Targets of Activity (part 2/3)

Notch regulation of Twist

One of the first steps in embryonic mesodermal differentiation is allocation of cells to particular tissue fates. In Drosophila, this process of mesodermal subdivision requires regulation of the bHLH transcription factor Twist. During subdivision, Twist expression is modulated into stripes of low and high levels within each mesodermal segment. High Twist levels direct cells to the body wall muscle fate, whereas low levels are permissive for gut muscle and fat body fate. Su(H)-mediated Notch signaling represses Twist expression during subdivision and thus plays a critical role in patterning mesodermal segments. This work demonstrates that Notch acts as a transcriptional switch on mesodermal target genes, and it suggests that Notch/Su(H) directly regulates twist, as well as indirectly regulating twist by activating proteins that repress Twist. It is proposed that Notch signaling targets two distinct 'Repressors of twist' - the proteins encoded by the Enhancer of split complex [E(spl)C] and the HLH gene extra machrochaetae (emc). Hence, the patterning of Drosophila mesodermal segments relies on Notch signaling changing the activities of a network of bHLH transcriptional regulators, which, in turn, control mesodermal cell fate. Since this same cassette of Notch, Su(H) and bHLH regulators is active during vertebrate mesodermal segmentation and/or subdivision, this work suggests a conserved mechanism for Notch in early mesodermal patterning (Tapanes-Castillo, 2004).

Analysis of Notch mutant embryos revealed that Notch signaling is essential for Twist regulation at mesodermal subdivision. However, comparison of Notch and Su(H) mutant embryos indicated that Notch regulates Twist differently from Su(H). At stage 10, uniform high Twist expression was maintained in Nnull mutants; by contrast, Su(H)null mutants have a wild-type-like Twist pattern. Furthermore, while constitutive activation of Notch represses Twist expression at stage 10, constitutive expression of a transactivating form of Su(H) [Su(H)-VP16] increases Twist expression. Despite these differences, double mutant analysis and rescue experiments demonstrate that Notch requires Su(H) to repress Twist. Moreover, further rescue experiments show that Notch signaling acts as a transcriptional switch, which alleviates Su(H)-mediated repression and promotes transcription. In addition, genetics, combined with promoter analysis, suggest that Notch and Su(H) have multiple inputs into twist. Notch/Su(H) signaling both directly activates twist and indirectly represses twist expression by activating proteins that repress Twist. Finally, the data indicate that Notch targets two distinct 'Repressors of twist' - E(spl)-C genes and Emc. It is proposed that Notch signaling activates expression of E(spl)-C genes, which then act directly on the twist promoter to repress transcription. Since removing groucho enhances the phenotype of the E(spl)-C mutant embryos, it is suggested that the corepressor, Groucho, acts with E(spl)-C proteins and the Hairless/Su(H) repressive complex to mediate direct repression of twist. The second 'Repressor of twist', Emc, mediates repression of Twist in an alternative fashion. It is hypothesized Emc activity inhibits dimerization of Da with itself or another bHLH protein. This, in turn, prevents Da from binding DNA and activating twist transcription. Since Emc is expressed in the embryo prior to stage 10, it is likely that the transition from uniform high Twist expression to a modulated Twist pattern involves Emc inhibition of Da activity at stage 9. In conclusion, this work uncovers how Notch signaling impacts a network of mesodermal genes, and specifically Twist expression. Given that Notch signaling directs cell fate decisions in many Drosophila embryonic and adult tissues and that Notch regulates Twist in adult flight muscles, these data may suggest a more universal mode of Notch regulation (Tapanes-Castillo, 2004).

The distinct mesodermal phenotypes of Notch and Su(H) mutants can be explained by Notch acting as a transcriptional switch. This aspect of Notch signaling has been described in other systems, and the early Drosophila mesoderm appears no different in this regard. However, these data suggest that there is more to the phenotypes; that is, additional layers of Notch regulation in the transcriptional control of twist (Tapanes-Castillo, 2004).

