Effects of Mutation or Deletion

Table of contents

Notch and wing morphogenesis

Growth and patterning of the Drosophila wing disc depends on the coordinated expression of the key regulatory gene vestigial both in the dorsal-ventral (DV) boundary cells and in the wing pouch. It is proposed that a short-range signal originating from the core of the DV boundary cells is responsible for activating Egfr in a zone of organizing cells on the edges of the DV boundary. Using loss-of-function mutations and ectopic expression studies, it has been shown that Egfr signaling is essential for vestigial transcription in these cells and for making them competent to undergo subsequent vestigial-mediated proliferation within the wing pouch (Nagaraj, 1999).

Unlike activation of Notch and Egfr, activation of the Wg pathway using similar experimental paradigms does not induce non-cell-autonomous proliferation in the wing pouch cells. A possible synergistic interaction between Wg and Egfr pathway in the control of Vg expression has not been ruled out, but unlike Egfr activation, Wg on its own is not able to induce high enough levels of Vg expression to cause cell proliferation. Wg does, however, have several important functions in the patterning of the wing. These include distinguishing the identity of the pouch cells from those of the notum; specifying the bristles along the anterior margin, and refining the DV boundary (Nagaraj, 1999 and references).

Activation of the Egfr pathway in cells adjacent to the DV boundary leads to the localized activation of MAPK in thin strips of cells flanking the DV boundary. These regions of MAPK activation are termed the competence zone (CZ). The activation of MAPK in this region is also dependent on a functional Notch signal at the DV boundary. The fact that Egfr signaling is operative in this zone is also supported by the earlier finding that argos and rhomboid are also expressed in this region. Also consistent with the hypothesis that Notch signal is essential for the activation of the Egfr in the CZ region, it has been reported that loss of Notch results in the loss of rho expression along the DV boundary even as the expression of rho in the vein regions is greatly expanded upon loss of the Notch signal. Localized Rhomboid expression has been implicated in Egfr signaling and could therefore account for the localized induction of Egfr activation at the DV boundary. Most importantly, these results show that a localized inactivation of the Egfr signal exclusively at the DV boundary results in dramatic loss of Vg in the remainder of the pouch. Thus, localized activation of the Ras pathway in cells flanking the DV boundary is important for the patterning of the entire pouch. Previous work has suggested that loss of Notch function at the DV boundary has a non-cell-autonomous effect on the expression of Vg in the pouch and the proliferation of cells in the rest of the pouch region. These results suggest that this effect is mediated through the Egfr pathway. It is hypothesized that high levels of Egfr signaling are required in these cells in order to provide them with competence to express Vg and therefore to proliferate (Nagaraj, 1999).

During vein differentiation dpp is expressed in the pupal veins under the control of genes that establish vein territories in the imaginal disc. Both dpp and thick veins are differentially expressed in vein territories during pupal development. dpp and tkv regulate one another by a feedback mechanism in which Tkv activity represses dpp expression. Dpp, acting through its receptor Thick veins, activates vein differentiation and restricts expression of both veinlet and the Notch-ligand Delta to the developing veins. Once Dpp is established in the veins, local activation of Tkv in these cells is required both for the maintenance of veinlet and Delta expression and for the veins to differentiate. In dpp mutants, the vein thickening observed in Notch mutants is elimated. Conversely, Notch gain-of-function alleles that lead to the truncation of veins results in very pronounced vein loss in combination with both dpp and tkv mutants. In dpp mutants, Delta and E(spl)mß, which normally takes place in vein territories, is lost. In summary, genetic combinations between mutations that increase or reduce Notch, veinlet and dpp activities suggest that the maintenance of the vein differentiation state during pupal development involves cross-regulatory interactions between these pathways (de Celis, 1997a).

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, 1996b)

The Drosophila Epidermal growth factor receptor (Egfr) is a key component of a complex signaling pathway that participates in multiple developmental processes. An F1 screen was performed for mutations that cause dominant enhancement of wing vein phenotypes associated with mutations in Egfr. With this screen, mutations were recovered in Hairless (H), vein, groucho (gro), and three apparently novel loci. All of the enhancers of Egfr mutations [E(Egfr)] identified show dominant interactions in transheterozygous combinations with one another and with alleles of N or Su(H), suggesting that they are involved in cross-talk between the N and Egfr signaling pathways. Further examination of the phenotypic interactions between Egfr, H, and gro reveals that reductions in Egfr activity enhances both the bristle loss associated with H mutations, and the bristle hyperplasia and ocellar hypertrophy associated with gro mutations. Double mutant combinations of Egfr and gro hypomorphic alleles leads to the formation of ectopic compound eyes in a dosage sensitive manner. These findings suggest that these E(Egfr)s represent links between the Egfr and Notch signaling pathways, and that Egfr activity can either promote or suppress Notch signaling, depending on its developmental context (Price, 1997).

