown factor(s). In the absence of H, the presumptive neuron gains thecogen fate. Therefore, H has an important role in protecting the neuron from the Notch signal. Since this signal does not emanate from Su(H), H must act through unknown component(s). This is the first unambiguous example of a Su(H)-independent function of H (Nagel, 2000).

Both in loss of function and gain of function combinations numb is epistatic to Su(H) within the pII cells, indicating that Su(H) acts downstream of numb. This is in agreement with a model, whereby numb antagonizes Notch signaling through direct interference with the Notch receptor. Within the pIIa progeny, however, Su(H) can override the inhibiting activity of ectopic numb protein. This interference might again be direct, because preliminary evidence from yeast interaction trap experiments shows that Numb and Su(H) physically interact. Furthermore, by inhibiting Notch signaling, numb might indirectly modulate the levels of Su(H) transcriptional activity. Thus, the conflicting epistasis data might reveal once more different sensitivities of the sensory organ cell lineage regarding Notch signaling, especially the preference of the pIIb over the pIIa fate (Nagel, 2000).

During the development of mechanosensory organs, Notch is required at two distinct steps: the singling out of the sensory organ precursor, SOP, and the correct specification of cell fates within the sensory organ lineage, SOL. Apparently, different subsets of Notch signaling components are used for these two processes. Whereas SOP selection in the process of lateral inhibition requires the 'classical' battery of Notch signaling components, namely Su(H), dx, mam, E(spl) bHLH and H, the subsequent asymmetric cell divisions require only certain Notch components plus the intrinsic activity of numb. Numb plays a major role in the distinction between the pII siblings. In the pIIa progeny, socket cell fate is enforced with the help of Su(H) and, to a lesser degree, dx, and the role of H is to protect the shaft from this fate. In the sub-epidermal progeny, Notch signaling determines sheath cell fate, promoted to some degree by dx and Su(H). However, since neither of the components, Su(H), dx nor E(spl) bHLH are strictly required for the selection of thecogen fate, the Notch signal is transduced by other factor(s), X. The role of mam in this process is as yet undecided, since the mutant cell clones are rather uninformative and appropriate overexpression constructs are unavailable. The neuron has to be protected from the Notch signal, and both numb and H play a pivotal role in this process. Apparently, the target of numb is the Notch receptor itself. It is not clear whether H acts at the same level, or whether it acts on a different target, maybe directly involving the presumptive signal transducer X. Although both mam and dx might be targets of H, no physical interactions were observed in the yeast interaction trap assay. Overall, H represents a key player in antagonizing the Notch signal and thus assures, that in the end all four different cell types of the mechano-sensory organ arise (Nagel, 2000).

A summary of Notch signaling during mechano-sensory organ development is presented. Notch signaling is required in the entire cell lineage, as is the antagonist Hairless. Whereas the lateral inhibition process uses the classical battery of Notch signaling components, the subsequent binary cell fate specifications rely only on a subset of these components and involve in addition the intrinsic antagonist Numb. In a first step SOP is singled out by lateral inhibition from a proneural cluster, protected through the activity of H. The surrounding cells are forced by the SOP into epidermal fate through the activation of the Notch receptor, implemented with the help of Su(H), dx, mam and E(spl) bHLH proteins (classical pathway). In a second step, Notch signal, promoted by Su(H) and dx, forces one SOP daughter cell into pIIa fate from which the pIIb cell is protected by the antagonists numb and H. The pIIb gives rise to the pIIIb and a glial cell. In a third step, the progeny of pIIa are socket and shaft cell. The socket cell receives the Notch signal via Su(H) and dx, the effector genes are unknown. The shaft cell is protected by H and numb from the Notch signal. In a fourth step, the progeny of the pIIIb are sheath cell and neuron. The sheath cell receives a Notch signal promoted by unknown factor X, whereas the neuron is protected by H and numb (Nagel, 2000).

Suppressor of Hairless signaling independent of Notch

Su(H)/CBF1 is a key component of the evolutionary conserved Notch signalling pathway. It is a transcription factor that acts as a repressor in the absence of the Notch signal. If Notch signalling is activated, it associates with the released intracellular domain of the Notch receptor and acts as an activator of transcription. During the development of the mechanosensory bristles of Drosophila, a selection process called lateral inhibition assures that only a few cells are selected out of a group to become sensory organ precursors (SOP). During this process, the SOP cell is thought to suppress the same fate in its surrounding neighbours via the activation of the Notch/Su(H) pathway in these cells. Although Su(H) is required to prevent the SOP fate during lateral inhibition, it is also required to promote the further development of the SOP once it is selected. Importantly, in this situation Su(H) appears to act independently of the Notch signalling pathway. Loss of Su(H) function leads to an arrest of SOP development because of the loss of sens expression in the SOP. These results suggest that Su(H) acts as a repressor that suppresses the activity of one or more negative regulator(s) of sens expression. This repressor activity is encoded by one or several genes of the E(spl)-complex. These results further suggest that the position of the SOP in a proneural cluster is determined by very precise positional cues, which render the SOP insensitive to Dl (Koelzer, 2003).

Thus Su(H) is required to promote SOP development. This is based on the fact that most cells of proneural clusters in the notum that lack Su(H) function do not express SOP markers such as Sens, Hindsight (Hnt) and partially neurA101-lacZ. Loss of neurA101-lacZ expression has been attributed to a 'general sickness' of the mutant discs, since the lack of neurA101-lacZ expression has only been observed in the late developing proneural clusters. The data argue against such an explanation: Presenilin (Psn) mutant wing imaginal discs exhibit a stronger neurogenic phenotype than do Su(H) mutants. Similar to Su(H) mutants, homozygous Psn mutant animals also die during the early pupal phase. Nevertheless, the cells of the proneural clusters of these mutants express all tested markers, indicating that SOP development is not affected. The same is true for kuzbanian (kuz) mutants, whose mutant phenotype is comparable with that of Su(H) mutants. Hence, general sickness of the wing imaginal disc cells is not likely to explain the arrest of SOP development in Su(H) mutants (Koelzer, 2003).

A role of Su(H) in development of the SOP is surprising, because it is a core element of the Notch signalling pathway and the activity of this pathway is required to prevent SOP development in cells of the proneural clusters. Importantly, in this new role, Su(H) seems to function independently of the Notch signalling pathway. This is indicated by the finding that the Su(H) mutant phenotype is epistatic over that of Psn mutants (Koelzer, 2003).

The data presented here indicate that Su(H) appears to be required to suppress the activity of one or more members of the E(spl)-C, that in turn suppress the expression of genes such as hnt and sens. This conclusion is based on: (1) the failure of Su(H)VP16 to activate sens; (2) the fact that Psn H double mutants display a similar loss or reduction of sens expression as Su(H) and Su(H); Psn double mutants, and (3) the fact that expression of a Su(H) construct that is unable to bind H (UAS Su(H)DeltaH) leads to an arrest of SOP development in Psn mutant wing imaginal discs. Several reports show that H is involved in Su(H)-related suppression of gene expression in the absence of Notch signalling. Recently, it has been shown that H acts as a bridge between Su(H) and the general co-repressors CtBP and Gro. It is therefore likely, that this Su(H)/H/Gro/dCtBP complex mediates the repressor function required during SOP development (Koelzer, 2003).

Repression by Su(H) is not strictly required in all proneural clusters to allow expression of sens and other late SOP markers. Examples are the clusters in the wing region, such as the clusters of the dorsal radius. However, even in these clusters, sens and hnt are not expressed in all cells that express early markers, such as neurA101. Therefore, it appears that the activity of Su(H) promotes SOP development also in these clusters. The clusters of the dorsal radius give rise to other types of sense organs, such as companiforme sensilla, and it is possible that there are different requirements for the activity of Su(H) for the development of the different types of sense organs (Koelzer, 2003).

The removal of one copy of the E(spl)-C is already sufficient to relieve the block in SOP development in Su(H) mutants, indicating that the arrest is probably caused by the abnormal expression of one or more members of the complex. Although the complex encodes for several well-characterized repressors of neural development, it was not possible to pinpoint the repressor function to any particular gene. Many studies by various groups have studied the regulation of the genes of the E(spl)-C. From these studies, it is clear that only three genes of the complex are expressed in the cells of Su(H) mutant proneural clusters. All other members are either not expressed in the notal region of the wing imaginal disc or their expression is lost in the mutant cells. Previous studies have shown that both bearded-like proteins that are expressed in Su(H) mutant proneural clusters promote SOP development. Hence, it is unlikely that the abnormal expression of these genes causes the observed arrest in SOP development. Surprisingly, it was found that the strongest candidate, the bHLH repressor encoded by E(spl)m8, is also abnormally expressed in Psn mutants, where SOP development proceeds and the Su(H)/H-containing complex is intact. The observation is interesting, because it suggests that the activity of the whole Notch pathway is required to switch off the expression of E(spl)m8 in the SOP, but it also indicates that abnormal expression of the gene cannot be the reason for the arrest in SOP development in Su(H) mutant cells. Thus, the repressor activity might not be encoded by a specific member of the E(spl)-C (Koelzer, 2003).

One possibility is that the combination of the three abnormally expressed genes of the complex generates the repressing activity. An alternative is that Su(H) controls the expression of other genes that act in combination with the upregulated members of the complex to suppress SOP development. Another possibility is that more genes of the complex are de-repressed in Su(H) mutants at a level not detectable by the currently available methods. In this scenario, the weak expression of several bHLH-encoding genes will sum up to a level of repressor activity sufficient to stop SOP development. Using currently available techniques, it is very difficult to discriminate between these possibilities (Koelzer, 2003).

In Su(H) mutant cell clones induced during the first larval instar stage, hnt is expressed in a fraction of cells of specific proneural clusters, such as the scutellar cluster, but absent or strongly reduced in other clusters. It was further found that in Su(H) mutant wing imaginal discs, expression of sens and hnt is either lost or strongly reduced when compared to mutant cell clones induced during the first larval instar (Koelzer, 2003).