Genetic experiments, as well as promoter analysis, raised the hypothesis that Notch signaling regulates twist directly, as well as indirectly by activating expression of a 'repressor of twist.' This indirect repression of twist concurs with the role of Notch in activating E(spl) transcriptional repressors. Moreover, a mechanism involving direct and indirect regulation is consistent with Su(H) mutant phenotypes. In Su(H)null embryos, neither twist nor repressor of twist (for example, emc) are repressed. The de-repression of both genes at the same time results in Twist expression appearing 'wild-type-like'. When a constitutively activating form of Su(H) is expressed, both twist and repressor of twist are activated. In these embryos, high Twist domains are expanded, but uniform high Twist expression is not observed because repressor of twist is expressed (Tapanes-Castillo, 2004).

However, simple direct and indirect regulation [through emc and E(spl)-C genes] by Notch still does not fully explain the phenotypes of Notch mutants. Both twist and repressor of twist should be repressed in Nnull embryos because Su(H) will remain in its repressor state. While the Nnull phenotype was consistent with repressor of twist being repressed, twist was still strongly expressed. Additionally, constitutive Notch activation should cause both twist and repressor of twist to be expressed. Consequently, Nintra was expected to cause a phenotype similar to that caused by Su(H)-VP16. Contrary to these predictions, panmesodermal expression of Nintra represses Twist, consistent with only repressor of twist being strongly expressed. Taken together, these results suggested that at stage 10, the twist promoter is less receptive to Notch/Su(H) activation than to Notch/Su(H) repression. As a result, constitutive activation of Notch represses twist, while loss of Notch activates twist ectopically (Tapanes-Castillo, 2004).

Notch regulates cut in trachea

A fundamental question in developmental biology is how tissue growth and patterning are coordinately regulated to generate complex organs with characteristic shapes and sizes. This study shows that in the developing primordium that produces the Drosophila adult trachea, the homeobox transcription factor Cut regulates both growth and patterning, and its effects depend on its abundance. Quantification of the abundance of Cut in the developing airway progenitors during late larval stage 3 revealed that the cells of the developing trachea had different amounts of Cut, with the most proliferative region having an intermediate amount of Cut and the region lacking Cut exhibiting differentiation. By manipulating Cut abundance, it was shown that Cut functioned in different regions to regulate proliferation or patterning. Transcriptional profiling of progenitor populations with different amounts of Cut revealed the Wingless (known as Wnt in vertebrates) and Notch signaling pathways as positive and negative regulators of cut expression, respectively. Furthermore, the gene encoding the receptor Breathless (Btl, known as fibroblast growth factor receptor in vertebrates) was identified as a transcriptional target of Cut. Cut inhibited btl expression and tracheal differentiation to maintain the developing airway cells in a progenitor state. Thus, Cut functions in the integration of patterning and growth in a developing epithelial tissue (Pitsouli, 2013).

Notch and the determination of appendage identity

The Notch signaling pathway defines an evolutionarily conserved cell-cell interaction mechanism that throughout development controls the ability of precursor cells to respond to developmental signals. Notch signaling regulates the expression of the master control genes eyeless, vestigial, and Distal-less, which in combination with homeotic genes induce the formation of eyes, wings, antennae, and legs. Therefore, Notch is involved in a common regulatory pathway for the determination of the various Drosophila appendages (Kurata, 2000).

The intracellular domain of the truncated Notch receptor reflects a constitutively activated state (Notch activated, Nact) and the extracellular domain of the truncated receptor mimics loss-of-function phenotypes representing a dominant negative form (Notch dominant negative, Ndn). To examine the role of Notch signaling in early eye development, these truncated forms were expressed in the early eye imaginal disc, using the GAL4 system with the eye-specific enhancer of the ey gene. This eye-specific enhancer induces target gene expression in the eye primordia of the embryo and maintains expression throughout eye morphogenesis. In contrast to ey expression in the wild-type eye-antennal disc, the enhancer-driven reporter gene expression is not down-regulated in the differentiating cells posterior to the morphogenetic furrow but it extends all over the eye disc and into the area of the antennal disc where the rostral membrane is going to be formed. However, the expression in the antennal disc is quite variable from disc to disc. Consistent with previous loss of Notch function studies, crossing ey enhancer-GAL4 (ey-GAL4) flies to a stock carrying Ndn under an upstream-activating sequence for GAL4 (UAS-Ndn) results in a strongly reduced eye phenotype in all transheterozygous flies similar to that of the ey2 mutant. Inhibition of Notch signaling by misexpression of Hairless (H) and dominant negative forms of Delta (Dl) and Serrate (Ser) also leads to a reduction or complete absence of the eye (Kurata, 2000).