Genetic interactions between the N and Egfr signaling pathways have been reported previously. There is a mutual enhancement between N gain-of-functions (gof) and Egfr loss-of-functon (lof) mutations and mutual suppression between lof alleles of Delta and Egfr. Egfr gof alleles enhance Notchspl in the eyes and Delta loss-of-function alleles in the wings of double mutant flies. Mutations in Egfr and two other Egfr pathway components (Son of sevenless and pointed) act as enhancers of N signaling in the eye. Mutations in both Hairless and groucho enhance L4 wing vein defects associated with mutations in both Egfr and vein. Egfr. In turn, Egfr mutations enhance the Hairless mutant-associated loss of macrochaetae and microchaetae from the head and thorax; therefore, Egfr and Hairless appear to cooperate in at least two developmental processes. Groucho appears to be required in contexts that appear to be distinct from its function in Notch signaling. Ectopic wing hairs are observed on the wings of Egfr, groucho and rolled;groucho double mutants that are similar to defects seen in hairy mutant flies or groucho/hairy transheterozygotes, and may indicate that Egfr mutations reduce Groucho's activity as a corepressor with Hairy. The spectrum of defects enhanced in Egfr;gro or rolled;groucho double mutants appears to reflect a reduction in most or all aspects of groucho activity. The simplest interpretation of these observations is that both Egfr and Rolled promote the activity of Groucho. Gro and its mammalian homolog, TLE1, are phosphorylated on serine residues and thus may be downstream targets of an Egfr-regulated phosphorylation cascade (Price, 1997 and references).

The Notch receptor signaling pathway regulates cell differentiation during the development of multicellular organisms. A number of genes are known to be either components of the pathway or regulators of the Notch signal. One candidate for a modifier of Notch function is the Drosophila Suppressor of deltex gene [Su(dx)]. Four new alleles of Su(dx) have been isolated and the gene has been mapped between 22B4 and 22C2. Loss-of-function Su(dx) mutations were found to suppress phenotypes resulting from Notch loss-of-function signaling and to enhance gain-of-function Notch mutations. Hairless, a mutation in a known negative regulator of the Notch pathway, is also enhanced by Su(dx). Phenotypes were identified for Su(dx) in wing vein development. Homozygous mutant flies for one allele [Su(dx)sp] have a wild-type vein pattern at 25 degrees C. However, when they are kept at 29 degrees, a recessive wing vein gap phenotype appears. The phenotype is manifested most often in veins L.IV and L.V, distal to the posterior cross-vein. Gaps are found frequently in L.II as well, but never in L.III. Three other alleles display wing vein gaps at 29 degrees when placed over Su(dx)sp. At 25°, the same combinations of alleles have intact longitudinal veins, but forked or incomplete cross-veins. A role was demonstrated for the gene between 20 and 30 hr after puparium formation. A temperature upshift after 28 hr after puparium formation allows normal development of the veins. This corresponds to the period when the Notch protein is involved in refining the vein competent territories (Fostier, 1998).

A number of observations indicate that the wild-type function of Su(dx) is as a negative regulator of the Notch pathway. The temperature-sensitive wing vein gap phenotype is similar to that observed for gain-of-function Abruptex alleles of Notch. Complementation tests over the deficiency have shown that the Su(dx) mutants described result in a loss of function of Su(dx). This is an important prerequisite for interpreting the wild-type function of Su(dx). The haplo-insufficient phenotype of Notch is suppressed by Su(dx) mutations, as is the mutation of Delta, the Notch ligand. In contrast, the gain-of-function AxE2 mutation of Notch is enhanced by Su(dx). This is similar to the known genetic interactions of Hairless with these Notch mutants. Hairless is a negative regulator of the Notch pathway, and it functions by binding to and inhibiting Suppressor of Hairless, a Notch-responsive transcription factor. The fact that Su(dx) enhanced the Hairless phenotype indicates that the two genes are regulating the Notch signal in the same direction. Similarly, the observed suppression of deltex is as expected. Because deltex is a positive regulator of Notch function, its mutation should be compensated by a mutant that leads to a hyperactivation of the Notch signal (Fostier, 1998).