A high stability of the Su(H) protein is a possible explanation for this discrepancy. In favor of this explanation is the observation that the maternal component of Su(H) is sufficient to allow the development of homozygous animals until early pupal stages. Furthermore, vestigial (vg), a target gene of the Notch/Su(H) pathway during wing development is expressed in the Su(H) mutant wing imaginal disc of the early third larval instar stage. This indicates the presence of Su(H) activity at this stage. This residual activity of Su(H) must be provided by the maternal component. Both observations suggest that the Su(H) protein is degraded slowly and thus persists in mutant cells for a long time. It is therefore likely that Su(H) mutant cells, induced at the first larval instar, contain a residual amount of Su(H). This residual amount of Su(H) might be sufficient to activate expression of late SOP marker in cells of certain proneural clusters (Koelzer, 2003).

An alternative explanation for the milder phenotype observed in the Su(H) mutant clones is that it requires time to accumulate a sufficient level of activity of the repressor(s) of the E(spl)-C to stop SOP development. Hence, in the case of the clonal analysis, the loss of Su(H) activity occurs later than in homozygous mutant wing imaginal discs and lower levels of repressor activity would be present in cells of the proneural clusters of the machrochaete (Koelzer, 2003).

The development of the machrochaete is a paradigm for the assignment of different fates to initially equivalent cells. Proneural genes are expressed in clusters of cells and confer on these cells the potential to become SOPs. Careful examination has revealed that the SOPs of the machrochaete arise at the same positions within the proneural cluster, indicating that the selection of the SOP is not random. It is thought that other factors, such as Extramachrochaete, Pannier and Wingless introduce small differences in proneural activity that favor cells at specific positions within the cluster to become the SOP. These small differences in proneural activity are enhanced through the activity of the Notch pathway: a cell with high proneural activity expresses high levels of Dl and is therefore potent to inhibit its neighbors. Cells with a high activity of Notch have less proneural activity and lower levels of Dl. Thus, they are less potent to inhibit their neighbors. In this scenario, the Notch pathway is required to amplify initially small differences in proneural activity among cells within a cluster. This amplification eventually results in the accumulation of high levels of activity in the SOP and loss of activity in the neighbors. In this way, the pathway acts to resolve a crude pre-pattern to the level of a single cell. According to this model, cells defective in Notch signal reception should be very potent in lateral inhibition. However, just the opposite was found: a cell that is located at the position where the SOP arises is able to adopt the SOP fate, even if surrounded by Su(H) mutant cells. It can do so despite the fact that the mutant neighbors accumulate high levels of proneural activity (indicated by the expression of the achaete-scute SOP enhancer), as well as Dl. Dl, expressed in the Su(H) mutant cells at high level, is active and can activate Notch signalling in wild-type cells with the exception of the SOP. Thus, although the SOP is adjacent to cells with an extremely high potency for lateral inhibition during the whole live of a proneural cluster, it has succeeded in adopting the SOP fate. It appears that the SOP is determined by positional cues that are much more precise than anticipated. These cues render the cell at the correct position in the cluster insensitive to lateral inhibition. This suggests that small differences in proneural activity are not the crucial bias imposed on cells within a proneural cluster and that lateral inhibition might not be required to resolve a crude pre-pattern (Koelzer, 2003).

Nevertheless, the big clusters of SOPs observed in other neurogenic mutants, indicate that the Notch pathway has a function in preventing the SOP fate in all cells of a proneural cluster and also in cells that are located further away from the SOP. Furthermore, the SOP sends a signal that activates the Notch pathway in its immediate neighbors. This lateral inhibition is relatively late, since it is observed only around SOPs that already express hnt. It also occurs only in the cells adjacent to the SOP (Koelzer, 2003).

Altogether, these observations suggest that the Notch pathway might have two separable functions during SOP development. During early phases of a proneural cluster, the activity of the pathway keeps the cells of the cluster undecided, perhaps by mutual repression. Owing to positional cues, one cell becomes insensitive to the inhibitory signal and adopts the SOP fate. Subsequently the SOP inhibits its immediate neighbours by sending an inhibitory signal through Dl (Koelzer, 2003).

Notch signaling independent of Suppressor of Hairless

The Notch receptor triggers a wide range of cell fate choices in higher organisms. In Drosophila, segregation of neural from epidermal lineages results from competition among equivalent cells. These cells express achaete/scute genes, which confer neural potential. During lateral inhibition, a single neural precursor is selected, and neighboring cells are forced to adopt an epidermal fate. Lateral inhibition relies on proteolytic cleavage of Notch induced by the ligand Delta and translocation of the Notch intracellular domain (NICD) to the nuclei of inhibited cells. The activated NICD, interacting with Suppressor of Hairless [Su(H)], stimulates genes of the E(spl) complex, which in turn repress the proneural genes achaete/scute. New alleles of Notch are described that specifically display loss of microchaetae sensory precursors. This phenotype arises from a repression of neural fate, by a Notch signaling distinct from that involved in lateral inhibition. The loss of sensory organs associated with this phenotype results from a constitutive activation of a Deltex-dependent Notch-signaling event. These novel Notch alleles encode truncated receptors lacking the carboxy terminus of the NICD, which is the binding site for the repressor Dishevelled (Dsh). Dsh is known to be involved in crosstalk between Wingless and Notch pathways. These results reveal an antineural activity of Notch distinct from lateral inhibition mediated by Su(H). This activity, mediated by Deltex (Dx), represses neural fate and is antagonized by elements of the Wingless (Wg)-signaling cascade to allow alternative cell fate choices (Raiman, 2001).

In a screen for flies associated with the loss of microchaetae, a number of mutations in Notch were isolated that result in a dominant loss of thoracic microchaetae, which are called NMcd, where Mcd stands for microchaetae defective. These mutations are lethal, and, for this reason, their behavior was analyzed in mosaics in which clones of mutant cells are juxtaposed with wild-type territories. In these mosaics, mutant cells are recognized by the use of both bristle and epidermal markers. All mutants behave genetically in a similar manner, the strongest alleles, NMcd1 and NMcd5 (collectively NMcd1/5), were chosen for further analysis. In clones for NMcd1 and NMcd5, 99% of the microchaetae are absent, whereas macrochaetae are not affected (Raiman, 2001).

Genetic analysis indicates that the dominant effects of the NMcd alleles are due to antagonism of the wild-type function of Notch. The mutant phenotype of NMcd is enhanced when N+ is lowered and is partially suppressed when N+ is increased. Thus, these gain-of-function alleles of Notch do not induce an aberrant function of the receptor (neomorphism), but rather produce receptors that are more active on the normal function of Notch. NAx alleles exhibit a similar genetic behavior and a similar phenotype to the NMcd alleles. However, several differences distinguish NAx from NMcd. The NAx mutant exhibits a variable loss of both thoracic microchaetae and macrochaetae, leading to irregular patterns. In contrast, NMcd affects only microchaetae. Furthermore, the remaining microchaetae of the NMcd/+ flies are arranged in fewer rows, which are organized in a regular pattern. Finally, NAx/+ flies exhibit broader wings with shortened veins. In contrast, the wings of the NMcd/+ flies appear as those of wild-type flies. In this study of the NMcd alleles, focus was placed on the bristle pattern (Raiman, 2001).

A further demonstration of the specificity of the NMcd mutations for microchaetae is seen by analysis of NMcd1/5clones with impaired function of either hairy or extramacrochaetae (emc), two negative regulators of ac/sc. Flies lacking hairy or its cofactor groucho (gro) exhibit ectopic microchaetae in the scutellum region of the thorax. In clones mutant for NMcd1/5 and lacking gro (NMcd1/5 gro-cells), ectopic microchaetae are absent. In contrast, the NAx mutants again behave differently, since, in Ax59b gro- cells, ectopic microchaetae form. The ectopic macrochaetae, which develop in emc1clones, also arise in NMcd1/5 emc1clones, even when their precursors differentiate simultaneously to those of the microchaetae (Raiman, 2001).

In the absence of any component of lateral inhibition, an excess of neural precursors occurs at the expense of epidermis. In Notch-, Su(H)-, and Dl-clones (mosaic animals), the neurogenic phenotype is extreme; all mutant cells adopt the neural fate, and no cells are left to form epidermis. The lack of epidermal mutant cells leads to a wound partially skinned up by wild-type epidermal-surrounding cells. In gro- and E(spl)-, as well as in the hypomorphic Dl clones, the neurogenic phenotype is less severe, and such clones can differentiate tufts of densely packed sensory bristles accompanied by few epidermal cells. Furthermore, mutant cells for loss-of-function alleles of Notch have an enhanced capacity to produce an inhibitory signal that forces neighboring wild-type cells to adopt the epidermal fate. This signal is mediated by Delta. Thus, along the borders of N mutant clones, no bristles are formed by wild-type cells (Raiman, 2001).

Alleles of Notch encoding constitutively activated receptors show the opposite phenotype, with wild-type bristles forming at the border of mutant territories that adopt epidermal fate. The phenotype of the NMcd mutants resembles that of classic gain-of-function alleles of Notch (among which are the NAx alleles) and therefore might result in an activation of the lateral inhibition function. If this were the case, removal of the function of some or all of the mediators of lateral inhibition will abolish the effects of the NMcdalleles. To test this, double-mutant clones were made using the loss-of-function mutations DlRevF10, Dl9P39, Df(3R)E(spl)b32.2, groE48, and Su(H)IB115. In this case, double-mutant clones for NMcd1,5 and components that mediate lateral inhibition [Delta; E(spl)-C; gro; Su(H)] would be predicted to inactivate lateral signaling; they would be predicted to display the neurogenic phenotypes characterized by the lack of mutant epidermal cells. Surprisingly, in all cases, the double-mutant clones display the NMcd1/5 phenotype with mutant epidermis and no microchaetae differentiated. Therefore, NMcdcells do not require Dl, Su(H), gro, or the E(spl)-C in order to adopt the epidermal fate. In contrast, neurogenic double-mutant clones are observed using Ax59bor AxSX1and at least with Dl, gro, and E(spl)-C. The NMcd Ser and NMcd Dl Ser clones display the NMcdphenotype, suggesting that the NMcdphenotype does not require Serrate, the other ligand of Notch (Raiman, 2001).