Activation of Notch signaling by crossing ey-GAL4 flies to a UAS-Nact line leads to significant pupal lethality, but all transheterozygotes that escape lethality show hyperplasia of the eyes with a significant increase in the number of facets. The disc overgrowth is found in all eye discs of ey-GAL4 UAS-Nact larvae, consistent with a role for Notch signaling in growth control of the eye imaginal disc. Furthermore, about 16% of the escapers form ectopic eyes on the rostral membrane of the head, which is derived from the antennal disc (Kurata, 2000).

Immunostaining of eye-antennal discs of ey-GAL4 UAS-lacZ UAS-Nact larvae using an ELAV antibody to identify the differentiating photoreceptor cells and a beta-galactosidase antibody to monitor the Nact protein shows that both the strong hyperplasia of the eye disc and ectopic eye formation in the antennal disc correlate with the expression of Nact. However, the time window for expression of the truncated receptors is critical. Transheterozygotes in which either Ndn or Nact were driven by the glass promoter GMR-GAL4, which drives expression in all cells posterior to the furrow only, show only a mild phenotypic effect. Ndn results in a roughening of the eye, whereas Nact produces a polished eye phenotype. Therefore, the timing of Notch signaling is of crucial importance (Kurata, 2000).

The reduced eye phenotype caused by expression of Ndn and the induction of ectopic eyes by the expression of Nact are similar not only to loss-and-gain mutants of ey but also resemble two other mutations acting downstream in the ey developmental pathway, eyes absent (eya) and dachshund (dac). Furthermore, a second Pax-6 gene of Drosophila, twin of eyeless (toy), was found to be an upstream regulator of ey capable of inducing ectopic eyes by inducing ey. To determine the epistatic relationship of Notch to those genes, the effect of Ndn on ectopic eye induction by ey and toy was studied (Kurata, 2000).

A dpp-enhancer GAL4 line (dpp-GAL4) was crossed to flies carrying both UAS-Ndn and UAS-ey or alternatively to UAS-Ndn and UAS-toy. Transheterozygotes from both crosses exhibit ectopic eyes on legs and wings in all flies. The size of the ectopic eyes is similar to those of the transheterozygous controls dpp-GAL4 UAS-ey and dpp-GAL4 UAS-toy, respectively, suggesting that Notch acts upstream of ey and toy. Double immunostaining of eye-antennal discs from transheterozygous ey-GAL4 UAS-Nact, UAS-lacZ using an anti-EY antibody to reveal EY protein and anti-beta-galactosidase antibody to indirectly reveal Nact demonstrates that ey expression is induced in all eye discs by the activation of Notch signaling. Moreover, strong ectopic expression of Ey protein has been observed. The ectopic expression pattern of Ey corresponds to that of lacZ reflecting the expression of Nact protein. Analysis of ey expression by in situ hybridization indicates that ey is induced at the transcriptional level. Similarly, ectopic expression of toy also is induced in the antennal discs of ey-GAL4 UAS-Nact larvae. Thus, activation of Notch signaling can induce toy and ey expression in antennal discs. Expression of Nact also correlates with the ectopic induction of photoreceptor cells as revealed by ELAV staining (Kurata, 2000).

Notch activation of ey and toy depends on the downstream effector of Notch, Suppressor of Hairless, because Su(H) mutant clones generated anterior to the morphogenetic furrow in the eye fail to produce adult structures, in agreement with a requirement for Notch signaling during eye morphogenesis (Kurata, 2000).

Consistent with the finding that ey acts downstream of Notch, the expression of Nact in an ey2 or eyR hypomorphic mutant background generates eyes of a reduced size. Approximately 72% of the ey-GAL4 UAS-Nact; ey2 flies that survived were found to have reduced eyes and about 15% of these flies had both a reduced original and a reduced ectopic eye. However, in addition to ectopic eyes Nact also induces ectopic antennae in 25% of these flies on the side of the head that is derived from the eye disc. Many of the induced ectopic antennae were complete with all three antennal segments and the arista (Kurata, 2000).