Activation of the Notch pathway can be mimicked by ectopic E(spl)mß expression in the wing, which results in gaps in the veins. The strength of this phenotype is dependent on the dosage of the expressed E(spl)mß, and the phenotype is enhanced in a Su(dx) mutant background. It is hypothesized that the Su(dx) mutation leads to an elevation of Notch signaling and increased expression of endogenous E(spl)mß, which augments the ectopically expressed protein levels. However, the alternative possibility that the enhanced phenotype may be caused by an upregulation of the downstream response to the activity of expressed E(spl)mß cannot be ruled out. Support for a negative regulatory function for Su(dx) also comes from comparison of Su(dx) phenotypes with those resulting from ectopic expression of activated Notch and wild-type deltex proteins. It is possible to make a constitutively activated Notch receptor by expressing a truncated form that lacks the extracellular domain. The Notch pathway can also be upregulated by overexpression of wild-type deltex. When activated Notch or wild-type deltex are expressed under control of a heat shock promoter 0-24 hr APF, a wing vein gap phenotype appears. In both cases, this phenotype is strongly enhanced in a heterozygous nd (a recessive Notch allele with a wing margin loss phenotype that is similar to the loss of one copy of Notch) background, similar to the interaction between Su(dx) mutants and nd. Thus, the Su(dx) mutation mimics an elevation of the Notch signal. Taken together, these data indicate a role for Su(dx) as a negative regulator of the Notch pathway. The existence of feedback regulatory loops in the control of Notch signaling makes the position of Su(dx) protein in the Notch pathway difficult to define through genetic analysis. Su(dx) mutants were first identified through their interaction with deltex. It cannot be concluded that the corresponding proteins interact directly, however, especially as there are genetic interactions between Su(dx) and a number of Notch pathway genes. The precise function of Su(dx) will only be resolved through cloning of the gene and analysis of its function at the molecular level, which is in progress. It is likely, therefore, that the further characterization of Su(dx) and its interacting mutations will be fruitful for the understanding of Notch pathway regulation (Fostier, 1998).

The function of extramacrochaetae is required during the development of the Drosophila wing in processes such as cell proliferation and vein differentiation. extramacrochaetae encodes a transcription factor of the HLH family, but unlike other members of this family, Extramacrochaetae lacks the basic region that is involved in interaction with DNA. Some phenotypes caused by extramacrochaetae in the wing are similar to those observed when Notch signaling is compromised. Furthermore, maximal levels of extramacrochaetae expression in the wing disc are restricted to places where Notch activity is higher, suggesting that extramacrochaetae could mediate some aspects of Notch signaling during wing development. The relationships between extramacrochaetae and Notch in wing development have been studied, with emphasis on the processes of vein formation and cell proliferation. Strong genetic interaction between extramacrochaetae and different components of the Notch signaling pathway have been observed, suggesting a functional relationship between them. The higher level of extramacrochaetae expression coincides with the domain of expression of Notch and its downstream gene Enhancer of split-m beta. The expression of extramacrochaetae at the dorso/ventral boundary and in boundary cells between veins and interveins depends on Notch activity. It is proposed that at least during vein differentiation and wing margin formation, extramacrochaetae is regulated by Notch and collaborates with other Notch-downstream genes such as Enhancer of split-m beta (Baonza, 2000).

Notch mutant cells show reduced viability, whereas activation of Notch signaling causes strong mitotic activity in the wing disc, independent of the activation of vestigial and wingless. These observations suggest that Notch, in addition to its function in the establishment of the D/V boundary is also directly involved in the control of cell proliferation. In this function of Notch the genes of the E(spl) complex are not required. emc is also involved in regulating cell proliferation during wing disc development, because emc mutant cells do not proliferate at all, and clones of cells of strong emc hypomorphic alleles reduce cell proliferation in intervein territories. Mutant cells for both emc and Notch have extremely poor viability, indicating that emc and Notch cooperate to promote cell proliferation. However, this interaction does not rely on Notch controlling emc transcription, because the basal level of Emc expression in the wing pouch is not affected in Notch mutant cells. Thus, it is proposed that during imaginal cell proliferation emc and Notch signaling act in parallel, possibly on the same set of downstream genes, to promote cell proliferation (Baonza, 2000).