The macrochaetae can differentiate normally in clones mutant for NMcd. In the absence of lateral signaling (double-mutant clones for NMcd1,5 and one of the components of lateral inhibition [Dl; E(spl)-C; gro; Su(H)]), mutant clones would be predicted to display tufts of macrochaetae (the neurogenic phenotype). Macrochaetae differentiating as single bristles are observed rather than as a neurogenic tuft. These results confirm that the NMcdmutants affect a function of Notch distinct from lateral inhibition (Raiman, 2001).

Loss-of-Su(H) alleles behave as dominant enhancers of the NMcd alleles. Dx is a cytoplasmic protein whose activity also relies on binding to the ankyrin repeats. The antagonism between Dx and Su(H) could be explained by a binding competition for the ankyrin repeats of the NICD. Thus, when Su(H) concentration is reduced, Dx signaling is increased and the NMcd phenotype is accentuated. This observation suggests that activity of the Notch receptor depends on the balance between Dx and Su(H) (Raiman, 2001).

Although Deltex has been interpreted as being involved in lateral inhibition, the results of this study make it more likely that it is associated with an alternative signaling event. Dx is a ubiquitous cytoplasmic protein that regulates Notch through binding to the NICD. During lateral inhibition, upon activation by the ligand Dl, the NICD is translocated to the nucleus where it interacts with Su(H) to regulate target genes. However, Su(H) is also present in the cytoplasm, where it displays antagonism with Dx, reflecting a competition to associate to the ankyrin repeats of Notch. Consistently, it has been suggested that Dx may maintain an activated state of Notch indirectly by interfering with the retention of Su(H) in the cytoplasm by virtue of its interaction with the ankyrin repeats of Notch. Moreover, loss-of-functions alleles of Su(H) and loss-of-functions alleles of dx behave, respectively, as dominant enhancers and dominant suppressors of the phenotype of NMcd/+ heterozygous flies. This observation demonstrates that Su(H) and Dx display antagonist activities during N signaling (Raiman, 2001).

The Notch signaling pathway is required to specify muscle progenitor cells in Drosophila

The organization and function of the Notch signaling pathway in Drosophila are best understood with respect to the role of this pathway in the process of selection of neural progenitor cells. However, there is evidence that, in addition to neurogenesis, the Notch signaling pathway is involved in several other developmental processes, one of which is the selection of muscle progenitor cells. Thus, the number of these progenitor cells is increased in neurogenic mutants. It has been proposed that muscle progenitor cells are selected from clusters of equivalent cells expressing genes of the achaete-scute gene complex (AS-C). Additional elements of the Notch signaling pathway participate in myogenesis. Gal4 mediated expression of a Notch variant, E(spl) and Hairless shows that the selection of muscle progenitor cells obeys principles apparently identical to those acting at the selection of neural progenitor cells (Giebel, 1999).

To test whether the Notch signaling pathway is involved in myogenesis, the effects of expression of a constitutively active Notch protein (Notchintra) were examined. A second chromosomal effector line with an UAS-Notchintra construct was used. This construct led to complete blocking of neural development upon activation with daG32. Embryos carrying that construct driven by daG32 or by 24B-Gal4, respectively, do not express any of the muscle founder cell markers S59 and Kruppel in the mesoderm. Therefore, it is assumed that no muscle progenitor cells are specified in these animals. Confirmation of this assumption is provided by the observation that no muscle fibers differentiate in these embryos, as shown by means of the expression of a myosin heavy chain (MHC) reporter gene. In mutants where fusion of myoblasts is blocked, founder cells express corresponding founder cell markers, while the non-founder myoblasts remain as undifferentiated rounded cells, which express certain muscle specific genes like myosin. Since Notchintra expressing mesodermal cells are rounded and many of them express the MHC reporter, it is assumed that the MHC expressing cells are non-founder myoblasts that have failed to undergo fusion due to the lack of muscle founder cells (Giebel, 1999).

Further evidence for a Notch pathway role in myogenesis was obtained by overexpressing UAS-E(spl) in the mesoderm. Following Gal4 mediated activation of UAS-E(spl), the number of S59 and Kruppel positive cells is strongly reduced. This correlates with a defect in the number of differentiated muscle cells, as shown by MHC reporter gene expression. Again these data fit well with the results obtained on the development of the neuroectoderm, in which Gal4 driven UAS-E(spl) expression leads to strong reduction of CNS and PNS structures (Giebel, 1999).

During neurogenesis Su(H) becomes active if Notchintra is expressed. During imaginal neurogenesis the function of Su(H) is antagonized by proteins encoded by Hairless; this effect is mediated by direct protein-protein interactions. During specification of imaginal sensory organ precursors, overexpression of Hairless counteracts the phenotypic effects of activated Notch. If Su(H) is involved in transducing the inhibitory signals mediated by activated Notch during myogenesis, Gal4 mediated expression of Hairless could theoretically weaken the effect of Notchintra on the course of myogenesis. To test the relationships between active Notch and Hairless during myogenesis, UAS-Hairless and UAS-Notchintra were expressed in the mesoderm of the same embryos. S59 and Kruppel positive cells are present in these embryos, although in much lower numbers than in wildtype embryos. Well differentiated muscles are also present in these embryos. Similar results were obtained in the course of embryonic neurogenesis. Embryos with daG32 driven UAS-Notchintra are completely aneural. After coexpression of UAS-Hairless, structures of the CNS as well as of the PNS differentiate as visualized with 22C10 and 44C11 antibody stainings. Gal4 mediated expression of UAS-Hairless alone leads to a slight increase in the number of S59 and Kruppel positive cells in the mesoderm. This correlates with an increase in the number of differentiated muscle fibers, shown by the expression of the MHC reporter gene. In correspondence to those data the neuroectodermal Gal4 mediated expression of UAS-Hairless leads to the development of a weakly hyperplasic nervous system, as shown by stainings using the neural antibodies 22C10 and 44C11 (Giebel, 1999).

Su(H) and heart development

During the formation of the Drosophila heart, a combinatorial network that integrates signaling pathways and tissue-specific transcription factors specifies cardiac progenitors, which then undergo symmetric or asymmetric cell divisions to generate the final population of diversified cardiac cell types. Much has been learned concerning the combinatorial genetic network that initiates cardiogenesis, whereas little is known about how exactly these cardiac progenitors divide and generate the diverse population of cardiac cells. In this study, the cell lineages and cell fate determination in the heart have been examined by using various cell cycle modifications. By arresting the cardiac progenitor cell divisions at different developing stages, the exact cell lineages for most cardiac cell types have been determined. Once cardiac progenitors are specified, they can differentiate without further divisions. Interestingly, the progenitors of asymmetric cell lineages adopt a myocardial cell fate as opposed to a pericardial fate when they are unable to divide. These progenitors adopt a pericardial cell fate, however, when cell division is blocked in numb mutants or in embryos with constitutive Notch activity. These results suggest that a numb/Notch-dependent cell fate decision can take place even in undivided progenitors of asymmetric cell divisions. By contrast, in symmetric lineages, which give rise to a single type of myocardial-only or pericardial-only progeny, repression or constitutive activation of the Notch pathway has no apparent effect on progenitor or progeny fate. Thus, inhibition of Notch activity is crucial for specifying a myogenic cell fate only in asymmetric lineages. In addition, evidence is provided that whether or not Suppressor-of-Hairless can become a transcriptional activator is the key switch for the Numb/Notch activity in determining a myocardial versus pericardial cell fate (Han, 2003).

Previous studies have suggested that Notch activity controls two distinct processes during the specification of cardiac cell fates. First, it is required to single initial progenitors out of a field of competence by supporting the selection and inhibiting surrounding cells from adopting the same fate. Subsequent to the progenitor specification, Notch is required again for the specification of alternative cell fates of sibling cells produced during asymmetric cell divisions. In this study, the cell autonomy of Notch was examined, by using eme-Gal4 (which confers expression in the mesodermal Eve lineage) to drive activated forms of Notch and Su(H) exclusively in the mesodermal Eve lineages. Conditional ubiquitous expression of activated Notch was used to examine its lineage-specific function in other cardiac lineages. Notch was found to be required for specification of a non-myogenic fate in both the Eve and the Svp lineages of the cardiac mesoderm. By contrast, activation or inhibition of the Notch pathway does not affect cell fate decisions within the symmetric lineages. This suggests a mechanism by which cell type diversity may be increased during evolution by co-opting the Notch pathway during cell division to distinguish between alternative fates of the daughter cells. The inability of activated Su(H) to autonomously influence cell fates in symmetric cardiac lineages further suggests that other factors or activities, not present in symmetric lineages, are crucial for the asymmetric lineage-specific functions of Notch and Su(H) (Han, 2003).

Interestingly, this influence of the Notch pathway on cell fate decision in asymmetric cardiac Eve and Svp lineages is not altered when cell division is arrested. Thus, cell division is not essential to distinguish between alternative cell fates. The data also suggest that the default cell fate of an asymmetrically dividing cardiac precursor in Drosophila is determined to assume a myogenic fate, owing to Numb-mediated inhibition of Notch, unless that fate is switched by the activation of target genes downstream of Su(H). Moreover, in a double mutant of Notch and numb one would expect to observe the same lineage phenotype as of Notch alone, i.e., a myogenic cell fate, since the primary role of Numb is to inhibit Notch signaling. Unfortunately, analysis of such double mutants is complicated by the earlier role of Notch in lateral inhibition (Han, 2003).

Another unresolved issue is the source of the Notch ligand that activates signal transduction within asymmetric cardiac lineages. If the myogenic cell were to produce the ligand for Notch activation in its pericardial sibling, then the undivided progenitor would have to secrete its own Notch ligand. This is unlikely, since production of the ligand is usually inhibited within the cell that experiences Notch signaling. In the asymmetric MP2 lineage of the Drosophila CNS, for example, ligand production appears to be required in cells outside the MP2 lineage. A similar scenario may be operating in the asymmetric cardiac lineages (Han, 2003).