Because Distal-less in combination with extradenticle (exd) and homothorax (hth) specifies the antennae, Dll expression was monitored in the eye discs that are capable of forming ectopic antennae. In wild-type larvae, Dll protein is expressed in the antennal but not in the eye disc. In all of the tested discs in ey-GAL4 UAS-Nactey2 animals that form ectopic antennae from the eye disc, significant Dll expression was detected ectopically. This indicates that Notch signaling directly or indirectly induces ectopic expression of Dll in the eye-antennal disc, leading to the ectopic induction of antennae (Kurata, 2000).

The observation that Nact can induce both ectopic eyes and, in a specific genetic background, antennae, led to a consideration of the possibility that Notch signaling also might induce the formation of other appendages in a different genetic context. To test this hypothesis, the activation of Notch signaling was combined with ectopic expression of Antennapedia (Antp). The latter is known to determine the identity of the second thoracic segment (T2), which on the dorsal side gives rise to a pair of wings and on the ventral side to a pair of second legs. For this purpose, transgenic flies of the constitution ey-GAL4 UAS-Nact UAS-Antp were generated. About 26 of the flies escaping pupal lethality were found to have ectopic wings on the head. Almost all ectopic wing structures consisted of dorsal and ventral wing blades bordered by bristles of the wing margin, but lacking wing veins. In contrast, in wing structures induced by the ectopic expression of vg, the wing margin is not formed, suggesting that Notch signaling and Antp are acting upstream of vg. Furthermore, about 17% of these flies show ectopic leg structures induced by secondary transformation of the ectopic antennal tissue into leg structures (e.g., arista into tarsus). Therefore, activation of Notch signaling when combined with the ectopic expression of Antp driven by ey-GAL4 is capable of inducing wing and leg structures on the head (Kurata, 2000).

In wild-type larvae, the vg gene is expressed in the wing but not in the eye disc. By contrast, in ey-GAL4 UAS-Nact UAS-Antp animals in which ectopic wing structures are induced in the eye disc all of the tested eye discs show significant ectopic expression of Vg protein. It therefore appears that activation of Notch signaling in the context of Antp expression induces vg expression in the eye discs and that there are synergistic effects between Notch signaling and Antp expression. Notch signaling pathway has been shown to be used to specifically activate the boundary enhancer of the vg gene necessary for dorso-ventral wing formation. The same enhancer also may be used for ectopic formation of the wing, a point that has to be investigated further. A dorso-ventral boundary also is established by Notch in the eye disc that controls growth and polarity in the Drosophila eye. In ey-GAL4 UAS-Nact UAS-Antp ectopic legs also are induced on the head; this is accompanied by Dll expression (Kurata, 2000).

In view of the above observations it is proposed that the effects of Notch signaling on the various appendages depend on the context provided by control genes such as ey and Antp. In the eye primordia, Notch signaling induces ey expression, which induces a cascade of downstream genes leading to eye morphogenesis. In conjunction with Antp, Notch signaling induces vg, leading to wing formation. At low levels of ey expression, Notch signaling induces Dll, leading to antenna morphogenesis. In the case of the leg, Notch also induces Dll expression that, in conjunction with Antp, leads to leg formation (Kurata, 2000).

Segmental identity is specified by the homeotic genes that are active in a particular combination in each segment. Within a given segment, the appendages are specified by a different set of subsidiary control genes; the eyes are specified by ey, the wings and haltere by vg; the legs by Dll and the antennae by Dll in combination with extradenticle (exd) and homothorax (hth). The results presented here indicate that they all are regulated by Notch signaling and that they share the same cell signaling pathway, which raises the possibility that the appendage specificity is provided by a combinatorial interaction between Notch and the homeotic and the subsidiary control genes (Kurata, 2000).

The repression of one control gene by the expression of another seems to be a widespread mechanism to ensure that the developmental pathways are mutually exclusive so that the formation of intermediary cell types is prevented. Similar to the repression of ey by Antp, ey directly or indirectly represses Dll. In hypomorphic ey mutants, the activation of Notch signaling leads to ectopic expression of Dll in the eye disc, suggesting that ey might repress Dll in the wild-type eye disc. In dpp-GAL4 UAS-ey transheterozygous flies Ey is expressed on the ventral side of the posterior half of the antennal discs under the control of the dpp-enhancer, whereas Dll is not detectable in this area. A similar mutually exclusive expression is found in the leg discs of these flies, suggesting that ey represses Dll expression (Kurata, 2000).