The activity of Notch is necessary for the formation and maintenance of the D/V boundary. Thus, loss of Notch prevents the formation of the wing margin and, conversely, ectopic Notch activity results in the formation of novel margin structures and wing outgrowths. During the third instar, Notch expression is maximal in the dorsal and ventral cells that form the D/V boundary. These cells also correspond to the places where E(spl)m beta, a Notch-downstream gene, is expressed, indicating high levels of Notch signaling there. The expression of emc at the D/V boundary is maximal in the same cells where Notch and E(spl)m beta genes are expressed, suggesting that Notch signaling could regulate emc expression. In fact, the expression of emc at the D/V border is eliminated in cells lacking Notch activity, whereas clones of cells expressing an activated form of N express ectopically high levels of Emc. Increased levels of Emc expression are also induced by the Notch ligands Dl and Ser in the dorsal and ventral compartments, respectively (Baonza, 2000).

The regulation of emc expression at the D/V boundary by Notch is not mediated by E(spl)m beta, since clones of E(spl)m beta-expressing cells do not affect the expression of emc. Elimination of E(spl)m beta or emc does not affect the formation of the wing margin, indicating that these Notch targets are not required for Notch activity in the formation of this structure. However, emc and E(spl) are required during the formation of the sensory organs characteristic of the wing margin. Thus, ectopic expression of emc [or E(spl)] throughout the wing pouch eliminates most of the sensory elements of the anterior wing margin. It is likely that this function of emc and E(spl) relies on the repression of the activity and expression of the Achaete and Scute proteins (Baonza, 2000).

The expression of several genes such as vein (ve) and blistered is restricted to either vein or intervein regions during imaginal development, indicating that at this stage the veins are being specified. A key component of vein specification is the activity of the Egfr signaling pathway, although it is not known which genes localize Egfr activation to vein territories. Both emc and Notch are required at this early stage to position vein territories and to define their extent, respectively, and it is likely that Notch and emc interact during the definition of vein territories in third instar wing discs. This interaction could be based in the regulation by Notch and Emc of similar target genes controlling the appearance and extent of vein-competent territories. However, the results suggest that in this initial establishment of vein territories the expression of emc and the activity of Notch are independent of each other, because the heterogeneity in emc expression related to developing veins observed in third instar discs is not modified in Notch mutant backgrounds. Furthermore, some characteristic phenotypes of emc clones, such as the appearance of ectopic veins of normal thickness, are never observed in Notch clones, indicating that emc and Notch are affecting independent processes during the initiation of vein development (Baonza, 2000).

After puparium formation the activity of Notch is continuously required to maintain the correct width of the vein, and at this stage Notch activation occurs in two stripes of cells adjacent to each vein. The accumulation of E(spl)m beta in these cells, as a consequence of Dl-mediated Notch activation, contributes to the restriction of ve expression to the vein, and prevents the differentiation as vein of the flanking pro-vein cells. Interestingly, the elimination of Notch or Dl activity results in the formation of thicker veins than elimination of E(spl)m beta, suggesting that additional elements are activated in response to Notch and participate in the repression of vein differentiation (Baonza, 2000).

Several arguments suggest that emc is one of these components that mediate Notch signaling during the pupal development of veins. (1) The expression of emc in pupal wings is maximal in the same cells that express E(spl)m beta, suggesting that Notch activity is responsible for the preferential accumulation of emc expression. This expression is modified when Notch activity is compromised; in such circumstances emc is detected in the novel flanking cells associated with the thickened Notch mutant veins. (2) Clones of emc mutant cells occasionally cause vein thickening, and this phenotype is greatly exaggerated in Notch and Dl mutant backgrounds, suggesting that in a situation of insufficient Notch activity, the levels of emc are critical to repress vein formation. The analysis of emc clones in l(1)N ts heterozygotes indicates that during the pupal stage cells are particularly sensitive to reduction in emc and Notch activities. In addition, clones of Dl-expressing cells induced during pupal development cause ectopic expression of emc, indicating that during this stage the activity of Notch is enough to increase the levels of emc. These results do not discard an earlier requirement for both genes in vein determination, but show that during pupal development emc and Notch do interact in the definition of vein thickness (Baonza, 2000).