Within the Eve lineages, Notch activation is mimicked by Su(H) fused to the VP16, a potent transcriptional activation domain. Recent studies suggest that in the absence of Notch activity, DNA-bound Su(H) prevents activators from promoting transcription. When Notch ligands, such as Delta, bind to its receptor, Notch is cleaved to produce an intracellular domain fragment, N(icd), which is thought to enter the nucleus and interact directly with Su(H) to recruit transcriptional co-activators and alleviate Hairless-mediated repression, thus promoting transcription. In support of this model, it has been found that Su(H) overexpression can mimic Notch activation only when linked directly to a transcriptional activator, but not in its wild-type form when it presumably associates with co-repressors, such as Hairless and Groucho, that prevent Su(H)-dependent transcriptional activation in the absence of Notch signaling (Han, 2003).

The role of the PTB-containing, membrane-associated protein Numb in preventing Notch activation in the nervous system is well established. To explore at which level Notch signaling is inhibited by Numb in the cardiac lineages, numb was overexpressed simultaneously with N(icd) or Su(H)vp16 within the mesodermal Eve lineages. Excess Numb was able to counteract activated Notch but not activated Su(H) function, suggesting that Numb can inhibit Notch activity after Notch has been cleaved, possibly by preventing its nuclear translocation, but is unlikely to prevent the transcriptional activator function of Su(H) directly. Recent data suggest that Numb is involved in stimulating endocytosis of Notch, thus removing it from the cell surface and inhibiting its function. It is not clear, however, if this inhibition by endocytosis is at the level of the entire Notch receptor, or (also) at the level of N(icd) after it is cleaved off. Experiments described here provide strong evidence that Numb can indeed interfere with N(icd) function, but it remains to be determined if endocytosis is an obligatory intermediate in this inhibition of activated Notch (Han, 2003).

The Notch pathway may also have a role in vertebrates in specifying pericardial and other non-myogenic cell fates within the dorsolateral cardiogenic region of the anterolateral plate mesoderm. As in the Eve and Svp lineage of the Drosophila heart, activation of the Notch pathway decreases myocardial gene expression and increased expression of a pericardial marker, whereas inhibition of Notch signaling resulted in an increase of cardiac myogenesis. Similar results were obtained with an activated form of RBP-J [a vertebrate homolog of Drosophila Su(H) fused to vp16, as in this study]. These data indicate that the Notch pathway may play a role in the specification of myocardial versus pericardial cell fates in both Drosophila and vertebrates. This raises the question of whether the mechanism of Notch mediated cell identity determination is also conserved between vertebrates and flies. Because it is not yet known if (Numb-controlled) asymmetric cell divisions are also involved in vertebrate heart development, the answer awaits future studies. However, recent studies on the role of Numb during cortical development suggest that it is likely to have a similar control function in cell fate specification in vertebrates as it does in flies (Han, 2003 and references therein).

Deciphering synergistic and redundant roles of Hedgehog, Decapentaplegic and Delta that drive the wave of differentiation in Drosophila eye development

In Drosophila, a wave of differentiation progresses across the retinal field in response to signals from posterior cells. Hedgehog (Hh), Decapentaplegic (Dpp) and Notch (N) signaling all contribute. Clones of cells mutated for receptors and nuclear effectors of one, two or all three pathways were studied to define systematically the necessary and sufficient roles of each signal. Hh signaling alone is sufficient for progressive differentiation, acting through both the transcriptional activator Ci155 and the Ci75 repressor. In the absence of Ci, Dpp and Notch signaling together provide normal differentiation. Dpp alone suffices for some differentiation, but Notch is not sufficient alone and acts only to enhance the effect of Dpp. Notch acts in part through downregulation of Hairy; Hh signaling downregulates Hairy independently of Notch. One feature of this signaling network is to limit Dpp signaling spatially to a range coincident with Hh (Fu, 2003).

The development of cells mutant for all three transcription factors, Mad, Su(H), and ci is a helpful starting point, since they may reflect a 'ground state' of eye development that requires extracellular signals to differentiate. Mad Su(H) ci cells fail to express the atonal or senseless genes that initiate R8 differentiation, and, consequently, fail to support retinal differentiation. This shows that the absence of Ci75 is not sufficient for differentiation. Dpp alone can induce Ato [e.g., in Su(H) ci clones], but N and Dpp signaling together are required to activate Atonal with normal kinetics, as occurs in ci-mutant cells. N signaling alone (in tkv ci clones) is insufficient. In the presence of Ci, prompt differentiation requires Hh to downregulate Ci75, and differentiation is delayed in Smo clones that lack this input. The normal role of Hh is not just to remove Ci75 thus permitting Dpp and N to work, because Atonal is turned on normally in Mad Su(H) clones that do not respond to Dpp or N signals. Such differentiation depends exclusively on Hh yet progresses normally, except that a neurogenic phenotype reflects dependence of lateral inhibition on Su(H). Hh depends positively on ci to drive differentiation in Mad Su(H) cells and, therefore, requires Ci155. The positive role of ci can also be inferred from the delayed differentiation of Su(H) ci clones in comparison with Su(H) clones (Fu, 2003).

Hairy is downregulated redundantly by Hh and N signaling. Prolonged Hairy expression is not sufficient to block differentiation completely but it does antagonize it (e.g., in Su(H) ci clones). Downregulation of Hairy in response to Hh as well as N explains why both ci and Su(H) mutant clones can differentiate promptly, and why N enhances differentiation in response to Dpp but is not required for differentiation in response to Hh (Fu, 2003).

Comparison between Mad Su(H) ci cells and Su(H) ci cells shows that Dpp signaling is sufficient to initiate eye differentiation in its normal location in the absence of Hh or N signals, but such differentiation is delayed. The normal timing of differentiation is restored by combined Dpp and N signals (in ci clones). This is the basis for the ectopic differentiation on co-expression of Dpp and Dl ahead of the furrow (Fu, 2003).

Superficially, these results differ from previous ectopic expression studies that concluded that Dpp signaling alone was not sufficient to induce ectopic differentiation in all locations. This discrepancy is probably explained by the baseline repressor activity of Su(H) protein. Previous work shows that without N signaling, repressor activity of Su(H) protein retards differentiation. Dpp signaling is sufficient for differentiation in the experiments where the Su(H) gene has been deleted. In the presence of the Su(H) gene, Dpp may be most effective at locations where there is little Su(H) repressor activity, such as close to the morphogenetic furrow where N signaling is active (Fu, 2003).

Comparison between Mad Su(H) ci cells, which do not differentiate, and Mad ci or tkv ci cells, which differentiate slowly or not at all, shows that Notch signaling alone is insufficient for differentiation. Premature differentiation reported when N is activated ectopically ahead of the furrow must reflect endogenous Dpp signaling at such locations (Fu, 2003).

These experiments reveal an outline of the mechanisms of Hh, Dpp and N redundancy. First, the results show that Mad and Ci independently reinforce differentiation, presumably through the transcription of target genes because Mad is sufficient for differentiation in the absence of Ci, and vice versa. The results show unequivocally that the transcriptional activator Ci155 activates differentiation in addition to Ci75 antagonizing differentiation (Fu, 2003).

It was surprising to find that Dpp stabilizes Ci155 in the absence of Smo, which suggests Dpp input into Hh signal transduction. Although the requirement for smo-dependent input through fused makes it unlikely that Ci155 is functional in smo clones, Ci155 accumulation might be associated with reduced Ci75 levels. Ci75 is shown to repress differentiation in smo clones because smo ci clones differentiation normally. Ci155 stabilization cannot be due to an indirect effect of Dpp signaling on Hh, Ptc or Smo expression levels because the effect is detected in the absence of smo, and, therefore, reflects an effect on Hh signal transduction components downstream of Smo. One idea is that Dpp signaling (or Dpp-induced differentiation) may replace SCFSlimb processing of Ci (which cleaves Ci155 to Ci75) with Cullin3-mediated Ci degradation, just as normally occurs posterior to the morphogenetic furrow. In a smo clone, Ci155 would accumulate because Smo is required for Cullin3 to degrade Ci. However, the SCFSlimb-to-Cullin3 switch may not be the only effect of Dpp on Ci processing, because Tkv slightly enhances Ci155 accumulation even when smo is present (Fu, 2003).

Finally, downregulation of Hairy by N requires the Su(H) gene. N also overcomes baseline repressor activity of Su(H) protein to promote progression of differentiation. This role of N must be independent of Hairy (Fu, 2003).

Dl, Hh and Dpp are generally thought to signal over very different distances. How can signals of such different range substitute for one another to permit normal eye development? Dpp is transcribed in response to Hh signaling and is produced where Ci155 levels are highest. Dl is regulated by Hh indirectly through Ato and Ato-dependent Egfr activity in differentiating cells. Hh is expressed most posteriorly of the three, in differentiating photoreceptors (Fu, 2003).

Eye differentiation uses Hh to progress through cells unable to respond to Dpp (tkv, Mad) or N (Su(H)). The range of Hh diffusion depends in part on the shape of the morphogenetic furrow cells. The Dpp that drives differentiation through ci-mutant cells unable to respond to Hh must diffuse from outside the ci clones because Dpp synthesis is Hh dependent. Large ci clones develop normally so Dpp diffusion cannot be limiting (dpp-mutant clones offer no information about the range of Dpp because they express and differentiate in response to Hh). Instead, the rate of progression in response to Dpp is controlled by Dl. Dl signals over (at most) one or two cell diameters at the morphogenetic furrow (Fu, 2003).

The previous view of eye patterning was influenced by the morphogen function of Hh and Dpp in other discs. It was thought that domains of Ato and Hairy expression reflected increasing concentrations of Hh and Dpp. The data shows that, in the eye, the combination of signals is important. Differentiation is triggered where Dl and/or Hh synergize with Dpp, regardless of where the source of Dpp is. The additional requirements limit Dpp to initiating differentiation at the same locations that Hh does (Fu, 2003).

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

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

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

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

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

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

Notch pathway and Friend of echinoid

echinoid (ed) encodes a cell-adhesion molecule (CAM) that contains immunoglobulin domains and regulates the Egfr signaling pathway during Drosophila eye development. Genetic mosaic and epistatic analysis, has suggested that Ed, via homotypic interactions, activates a novel, as yet unknown pathway that antagonizes Egfr signaling. Alternatively, later studies indicate that Ed inhibits Egfr through direct interactions. Another body of work suggests that Ed functions as a homophilic adhesion molecule, and also engages in a heterophilic trans-interaction with Drosophila Neuroglian (Nrg), an L1-type CAM. Co-expression of ed and nrg in the eye exhibits a strong genetic synergy in inhibiting Egfr signaling. This synergistic effect requires the intracellular domain of Ed, but not that of Nrg. A model for this interaction suggest that Nrg acts as a heterophilic ligand and activator of Ed, which in turn antagonizes Egfr signaling (Spencer, 2003 and references therein; Islam, 2003 and references therein).