The molecular basis of this interaction is unclear; so far there is no emc-target gene identified affecting vein formation. By analogy to the function of emc in antagonizing the activity of proneural proteins, it is postulated that Emc modulates the function of some protein involved in promoting vein formation. Interestingly, when both emc and E(spl)m beta are overexpressed, an enhancement of the E(spl)m beta overexpression phenotype of loss of veins is observed, suggesting that the combination of high levels of both emc and E(spl)m beta results in more effective repression of vein differentiation. Thus, it is proposed that Notch signaling, in addition to activating the expression of E(spl)m beta, induces high levels of emc expression in flanking cells, and that the combination of emc and E(spl)m beta is more efficient in suppressing vein formation than E(spl)m beta alone. Emc and E(spl) do not physically interact with each other, and therefore it is unlikely that emc contributes to E(spl)mb activity. Therefore it is suggested that Emc and E(spl)mbeta contribute to the regulation of the activity and expression of a vein-promoting protein and gene, respectively, thus explaining the observed synergy between Notch signaling and emc function in vein formation (Baonza, 2000).

During development of multicellular organisms, cells are often eliminated by apoptosis if they fail to receive appropriate signals from their surroundings. Short-range cell interactions support cell survival in the Drosophila wing imaginal disc. Evidence is presented showing that cells incorrectly specified for their position undergo apoptosis because they fail to express specific proteins that are found on surrounding cells, including the LRR transmembrane proteins Capricious and Tartan. Interestingly, only the extracellular domains of Capricious and Tartan are required, suggesting that a bidirectional process of cell communication is involved in triggering apoptosis. Evidence showing that activation of the Notch signal transduction pathway is involved in triggering apoptosis of cells misspecified for their dorsal-ventral position (Milán, 2002).

Cells in the wing disc often die in small groups, raising the possibility that death signals may not be targeted precisely at the defective cell. Although the nature of the proposed death signal is not known, the results have implicated activation of the Notch signaling pathway in elimination of cells mispositioned with respect to DV identity. Blocking Notch activation in these cells using the dominant-negative NotchECD receptor or using a dominant-negative form of the Notch effector Mastermind is sufficient to prevent removal of these cells by apoptosis. This indicates that loss of cells is due to activation of the conventional Notch signaling pathway. There is a similar requirement for Notch activation in programmed cell death in the eye imaginal disc. It is clear that Notch signaling is not dedicated to elimination of cells. On the contrary, wing disc cells unable to transduce the Notch signal are lost. Thus, it is evident that Notch signaling is used to cause apoptosis in a specific context, in conjunction with other signals. Cells may die when they receive a combination of signals that indicate incorrect position. Dorsal cells expressing dLMO lack Caps and Tartan, which mediate dorsal cell interactions, as well as Serrate and Fringe, which influence Notch signaling. Restoring either category of cell interaction is sufficient to suppress apoptosis of these cells (Milán, 2002).

A P-element line (P0997) of Drosophila in which the P element disrupts the Drosophila homolog of the Saccharomyces cerevisiae gene APG4/AUT2 was identified during the course of screening for cut (ct) modifiers. The yeast gene APG4/AUT2 encodes a cysteine endoprotease directed against Apg8/Aut7 and is necessary for autophagy. The P0997 mutation enhances the wing margin loss associated with ct mutations, and also modifies the wing and eye phenotypes of Notch (N), Serrate (Ser), Delta (Dl), Hairless (H), deltex (dx), vestigial (vg) and strawberry notch (sno) mutants. These results therefore suggest an unexpected link between autophagy and the Notch signaling pathway (Thumm, 2001).

Notch inhibits yorkie activity in Drosophila wing discs

During development, tissues and organs must coordinate growth and patterning so they reach the right size and shape. During larval stages, a dramatic increase in size and cell number of Drosophila wing imaginal discs is controlled by the action of several signaling pathways. Complex cross-talk between these pathways also pattern these discs to specify different regions with different fates and growth potentials. This study shows that the Notch signaling pathway is both required and sufficient to inhibit the activity of Yorkie (Yki), the Salvador/Warts/Hippo (SWH) pathway terminal transcription activator, but only in the central regions of the wing disc, where the TEAD factor and Yki partner Scalloped (Sd) is expressed. This cross-talk between the Notch and SWH pathways is shown to be mediated, at least in part, by the Notch target and Sd partner Vestigial (Vg). It is proposed that, by altering the ratios between Yki, Sd and Vg, Notch pathway activation restricts the effects of Yki mediated transcription, therefore contributing to define a zone of low proliferation in the central wing discs (Djiane, 2014).