Complicating the picture even further is an analysis of a paralogue of Ed termed friend of echinoid (fred). ed and fred transcription units are adjacent to one another, approximately 100 kilobases apart on chromosome arm 2L, but they are divergently transcribed in opposite directions. Fred acts in close concert with the Notch signaling pathway. Suppression of fred function results in specification of ectopic SOPs in the wing disc and a rough eye phenotype. Overexpression of N, Su(H), and E(spl)m7 suppresses the fred RNAi phenotypes. Accordingly, decreasing Su(H) or overexpression of Hairless enhances the fred RNAi phenotypes. Thus fred, a paralogue of ed, shows close genetic interaction with the Notch signaling pathway. The weak genetic interaction observed between fred and components of the Egfr pathway also links fred to the Egfr pathway; however, analysis of additional components of the Egfr pathway are necessary to determine Fred's role in the Egfr signaling (Chandra, 2003).

In order to study the function of fred, the heritable and inducible double-stranded RNA-mediated interference (RNAi) method was used. For this study, transcript sequence of fred was cloned as a dyad symmetric molecule in the pUAST vector and transgenic lines established. Expression of the construct was induced by crossing the transgenic lines to tissue- and/or stage-specific GAL4 driver lines. Transcription of a dyad symmetric molecule results in a RNA that snaps back to give rise to a dsRNA with a hairpin loop; this mediates the degradation of the corresponding endogenous mRNA. A 638-bp region of fred was selected for this analysis based on minimal similarity to ed sequence (Chandra, 2003).

The Notch signaling pathway is involved in limiting the SOP fate to a single cell within each proneural cluster. Since degradation of fred mRNA leads to formation of ectopic SOPs, it was of interest to see if the Notch signaling pathway genes functionally interact with fred in this process and, thus, may modulate the fred RNAi phenotype. To this end, four Notch pathway genes, Notch (N), Suppressor of Hairless [Su(H)], Hairless (H), and E (spl) m7 were tested for genetic interactions with fred (Chandra, 2003).

Following Notch activation, the Nicd translocates to the nucleus, where it forms a complex with the transcription factor Su(H) and switches on the transcription of E (spl) complex. Loss of Su (H) results in the formation of ectopic sensory bristles, while overexpression results in suppression of sensory organ specification. Ectopic expression of Su(H), using the pnr-GAL4 driver, results in the absence of sensory organs in the medial region of the notum. Simultaneous expression of both Su(H) and the fred RNAi construct in the pnr domain produces flies that are similar to the UAS-Su(H); pnr-GAL4 flies. Moreover, the ectopic cell death associated with fred suppression was alleviated by Su(H) overexpression. Thus, ectopic expression of Su(H) effectively suppresses the phenotype associated with the reduction of fred function. The effect was tested of loss of Su(H) function on the fred RNAi phenotype. Reduction of one functional copy of Su (H) in UAS-fredRNAi/pnr-GAL4 flies did not show a consistent modulation of the phenotype, indicating that this assay might not be sensitive enough. However, eye morphogenesis has been proven to be very sensitive to dosage-sensitive interactions. Therefore, the effect of loss of one functional Su (H) copy on the rough eye phenotype generated by expression of UAS-fred RNAi was tested in eye with the GMR-GAL4 driver. A consistent enhancement of the fred RNAi induced rough eye phenotype was observed upon decreasing Su (H) function (Chandra, 2003).

The observations that changes in the activity of four genes of the Notch signaling pathway can either suppress or enhance the phenotypes associated with the suppression of fred function suggest that fred is functioning in close concert with the Notch signaling pathway. Reduction in the activity of a Notch signaling pathway gene, Su(H) results in an enhancement of the fred RNAi phenotype. In contrast, ectopic expression of Notch signaling pathway genes, Notch, Su(H), and E(spl)m7 suppresses, to different degrees, different aspects of the fred RNAi phenotype in the developing wing, thorax, and eye. In contrast, overexpression of Hairless (a negative regulator of the Notch pathway) enhances the phenotypes induced by Fred suppression. It is presently not clear whether Fred defines a separate pathway for SOP determination or if it shares downstream components of the Notch signaling pathway. The remarkable degree to which ectopic expression of an E(spl) complex bHLH transcription factor results in a nearly complete suppression of phenotypes associated with fred degradation strongly supports the idea of very close functional interactions. These observations, furthermore, raise the possibility that E(spl) complex genes and/or other genes of the Notch signaling pathway act downstream of fred function (Chandra, 2003).

Genetic interactions between deltex and Suppressor of Hairless

Notch is a single-pass transmembrane receptor. The N signaling pathway is an evolutionarily conserved mechanism that controls various cell-specification processes. Drosophila Deltex (Dx), a RING-domain E3 ubiquitin ligase, binds to the N intracellular domain, promotes N’s endocytic trafficking to late endosomes, and has been proposed to activate Suppressor of Hairless [Su(H)]-independent N signaling. However, it has been difficult to evaluate the importance of dx, because no null mutant of a dx family gene has been available in any organism.This study reports the first null mutant allele of Drosophila dx. dx is involved only in the subsets of N signaling, but is not essential for it in any developmental context. A strong genetic interaction exists between dx and Su(H); this suggests that dx might function in Su(H)-dependent N signaling. These epistatic analyses suggested that dx functions downstream of the ligands and upstream of activated Su(H). A novel dx activity has been uncovered that suppresses N signaling downstream of N (Fuwa, 2006).

A null allele of dx, dx152 was generated to elucidate the functions of the dx gene during development. Homo- and hemi-zygotes of dx152 are viable and fertile, although the recessive phenotypes coincide with a subset of weak phenotypes observed in N mutants. Thus, it is concluded that dx function is not required for N signaling in any context. These results also show that the involvement of dx in N signaling is tissue-specific. In contrast, it is noted that dx seems to play a role in tissues that are not affected in the dx null mutant. For instance, dx152 /Y ;; Dlrev10 /+ shows fusion of the second and third tarsal segments of the foreleg, while neither dx152 /Y nor Dlrev10 /+ has this defect. Thus, dx is involved in the development of the tarsal segment. However, even in the null dx background, N signaling activity is normally above the threshold required for wildtype leg development. In the Drosophila genome, no other genes homologous to dx are found. Nevertheless, it is possible that some protein functionally related to Dx compensates for the absence of the dx gene functions. It was also found that the expressivity of dx152 phenotypes varies greatly among tissues. Compensation by another protein, which could have some tissue specificity, may also account for the variation in the expressivity of dx152 phenotypes. It is noted that, to the extent they have been tested; the genetic behavior of dx152 is similar to that of dx24, which has been used in previous studies (Fuwa, 2006).

This study shows that the ability of Dl/Ser ligands to activate N signaling is partially reduced in the wing discs of the dx152 mutant. This result suggests that dx acts downstream of the Dl/Ser ligands to activate N signaling. When Dx is overexpressed, N signaling is induced independent of the presence of the Dl/Ser ligands. It is therefore possible that artificially elevated levels of Dx somehow overcome the Ser/Dl requirement for N activation. However, it is notable that both Dl and Ser showed a substantial ability to stimulate N signaling in the dx152 mutant disc, but they failed to activate N signaling in Su(H) mutant clones. Therefore, both dx and Su(H) play roles in ectopic vgBE activation, but while Su(H) is indispensable, Dx is required only for strong signal induction (Fuwa, 2006).

Genetically, dx has been considered a positive regulator of N signaling in Drosophila. Furthermore, mammalian homologues of dx activate the reporter genes of N signaling target genes. Also, it was shown that Dx and a dominant-negative form of Nedd4 activate the E(spl)mγ promoter in Drosophila cultured cells. However, in contrast, a human Dx homolog antagonizes N signaling in cortical neurons. This discrepancy could be explained by the finding that dx both activates and suppresses N signaling (Fuwa, 2006).

In this study, a novel genetic interaction involving dx was uncovered. It has been reported that N shows a dominant lethal interaction with hypomorphic alleles of dx. Here, it was found that Ser94c shows a dominant lethal interaction with dx152. This result was unexpected, because Dl, which functions more broadly than does Ser, did not show a lethal interaction. Thus, it is possible that dx and Ser have common tissue specificity. However, the developmental and molecular basis of this interaction between dx and Ser remains to be addressed (Fuwa, 2006).

Another curious genetic interaction was found between dx and Su(H). The wing-vein phenotype of dx152 was suppressed dominantly by Su(H)Δ47, a null mutation of Su(H). This dx152 phenotype is suppressed by a hypomorphic allele of Su(H), Su(H)SF8. A similar suppression was also reported previously with the combined hypomorphic alleles of dx and Su(H). It is known that Su(H) acts both as a repressor and activator of N target genes. Thus, the lack of this repressor function in Su(H) mutants probably explains the suppression of dx152 phenotypes in combination with Su(H)/+. It has been demonstrated that Dx ectopically activates vgBE in Su(H) null mutant clones, which suggests Dx is involved in Su(H)-independent N signaling. This study showed that vgBE is activated along the A/P boundary during the late third-instar in a Su(H)- and Dx-independent manner, indicating that this activation is irrelevant to N signaling. However, this activation is not detected during the middle third-instar. Thus, previous experiments, which were carried out in middle third-instar larvae, were not affected by this background activation of vgBE (Fuwa, 2006).

It has been shown that dx modulates the endocytic trafficking of N. However, most experiments in these studies rely on the overexpression of dx. Therefore, the dx null mutant should offer the opportunity to study the requirement for dx in N transportation (Fuwa, 2006).

Regulation of expression of Vg and establishment of the dorsoventral compartment boundary in the wing imaginal disc by Suppressor of Hairless

The transcription factor Suppressor of Hairless [Su(H)] belongs to the CSL transcription factor family, the main transcriptional effector of the Notch-signaling pathway. Su(H) is the only family member in the Drosophila genome and should therefore be the main transcriptional effector of the Notch pathway in this species. Despite this fact, in many developmental situations, the phenotype caused by loss of function of Su(H) is too weak for a factor that is supposed to mediate most or all aspects of Notch signaling. One example is the Su(H) mutant phenotype during the development of the wing, that is weaker in comparison to other genes required for Notch signaling. Another example is the complete absence of a phenotype upon loss of Su(H) function during the formation of the dorsoventral (D/V) compartment boundary, although the Notch pathway is required for this process. Recent work has shown that Su(H)/CBF1 has a second function as a transcriptional repressor, in the absence of the activity of the Notch pathway. As a repressor, Su(H) acts in a complex together with Hairless (H), which acts as a bridge to recruit the co-repressors Groucho and CtBP, and acts in a Notch-independent manner to prevent the transcription of target genes. This raises the possibility that a de-repression of target genes can occur in the case of loss if function of Su(H). This study shows that the weak phenotype of Su(H) mutants during wing development and the absence of a phenotype during formation of the D/V compartment boundary are caused by the concomitant loss of the Notch-independent repressor function. This loss of the repressor function of Su(H) results in a de-repression of expression of target genes to a different degree in each process. Loss of Su(H) function during wing development results in a transient de-repression of expression of the selector gene vestigial (vg). This residual expression of vg is responsible for the weaker mutant phenotype of Su(H) in the wing. During the formation of the D/V compartment boundary, de-repression of target genes seems to be sufficiently strong, to compensate for the loss of Su(H) activity. Thus, de-repression of its target genes obscures the involvement of Su(H) in this process. Furthermore, evidence is provided that Dx does not signal in a Su(H)-independent manner as has been suggested previously (Koelzer, 2006).

This work provides an answer to the observation that the patterning defects of Su(H) mutant wing imaginal discs is weaker than anticipated for a gene that encodes a factor that mediates most of the transcriptional activity of the Notch-signaling pathway. Su(H) is required for the formation of the D/V compartment boundary despite any obvious defect in this process in the absence of its function. In both processes, the explanation for the phenotype of Su(H) mutants is the loss of its function as repressor of transcription along with its function as an activator (Koelzer, 2006).

Loss of function of Su(H) leads to an arrest in the development of the sensory organ precursor cell of the bristle sense organ. Although it was possible to demonstrate genetically that de-repression of expression of some genes of the Enhancer of split-complex are responsible for the arrest, it was not possible to detect the expression of any of these genes directly. This work shows that de-repression of vg is a consequence of loss of Su(H) function during wing development. Although this de-repression is weak and transient, it is sufficient to establish more distal elements than in mutants of other genes necessary for Notch signaling. The results are in agreement with two reports that show de-repression of target genes in Su(H) mutants in other developmental processes such as mesectoderm specification and bristle development. Thus, de-repression of target genes appears to be a common phenomenon during Drosophila development, if Su(H) function is lost. Importantly, this de-repression can even become strong enough to obscure an involvement of Su(H) in a developmental process, the formation of the D/V compartment boundary. De-repression of target genes upon loss of the repressor function of Su(H) is an attractive explanation for the paradox that loss of Notch function during the first larval instar stage is cell lethal, but loss of Su(H) function is not. Presumably, the de-repression of expression of target genes that are required for cell survival guarantees the survival of Su(H) mutant cells. In contrast, a similar de-repression cannot occur in Notch mutant cells, and the cells undergo apoptosis. Although the repressor function has been initially found in cell culture experiments with the vertebrate ortholog CBF1, reports analyzing the consequences of loss of its repressor function during vertebrate development are missing. The presented results should encourage researchers to search for such an effect in their vertebrate model systems (Koelzer, 2006).

The results have important implications on the use of various mutants in order to analyze the function of the Notch pathway in a particular developmental process. They show that the phenotype of loss of function of Su(H), or its vertebrate ortholog CBF1, is not necessarily identical to that of loss of the Notch-signaling activity. It is possible that de-repression of Notch target genes occurs upon loss of function of Su(H) but not upon inactivation of the pathway by other means. Previous work indicates that only a subset of genes might be de-repressed in a developmental process if Su(H) is absent. For example, de-repression of expression of wg along the D/V compartment boundary has never been observed upon loss of Su(H) function. The de-repression of only a subset of target genes could cause a phenotype that is difficult to interpret. Thus, it is better to use alleles of genes such as Psn, kuz or nic, which do not affect the repressor function of Su(H), to determine the function of the Notch pathway within a process of interest (Koelzer, 2006).

The weaker phenotype of Su(H) mutants during wing development was considered an argument for the existence of a Su(H)-independent mechanism of Notch signal transduction. The current findings strongly argue against the existence of such a mechanism in the analyzed processes. Evidence has been provided for the existence of a Su(H)-independent Notch-signaling pathway that is mediated by Dx. Since the existence of such a pathway has been excluded in the two other situations, it was of interest to discover whether an alternative explanation might exist for observations on the role of Dx. Indeed no evidence was found that Dx participates in a Su(H)-independent Notch signal during wing development. The results suggest that in this case, the confusion came from analyzing a domain of the vgBE (domain 2) that appears not to be completely dependent on the function of Su(H). Using the MARCM technique to generate Dx expressing Su(H) mutant cell clones, it was clearly show that Dx depends on the function of Su(H) to induce target gene expression in ectopic places as well as along the D/V boundary. Thus, the results abolish three arguments for the existence of a Su(H)-independent signal transduction mechanism during wing development. However, this does not imply that such a pathway does not exist. Indeed, evidence exists that during dorsal closure of the embryo, Notch acts independently of Su(H), through the JNK pathway (Koelzer, 2006).

Recent work indicates that cell-cell interactions are required for the establishment of both the A/P as well as the D/V compartment boundaries. While it is clear that a transcriptional response mediated by the transcription factor Cubitus interruptus (Ci) is necessary to establish the A/P boundary, the situation at the D/V boundary was unclear. The possibility has been raised of a Su(H)-independent mechanism that is used to establish the D/V boundary. This mechanism might not even require a transcriptional response to the Notch signal. The results demonstrate that this is not the case: similar to the formation of the A/P boundary compartment boundary, a transcriptional response to the Notch signal is required for the segregation of dorsal and ventral cells, and this response is mediated by Su(H). Similar to Ci, Su(H) acts as a transcriptional activator at the D/V boundary, where Notch is active and as a transcriptional repressor in a complex with H, and probably Groucho and dCtBP away from the boundary. The results suggest that the loss of this repressor function results in the de-repression of the relevant target genes in a manner sufficient to allow the formation of the D/V compartment boundary even in absence of Su(H). Overall the scenario at the D/V boundary seems to be very similar to that proposed for the formation of the A/P compartment boundary. In this situation, En endows the posterior fate and regulates the expression of Hedgehog that signals to anterior cells. As a response to Hh, the transcription factor Ci is transformed from a repressor to an activator of transcription and activates the expression of target genes in a stripe along the anterior side of the A/P boundary. The results suggest a similar scenario for the formation of the D/V compartment boundary: similar to En, Ap imposes the dorsal fates on cells and activates the expression of Ser. Ser signals to the ventral cells at the D/V boundary. Similar to Hh transforming Ci from a repressor into an activator of transcription, Ser induced activation of the Notch pathway transforms Su(H) from a repressor into an activator. In analogy to En, it was found that Ap has a second, Notch-independent function during D/V boundary formation. As in the case for En, an attractive possibility is that Ap acts to repress activation of the relevant target genes of Su(H) in dorsal cells. This repression creates a strong difference in expression of these genes at the D/V boundary and eventually leads to a strong difference in adhesion between the dorsal and ventral cells. This repressor function of Ap would also explain why the compartment boundary can form in the absence of Su(H) function, since the de-repression of target genes of Su(H) would be still restricted to ventral cells leading to a similar, albeit weaker difference in expression of these genes and in adhesion at the D/V boundary. Furthermore, it explains why the formation of the boundary fails in the absence of the function of ap and Su(H), since in this case no strong difference in expression of target genes will be created (Koelzer, 2006).

It appears that very similar strategies are exploited at both compartment boundaries to achieve segregation of the cell lineages. However, in each situation, a set of different but mechanistically similar acting signaling molecules are used to achieve the segregation of cell populations and formation of a compartment boundary (Koelzer, 2006).

Complex interplay of three transcription factors in controlling the tormogen differentiation program of Drosophila mechanoreceptors

This study has investigated the expression and function of the Sox15 transcription factor during the development of the external mechanosensory organs of Drosophila. Sox15 is expressed specifically in the socket cell, and the transcriptional cis-regulatory module has been identified that controls this activity. Suppressor of Hairless [Su(H)] and the POU-domain factor Ventral veins lacking (Vvl) bind conserved sites in this enhancer and provide critical regulatory input. In particular, Vvl contributes to the activation of the enhancer following relief of Su(H)-mediated default repression by the Notch signaling event that specifies the socket cell fate. Loss of Sox15 gene activity was found to severely impair the electrophysiological function of mechanosensory organs, due to both cell-autonomous and cell-non-autonomous effects on the differentiation of post-mitotic cells in the bristle lineage. Lastly, it was found that simultaneous loss of both Sox15 and the autoregulatory activity of Su(H) reveals an important role for these factors in inhibiting transcription of the Pax family gene shaven in the socket cell; shaven serves to prevent inappropriate expression of the shaft differentiation program. These results indicate that the later phases of socket cell differentiation are controlled by multiple transcription factors in a collaborative, and not hierarchical, manner (Miller, 2009).

After Su(H), Sox15 is the second transcription factor gene known to be activated specifically in the postmitotic socket cell of the Drosophila external sensory organ lineage. Three observations reported here indicate that although both genes come to be expressed at high levels in this cell, the underlying regulatory logic may be quite different (Miller, 2009).

The first is the distinct dynamics of autoregulatory socket enhancer (ASE)-stimulated Su(H) transcription versus Sox15 expression. Su(H) is immediately activated at high levels following the specification of the socket cell, due at least in part to the establishment of an autoregulatory loop working through the Su(H) ASE. Sox15 expression, however, exhibits a significant delay between socket cell specification and the time peak levels of transcript accumulation are achieved (Miller, 2009).