In order to investigate the possibility of cross talk between the Notch and Sav/Warts/Hippo (SWH) pathways, the expression pattern of ex-lacZ, a reporter of Yki activity, which reveals the places where SWH activity is lowest was compared with NRE-GFP, which gives a direct read out of Notch activity. In the wing pouch these reporters direct expression in patterns that are complementary. Thus, ex-lacZ expression is completely absent from the dorso-ventral boundary where Notch activity, reported by NRE-GFP, is at its highest. Conversely, in late stage discs, ex-lacZ expression is higher in pro-vein regions where Notch activity (NRE-GFP) is low. Because ex-lacZ gives a mirror image of SWH activity, these results suggest that both Notch and SWH pathways are active together in the D/V boundary and are largely inactive in the pro-veins (Djiane, 2014).

The consequences of modulating Notch activity on the expression of ex-lacZ was tested as an indicator of its effects on SWH pathway. Expression of Nicd, the constitutively active form of the Notch receptor, promoted a strong down-regulation of ex-lacZ in the wing pouch. This effect was stronger in the region surrounding the D/V boundary and weaker towards the periphery. Little down-regulation occurred outside the pouch. Conversely, when Notch activity was impaired, through RNAi mediated knock-down in randomly generated overexpression clones, ex-lacZ levels were up-regulated. This effect was also only evident within the wing-pouch. Notch activity is therefore necessary and sufficient for the inhibition of ex-lacZ in the wing pouch, suggesting that it contributes to the normal down-regulation of ex-lacZ at the D/V boundary (Djiane, 2014).

In the wing pouch, ex-lacZ expression requires Yki. Therefore, to mediate the observed inhibition of ex-lacZ expression, Notch could either exert its actions upstream of Yki, by activating the SWH pathway, or downstream of Yki, by inhibiting Yki's transcriptional activity. To determine which of these alternatives is correct, the consequences of Notch activity on ectopic Yki expression were assessed. When over-expressed in a stripe of cells along the A/P boundary, Yki was able to promote strong expression of ex-lacZ at the periphery of the wing pouch. Strikingly, the high levels of Yki were not able to force ex-lacZ expression at the D/V boundary where Notch activity is highest. These results suggest that the actions of Notch, ie ex-lacZ down-regulation, are epistatic to Yki. This was further verified when high levels of Yki were expressed together with high levels of Nicd. In this case, Nicd suppressed the ex-lacZ expression, demonstrating that it wins out over Yki in the wing pouch. However, at the periphery of the discs, Nicd was unable to modify the effects of Yki over-expression on ex-lacZ levels. Taken together these results suggest that Notch-mediated down-regulation of ex-lacZ occurs at the level or downstream of Yki (Djiane, 2014).

Since a major output of Notch pathway activity is the up-regulation of gene expression, whether any of the directly regulated Notch target-genes could be responsible for antagonizing Yki was assessed. Amongst the direct Notch targets identified in wing discs, several are predicted to encode transcriptional repressors. These include members of the HES family, E(spl)mβ, E(spl)m5, E(spl)m7, E(spl)m8 and Deadpan (dpn), as well as the homeodomain protein Cut. All of these proteins are normally expressed at high levels along the D/V boundary, in response to Notch activity, and hence are candidates to mediate the repression of ex-lacZ (Djiane, 2014).

Over-expression of E(spl)mβ, E(spl)m5 or E(spl)m7 repressors had no effect on ex-lacZ expression. In contrast, over-expression of either E(spl)m8 or of dpn resulted in a robust down-regulation of ex-lacZ. The effect differed slightly from that from Nicd expression, in that ex-lacZ expression was not completely abolished and low levels persisted throughout the wing pouch domain. These results indicate that a subset of the HES bHLH proteins have the capability to repress ex-lacZ, and hence are candidates to antagonize Yki. Previous experiments have demonstrated that the E(spl)bHLH genes and dpn have overlapping functions, especially at the D/V boundary. Therefore to determine whether these factors normally contribute to the repression of ex-lacZ, it was necessary to eliminate all of the E(spl)bHLH genes in combination with dpn. To achieve this a potent RNAi directed against dpn was expressed in MARCM clones that were homozygous mutant for a deficiency removing the entire E(spl) complex. No derepression of ex-lacZ was detectable in such clones, suggesting that none of the E(spl)bHLH/dpn genes can account for the repression of ex-lacZ at the D/V boundary or in the wing pouch. Therefore even though E(spl)m8 and dpn expression is sufficient for ex-lacZ repression, they do not appear to be essential in the context of the wing pouch (Djiane, 2014).

An alternative candidate was Cut, which encodes a transcriptional repressor and is expressed at the D/V boundary in response to Notch signaling. Similar to some of the HES genes, over-expression of Cut promoted a down-regulation of ex-lacZ. This was most clearly evident at early developmental stages because Cut induced a strong epithelial delamination at later stages, confounding the interpretation. However no up-regulation of ex-lacZ was detectable when Cut function was ablated, using RNAi, even though Cut levels where efficiently reduced. Thus, as with the HES genes, Cut is capable of inhibiting ex-lacZ expression but does not appear to be essential for the regulation of ex under normal conditions in the wing pouch (Djiane, 2014).

Recent studies have demonstrated that, in the absence of Yki, several SWH target genes are kept repressed by Sd, the DNA-binding partner of Yki. This so-called 'default repression' requires Tondu-domain-containing growth inhibitor (Tgi), an evolutionarily conserved tondu domain containing protein, which acts as a potent co-repressor with Sd. There is no evidence that Drosophila tgiM is a target of Notch in the wing disc, making it an unlikely candidate to mediate the inhibitory effects on Yki-mediated ex-lacZ expression. However, vg, which encodes another Sd binding-partner with a tondu domain, is directly regulated by Notch in the wing pouch. It was therefore hypothesized that Vg could mediate the effects of Notch on Yki function and ex-lacZ down-regulation (Djiane, 2014).

In agreement with the hypothesis, when Vg was over-expressed it strongly inhibited ex-lacZ expression in the pouch and promoted a modest overgrowth of the tissue. This overgrowth is somewhat puzzling since it appears that Yki activity, as monitored by ex-lacZ, is lowered in the presence of excess Vg. How over-expressed Vg triggers overgrowth remains poorly understood, but has been proposed to involve a cross-talk with the wg pathway. More recently, it has been shown that the expansion of the pouch region is achieved by Vg activating transiently and non-autonomously Yki in cells not expressing Vg. These cells are then recruited to become wing pouch cells and turn on vg expression. This model predicts a wave of Yki activation around Vg positive cells. Therefore, the overgrowth seen when Vg is over-expressed, could be due to a non-autonomous effect where more cells are recruited as pouch cells at the expense of more peripheral cells. Alternatively, Vg could promote proliferation of the pouch cells by an as yet unidentified mechanism, independent of Yki (Djiane, 2014).

Conversely to over-expressed Vg inhibiting ex-lacZ expression, lowering the levels of vg using RNA interference in the whole posterior compartment resulted in a significant up-regulation of ex-lacZ. Vg knock down has proven difficult to achieve in small populations of cells, due to their elimination from the wing pouch, probably by cell competition. Thus, unlike the other factors tested, Vg is required for the repression of ex-lacZ in the wing pouch. It was further shown that, co-expressing with NICD a vg RNAi transgene in the patched domain, suppresses the NICD mediated ex-lacZ repression in the wing pouch. Taken together, these results suggest that Vg mediates the repressive effects of Notch on expanded expression (Djiane, 2014).

If the involvement of Vg downstream of Notch is a general mechanism for cross-talk between Notch and Yki, other targets of the Sd-Yki complex should be inhibited by Notch in a similar manner to ex-lacZ. However, apart from expanded, all other known Yki targets in the wing pouch, such as thread/DIAP1, diminutive/myc, and Cyclin E are also direct Notch targets. Their final expression patterns are therefore a reflection of the balance between different transcriptional inputs, in particular Notch and Yki. A model predicts that Notch could have a dual effect on the expression of genes: a positive direct effect through the NICD/Su(H) complex when bound in their promoters, but also a negative effect through the induction of Vg, which prevents the positive effect of Yki on Sd bound promoters (Djiane, 2014).

In agreement with this model, thread/DIAP1 and diminutive/myc, two well established Yki targets in wing discs, which are normally refractory to Notch mediated activation in the centre of the pouch, become susceptible to Nicd when Vg or Sd levels are lowered through RNAi (Djiane, 2014).