The second observation concerns the role played by Vvl in the activation of the Sox15 socket enhancer and the Su(H) ASE (Barolo, 2000). Conserved within the ASE lies a motif, CATAAAT, that might act as a weak Vvl binding site, suggesting the possibility that Vvl could play a part in the high-level activation of Su(H) in the socket cell. However, this appears not to be the case, since ASE-GFP is activated within the same temporal window, and just as strongly, in vvl mutant clones as in neighboring wild-type tissue. By contrast, while the long reporter fragment Sox7.5 > GFP, covering the whole intron, is also activated in vvl mutant sensory organs, there is a substantial delay in this expression, which is often not detectable until the socket cell has begun to divide aberrantly. At this time, neighboring wild-type sensory organs are already strongly expressing Sox7.5 > GFP. Vvl thus appears to be one factor present in the socket cell that is necessary for the full activation of Sox15, but not of Su(H) (Miller, 2009).

Finally, there is the observed role of N-activated Su(H) in contributing to the transcriptional activation of the Sox15 socket enhancer versus the Su(H) ASE. A major difference between the two genes is made apparent by the contrasting effects on reporter gene expression of mutating the high-affinity Su(H) site(s) in their respective socket cell enhancers. In the case of the Su(H) ASE, mutation of the Su(H) sites causes a strong reduction in socket cell activity at early times, along with ectopic activity in the shaft cell; by the adult stage, the mutant enhancer is inactive. Thus, N-activated Su(H) contributes critically to the transcriptional activation of the Su(H) ASE. The Su(H)-site-mutant Sox15 enhancer, on the other hand, shows no apparent diminution of its socket cell activity early (when it also drives ectopic expression in the shaft cell), and remains fully active in the pharate adult. In the case of Sox15, then, activation of Su(H) by the N signaling event appears to serve only the purpose of relieving Su(H)-mediated default repression; activation of the enhancer is evidently accomplished entirely through the action of other factors such as Vvl. This distinction in the role of N signaling in enhancer activation has been referred to as 'Notch instructive' [Su(H) ASE] versus 'Notch permissive' (Sox15 socket enhancer) (Miller, 2009).

This investigation of the loss-of-function phenotype of Sox15 has revealed that, like Su(H), it has an important role in controlling the socket cell differentiation program. Comparison of the phenotypic effects of losing Sox15 function, Su(H) function, or both, suggests an incomplete overlap in the target gene batteries regulated by the two factors. Loss of either Sox15 or Su(H) ASE activity causes a serious defect in mechanosensory organ function. The lack of Su(H) ASE activity confers the more severe phenotype, including significant reductions of both transepithelial potential (TEP) and mechanoreceptor current (MRC). The TEP defect signifies an inability of the socket cell to establish the receptor lymph cavity itself, the proper ionic composition of the receptor lymph, or a combination of the two. The genes required for these events have yet to be identified, but it is likely that Su(H) plays a role in regulating their expression in the socket cell. Sox15, on the other hand, does not appear to share this role, based on the apparent lack of a major TEP defect in Sox15 mutants. Instead, Sox15 appears to regulate targets that contribute to socket cell viability. Without these target factors, the cell eventually becomes necrotic. In addition, the principal physiological phenotype of Sox15 mutants is the MRC defect, which is also conferred by loss of Su(H) ASE function. Loss of MRC is indicative of a failure in neuronal function, yet both Sox 15 and the Su(H) ASE are active specifically in the socket cell. This apparent paradox indicates an important role for the socket cell as a support cell for the mechanosensory neuron. To date three proteins - Sox15 (this paper), Su(H), and the cytochrome P450 Cyp303a1 - expressed in and required specifically for socket cell differentiation appear to contribute to neuronal function in mechanosensation. Given that the socket cell envelops the other cells of the sensory organ as it develops, the socket may be intimately involved in their normal differentiation and in the establishment of structural and functional connectivity between them. Defects in these processes could readily manifest themselves in an MRC phenotype. Thus, the abnormal microtubule bundling in the sensory dendrite in Sox15 mutants may very well be the result of a defect in the socket cell's ability to contribute as it should to the neuron's normal development. It is unclear at this point if the dendrite defect is due to a failure to activate Sox15-dependent target genes directly involved in the socket cell's support function, or if it is an indirect consequence of the degeneration of the socket cell (Miller, 2009).

Previous studies have established that both daughters of the pIIa secondary precursor division are bipotent cells that can adopt either the shaft or socket cell fate. Asymmetric N signaling specifies that the posterior daughter expresses only the signal-dependent socket fate and the anterior daughter only the signal-independent shaft fate. Correspondingly, investigation of socket cell fate specification has largely focused on its positive aspects; i.e., those ways in which the N signaling event promotes the socket cell from the 'default' (signal-independent) shaft fate to the alternative fate, triggering its execution of the distinctive socket differentiation program. This study has shown that socket cell-specific activation of Sox15 expression is an important component of this program. But the present study has also revealed the other side of the coin, by showing that the N signaling event also results in the activation of a mechanism for suppressing in the socket cell the capacity to execute the shaft differentiation program. This suppression mechanism involves the combined action of Sox15 and Su(H) in inhibiting transcription of the sv gene, which encodes a Pax transcription factor that is a high-level activator of the shaft differentiation program. Without this inhibition, the socket cell generates both socket and shaft cuticular structures. It is clear, then, that much of the network circuitry necessary for the execution of the shaft differentiation program remains intact in the socket cell even after its fate has been specified. These results show that robust N-mediated cell fate specification in the mechanosensory bristle lineage involves not only promoting the signal-dependent fate, but also actively inhibiting the alternative program (Miller, 2009).

It is likely that at least Su(H)'s role in inhibiting sv expression in the socket cell is indirect, and occurs via an as yet unidentified repressor. An attractive candidate for this factor X would be one or more basic helix-loop-helix (bHLH) repressors encoded in the Enhancer of split Complex [E(spl)-C]. Multiple E(spl)-C bHLH repressor genes are activated directly by Su(H) in response to N signaling in a variety of developmental contexts. Consistent with this possibility, it was observed that socket cell-specific overexpression of E(spl)m7-VP16, a form of the E(spl)m7 bHLH repressor that has been converted to a strong activator, phenocopies the ectopic-shaft effect of sv overexpression in the same cell (Miller, 2009).

The results of this and earlier studies afford a glimpse of the regulatory architecture of the socket differentiation program, which is set in motion by the N signaling event that specifies the socket cell fate. It seems useful to distinguish two broad phases of this program, which no doubt overlap each other in time and are also very likely to share at least some components of the regulatory network. These two phases might be referred to as the earlier 'morphogenetic' and the later 'physiological' subdivisions of the socket program. The distinction is prompted by the observations of the phenotypes conferred by loss of the two socket cell-specific transcription factor activities identified so far, Su(H) and Sox15. In both cases, it was found that many characteristic aspects of the socket's cellular differentiation proceed completely normally, most notably the construction of the complex socket cuticular structure that surrounds the shaft structure (morphogenesis). By contrast, loss of Su(H) or Sox15 function in the socket cell results in major deficits in the electrophysiological capacity of the sensory organ (physiological differentiation). As described above, the specifics of these deficits differ for Su(H) versus Sox15 mutants, and include distinctive cell-autonomous defects in the socket cell and defects in other cells likely due to the failure of some aspects of the socket cell's support function. But the phenotypic commonalities (emphasizing the physiological and not the morphogenetic) are striking nonetheless. It is perhaps reasonable to speculate that transcription factors like Su(H) and Sox15 that are activated for the first time in the sensory organ lineage specifically in the socket cell will tend to function primarily in the later physiological phase of the differentiative program. By contrast, it may be expected that the earlier morphogenetic phase is controlled primarily by factors first expressed earlier in the lineage, at least in the pIIa precursor cell and perhaps in the SOP. Vvl exemplifies this notion: It is first expressed in the SOP, and loss of its activity causes visible defects in the socket cuticular structure, as well as aberrations in the mitotic status of the normally postmitotic socket cell. Investigation of the roles of additional transcriptional regulators in directing the socket differentiation program will test the viability of this broad conceptual framework (Miller, 2009).

Overall, this comparison of the roles of Sox15, Su(H), and Vvl in controlling aspects of the socket differentiation program indicates that they function largely in parallel, and collaboratively, rather than in a hierarchical fashion. This may suggest that the socket program will prove to be characterized by an ensemble of such parallel regulatory inputs that collectively direct the complex differentiation of the cell. It is perhaps useful to note that this picture contrasts already with what is known about the control of the shaft differentiation program, which is dominated by the function of Sv as a high-level regulator. Whether this reflects some important difference in how the differentiative programs of N-responsive versus N-non-responsive cell types are controlled will become clearer as more is learnt about the gene regulatory network that underlies mechanosensory organ development (Miller, 2009).

Notch signaling regulates neuroepithelial stem cell maintenance and neuroblast formation in Drosophila optic lobe development

Notch signaling mediates multiple developmental decisions in Drosophila. This study examined the role of Notch signaling in Drosophila larval optic lobes development. Loss of function in Notch or its ligand Delta leads to loss of the lamina and a smaller medulla. The neuroepithelial cells in the optic lobe in Notch or Delta mutant brains do not expand but instead differentiate prematurely into medulla neuroblasts, which lead to premature neurogenesis in the medulla. Clonal analyses of loss-of-function alleles for the pathway components, including N, Dl, Su(H), and E(spl)-C, indicate that the Delta/Notch/Su(H) pathway is required for both maintaining the neuroepithelial stem cells and inhibiting medulla neuroblast formation while E(spl)-C is only required for some aspects of the inhibition of medulla neuroblast formation. Conversely, Notch pathway overactivation promotes neuroepithelial cell expansion while suppressing medulla neuroblast formation and neurogenesis; numb loss of function mimics Notch overactivation, suggesting that Numb may inhibit Notch signaling activity in the optic lobe neuroepithelial cells. Thus, these results show that Notch signaling plays a dual role in optic lobe development, by maintaining the neuroepithelial stem cells and promoting their expansion while inhibiting their differentiation into medulla neuroblasts. These roles of Notch signaling are strikingly similar to those of the JAK/STAT pathway in optic lobe development, raising the possibility that these pathways may collaborate to control neuroepithelial stem cell maintenance and expansion, and their differentiation into the progenitor cells (Wang, 2011).

This study find that Notch signaling plays an essential role in the maintenance and expansion of neuroepithelial cells in the optic lobe; it also inhibits medulla neuroblast formation. Clonal analyses of several pathway components indicate that this dual function bifurcates downstream of Su(H) with E(spl)-C only partly involved in the inhibition of medulla neuroblast formation but not the maintenance and expansion of neuroepithelial stem cells (Wang, 2011).

In the optic lobe, Notch signaling plays a role analogous to lateral inhibition during embryonic CNS development. However, the selection of neuroblasts in the OPC neuroepithelium is an all-or-none process rather than selecting individual neuroblasts from the neuroepithelium. Medulla neuroblasts are generated in a wave progressing in a medial to lateral direction in the OPC neuroepithelium with all cells at a particular position along the medial-lateral axis differentiating into neuroblasts. Interestingly, this wave of medulla neuroblast formation coincides with the down-regulation of both Delta and Notch expression in the medial cells in the OPC, which might reduce Notch signaling activity, thereby allowing medulla neuroblasts to form. What factors drive the recession of both Delta and Notch expression in the OPC neuroepithelium along the medial-lateral axis is not known. When Notch signaling is inactivated, neuroepithelial cells in the OPC change cell morphology and differentiate into medulla neuroblasts prematurely. The results indicate that Notch signaling actively controls neuroepithelial integrity, possibly by regulating the adherens junction (AJ), since in Notch pathway mutant mosaic clones in the OPC, the apical determinants PatJ, Crumbs and aPKC are cell autonomously reduced or lost and the mutant cells change to rounded or irregular morphology. Further experiments will be needed to determine how Notch signaling activity affects the maintenance of neuroepithelial integrity, particularly the stability of the adherens junction (Wang, 2011).

Is neuroblast formation also actively inhibited by Notch signaling or simply a default state of neurogenic epithelial cells? In the latter model, Notch signaling may only maintain neuroepithelial integrity and promote their expansion while medulla neuroblasts form when the neuroepithelial integrity is disrupted. The argument against this model is that changes in neuroepithelial integrity are not always accompanied with cell fate changes. In N, Dl or Su(H) mosaic clones located in the OPC neuroepithelium, it was found that in about 25% of the clones, the mutant cells changed morphology or lost apical marker expression but did not become neuroblasts (Dpn-negative), whereas in E(spl)-C mosaic clones, Dpn+ cells were prematurely induced, which indicate that the cells begin to differentiate into neuroblasts, but these cells still retained columnar epithelial cell morphology and apical marker expression. This suggests that the suppression of neuroblast formation by Notch signaling activity is separable from the maintenance of neuroepithelial integrity and that medulla neuroblast formation is actively suppressed by Notch signaling. A possible scenario is that activation of the Notch pathway turns on the E(spl)-C genes, which in turn suppress proneural gene expression in the optic lobe neuroepithelia. Indeed, at least one member in the E(spl)-C genes, E(spl)m8, appears to be activated in the neuroepithelial cells by the Notch pathway, as the E(spl)m8-lacZ reporter is expressed in a pattern similar to Delta and Notch expression in the OPC and IPC. E(spl)m8 protein and possibly additional members of the E(spl)-C may suppress the expression of proneural genes in the optic lobe. The proneural genes of the achaete-scute complex (as-c) comprise four members, achaete, scute, L'sc, and asense. achaete is not expressed in the optic lobe, but scute is expressed in both the neuroepithelial cells and neuroblasts in the OPC implying that scute expression in the neuroepithelial cells is not suppressed by Notch signaling activity. By contrast, asense is only expressed in the neuroblast and GMCs and L'sc is transiently detected in an advancing stripe of neuroepithelial cells of 1-2 cells wide that are just ahead of newly formed medulla neuroblasts. Thus, E(spl)-C proteins may suppress L'sc and/or ase expression, the release of this suppression may allow the neuroepithelial cells to begin to differentiate into medulla neuroblasts. It should be noted, however, that the removal of the E(spl)-C activity does not seem to be sufficient to allow full differentiation of neuroepithelial cells into medulla neuroblasts, suggesting that additional factors downstream of Notch signaling may be involved in the suppression of medulla neuroblast formation (Wang, 2011).

The phenotypes of Notch pathway mutants are reminiscent of those of JAK/STAT mutants. For example, inactivation of either pathway led to early depletion of the OPC neuroepithelium; either pathway inhibits neuroblast formation, and ectopic activation of either pathway promotes the growth of the OPC neuroepithelium. The remarkable phenotypic similarities in Notch and JAK signaling mutant brains suggest that these pathways may act in a linear relationship such that activation of one pathway is relayed to the second, perhaps by inducing the expression of a ligand. Alternatively, these pathways may act in parallel and converge onto some key downstream effectors or target genes. Further experiments will be needed to test whether Notch interacts with JAK/STAT and if it does, to find out where the interaction occurs during the development of the optic lobe (Wang, 2011).

The roles of Notch signaling in mammalian brain development have been studied intensely. Many Notch pathway components have been examined in knockout mice, which showed defects in brain development. Mice deficient for Notch1 or Cbf all display precocious neurogenesis during early stages of nervous system development. This has led to the view that the role of Notch signaling in the mouse brain is to maintain the progenitor state and inhibit neurogenesis. However, it is not clear from these studies whether the premature neurogenesis in Notch signaling mutant mice was caused by premature differentiation of neuroepithelial stem cells into neurons or by premature differentiation of neuroepithelial stem cells into progenitor cells, which then generated neurons. In fact, it has been proposed that Notch activation can promote the differentiation of neuroepithelial stem cells into radial glial cells, the progenitor cells that generate the majority of neurons in the cerebral cortex. This is based on the observation that ectopic Notch activation using activated forms of Notch1 and Notch3 (NICD) caused an increase in radial glial cells as compared to control. The radial glial cells resemble medulla neuroblasts in the Drosophila optic lobe in that they are both derived from neuroepithelial stem cells and undergo asymmetric division to self-renew and generate neurons, although morphologically radial glial cells are still polarized while medulla neuroblasts have lost epithelial characters and are rounded in shape. Based on the current results, it is suggested that Notch signaling maintains the pool of neuroepithelial stem cells and promotes their expansion in both Drosophila and mammals and that the precocious neurogenesis in Notch signaling mutant brains arise due to premature differentiation of the neuroepithelial stem cells into the progenitor cells (Wang, 2011).

However, ectopic Notch activation may indeed promote progenitor cell proliferation in the brain. Ectopic neuroblasts were observed in the medulla cortex when NACT was ectopically expressed by the neuroblast/GMC driver insc-Gal4, by ubiquitous expression using hs-Gal4, or when numb15 mosaic clones were induced at later larval stages when neuroblasts normally begin to form. Since the results have shown that the Notch pathway is not essential for medulla neuroblast formation or self-renewal, the ectopic neuroblasts are a novel phenotype solely induced by ectopic Notch signaling activity. This is consistent with Notch activation promoting ectopic neuroblast formation in the central brain and VNC without being required for neuroblast self-renewal in these regions of the CNS; and Notch has been shown to be an oncogene in mammals. Since the sizes of the ectopic neuroblasts were in the range of GMC or neurons, they may resemble the transit-amplifying (TA) neuroblasts that are found in the dorsal-medial region of the central brain. The origin of these ectopic neuroblasts in the medulla cortex is not clear, but it is unlikely that they are derived from differentiated medulla neurons as ectopic expression of NACT using elav-Gal4, which is active in medulla neurons, did not result in ectopic neuroblasts and by the fact that ectopic neuroblasts can be induced in numb15 mosaic clones, which could only arise from mitotically active cells that include neuroepithelial cells, medulla neuroblasts, and ganglion mother cells (GMCs), but not neurons. The ectopic neuroblasts could be generated by a transformation of GMCs into a neuroblast identity as suggested for ectopic neuroblasts in brat mutant central brains. Ectopic Notch signaling activity may even directly promote the expansion of neuroblasts after they have differentiated from the neuroepithelial cells in the OPC. In either case, ectopic Notch signaling activity may block the normal path of neuronal differentiation and lock the cells in a proliferative state. This is indeed what was observed in numb15 mosaic clones in which numerous ectopic neuroblasts were induced in the medulla cortex without generating medulla neurons. Perhaps ectopic Notch signaling activity may also promote the proliferation of neural progenitors in vertebrates, such as the radial glial cells in the mouse brain (Wang, 2011).

Local overexpression of Su(H)-MAPK variants affects Notch target gene expression and adult phenotypes in Drosophila

In Drosophila, Notch and EGFR signalling pathways are closely intertwined. Their relationship is mostly antagonistic, and may in part be based on the phosphorylation of the Notch signal transducer Suppressor of Hairless [Su(H)] by MAPK. Su(H) is a transcription factor that together with several cofactors regulates the expression of Notch target genes. This study addresses the consequences of a local induction of three Su(H) variants on Notch target gene expression. To this end, wild-type Su(H), a phospho-deficient Su(H)MAPK-ko and a phospho-mimetic Su(H)MAPK-ac isoform were overexpressed in the central domain of the wing anlagen. The expression of the Notch target genes cut, wingless, E(spl)m8-HLH and vestigial, was monitored. For the latter two, reporter genes were used (E(spl)m8-lacZ and vgBE-lacZ). In general, Su(H)MAPK-ko induced a stronger response than wild-type Su(H), whereas the response to Su(H)MAPK-ac was very weak. Notch target genes cut, wingless and vgBE-lacZ were ectopically activated, whereas E(spl)m8-lacZ was repressed by overexpression of Su(H) proteins. In addition, in epistasis experiments an activated form of the EGF-receptor (DERact) or the MAPK (rlSEM) and individual Su(H) variants were co-overexpressed locally, to compare the resultant phenotypes in adult flies (thorax, wings and eyes) as well as to assay the response of the Notch target gene cut in cell clones (Auer, 2015).

Suppressor of Hairless: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | References

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