Focusing on DIAP1, it was decided to separate the Notch and Yki direct inputs on transcription by isolating the Hippo pathway Responsive Elements (HREs) from any potential Notch Responsive Elements (NREs). IAP2B2C-lacZ is a previously described DIAP1-HRE driving lacZ reporter expression that does not contain any NRE, at least based on Su(H) ChIP data and bio-informatics prediction of Su(H) binding sites. The model predicts that this IAP2B2C-lacZ reporter should be inhibited by Vg. In control wing discs, it was confirmed that IAP2B2C-lacZ is expressed at uniform low levels with a slight increase at the periphery of the pouch, where Vg protein levels have been shown to fade. The D/V boundary expression of DIAP1 is not reported by IAP2B2C-lacZ confirming that the NRE is absent in this reporter (Djiane, 2014).

Vg levels were lowered using moderate RNAi knocked down in the whole posterior compartment using the hh-Gal4 driver. In this experimental set-up, the posterior compartment is smaller than normal, and vg knock-down induced a 12% up-regulation of IAP2B2C-lacZ expression when compared to IAP2B2C-lacZ levels in the anterior control compartment, demonstrating that Vg has a negative effect on this reporter activity (there was no difference in IAP2B2C-lacZ expression between the anterior and posterior compartment in the pouch region of control discs). It is noted that IAP2B2C-lacZ expression was up-regulated in a small stripe of cells in the anterior compartment just at the boundary with vg depleted cell. This region was excluded from the quantifications, but suggests that the IAP2B2C-lacZ reporter fragment could be sensitive to a non-autonomous input acting around the boundary of cells with different Vg levels (Djiane, 2014).

It appears therefore, that at least for the two Yki targets ex-lacZ and IAP2B2C-lacZ, Vg inhibits their expression in the wing pouch. Previous studies reported independent roles of Vg and Yki on the activation of their targets, and could appear to contradict this newly described inhibitory role of Vg on Yki targets. However, in these previous studies, it was demonstrated that Vg and Yki do not require each other to promote wing pouch cell survival and to activate their respective targets, which does not rule out any negative cross regulation, as shown in this report (Djiane, 2014).

This analysis brings therefore new evidence of the central role of Vg in the complex network regulating wing disc growth, adding a new level of complexity in its interaction with the SWH pathway effector Yki. Thus, Notch induced expression of Vg could give rise to an Sd-Vg repressive complex that prevents expression of Yki targets. In situations where SWH signaling is lowest, Yki levels may be sufficiently high to overcome this repression. This suggests that in the wing pouch, Notch and SWH would act co-operatively rather than antagonistically (Djiane, 2014).

Outside of the pouch, at the wing disc periphery, sd and vg expressions are not promoted by Notch activity. Furthermore, other binding partners for Yki, such as Homothorax are expressed there and might substitute for Sd to control the expression of Yki targets in a way similar to what has been described in the Drosophila eye. The differential expression of these transcription factors in the disc could explain why Notch only has an inhibitory effect on Yki targets in the wing pouch. Furthermore, it is also worth noting that Notch has very different effect outside of the pouch, where it promotes Yki stabilization non-autonomously via its regulation of ligands for the Jak/Stat pathway (Djiane, 2014).

In summary, the evidence demonstrates that Notch activity can inhibit Yki under circumstances where Yki acts together with Sd. It does so by promoting the expression of Vg, a co-factor for Sd, counteracting the effects of Yki. This cross talk potentially extends to mammalian systems as the active form of NOTCH1, NICD1 promotes the up-regulation of VGLL3 (a human homologue of vg) in MCF-10A breast cancer derived cells. Thus, similar mechanisms may also be important in mediating interactions between the NOTCH and SWH pathways in human diseases (Djiane, 2014).

Because the end-point of SWH pathway activity is to prevent Yki function, the inhibitory effects of Notch on Yki could provide an explanation for those cellular contexts where the two pathways act co-operatively, as at the D/V boundary in the wing discs. Similar co-operative effects have been noted in the Drosophila follicle cells. However, in this case it is the SWH activity that is involved in promoting the expression of Notch targets. In other contexts, such as the mouse intestine, accumulation of Yap1, the mouse Yki homolog, and therefore inhibition of the SWH promotes Notch activity. These examples demonstrate that the interactions between Notch and the SWH are highly dependent on cellular context. The results suggest that some of these differences may be explained by the nature of the target genes that are regulated and by which Yki co-operating transcription factors are present in the receiving cells (Djiane, 2014).

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