Based on these findings, a model is proposed to explain the difference between the eye and antennal pathway starting from a common signaling mechanism. Notch signaling induces the expression of both ey and Dll. However, in the eye primordia ey represses Dll and induces eye morphogenesis. By contrast, in the antennal disc ey is repressed by a repressor, resulting in Dll expression that confers antennal (ventral appendage) specificity. Two of the possible candidates for the repressor are the homeobox genes exd and hth, because both exd- and hth- mutant clones in the rostral membrane region of the antennal disc can result in ectopic eye development, which presumably is caused by derepression of ey. Both exd and hth also may function in conjunction with Dll, serving as corepressors. The present study extends the fundamental role of Notch by indicating that the implementation of entire developmental programs leading to appendage formation and organogenesis may be controlled by Notch activity (Kurata, 2000).

AP-2 is expressed in the presumptive joints under control of the Notch signaling pathway. AP-2 is required for formation of joints and is sufficient to induce supernumerary joints when ectopically expressed. Unlike Notch mutants, strong AP-2 mutants are viable and produce flies with short legs. The activity of AP-2 in the presumptive joints is required to support survival of cells in the interjoint region. On the basis of clonal analysis it has been inferred that Notch activity in the joints is required for development of the interjoint region and that joints are centers of growth control in the leg. Experiments reported in this study suggest that Notch acts via AP-2 to support survival of cells in the interjoint region of the leg segments. Clonal analysis has shown that AP-2 activity is not required by the interjoint cells themselves, therefore it is suggested that AP-2 might control expression of a secreted factor that is produced by the joint cells and acts non-autonomously to support survival of nearby cells. These observations suggest that AP-2 is an important mediator of Notch signaling in joint formation and leg segment development (Kerber, 2001).

Several observations indicate that AP-2 does not mediate all of the effects on Notch in the leg. (1) AP-2 is not sufficient to induce joints in a Notch mutant leg; (2) other Notch-dependent target genes, including big brain and E(spl) are induced normally in AP-2 mutant leg discs; (3) ectopic activation of the Notch pathway produces supernumerary joints that are often associated with outgrowths of the leg. Like Notch, ectopic AP-2 induces supernumerary joints, but does not cause outgrowths. These observations indicate that AP-2 mediates some, but not all of the activities of Notch in the leg. For example, Fringe is expressed at high levels in the interjoint region. It has been shown that ectopic expression of Fringe can inhibit joint formation. It is possible that the presence of Fringe influences Notch activity to limit joint formation by AP-2. This may provide an explanation for the clustering of ectopic joints when AP-2 is misexpressed (Kerber, 2001).

Ectopic activation of the Notch pathway can induce leg repatterning, outgrowths and ectopic joint structures in tarsal segments. Since AP-2 is required downstream of Notch for joint formation it was asked whether ectopic expression of AP-2 would be sufficient to induce ectopic joints. AP-2 was misexpressed in a stripe of cells along the proximal-distal axis of the leg using the patchedGal4 driver (i.e. crossing the endogenous AP-2 rings). Legs from patchedGal4/UAS-AP-2 flies contain many ectopic joints in the tarsal segments. It was note that AP-2 does not produce the other pattern abnormalities associated with expression of activated Notch. The ectopic joints induced by AP-2 in the tarsal region have wild-type morphology. The supernumerary joints tend to be clustered together and not uniformly distributed along the segment. The significance of this observation is unclear. To ask whether AP-2 is sufficient to mediate all of the activities of Notch in joint formation, UAS-AP-2 was expressed using patchedGal4 in a Notchts mutant background. Under these conditions, joints do not form. This indicates that, while AP-2 is required for joint formation it is not able to induce joints in the absence of Notch activity. To further test the requirement for AP-2, Notchintra was expressed with the dppGal4 driver in AP-2 mutant larvae. In the absence of AP-2 activity, activated Notch is not able to rescue joint formation. Together, these observations indicate that whereas AP-2 expression is regulated by Notch signaling, AP-2 is not the only mediator of Notch activity in joint formation and the requirement for AP-2 function cannot be overcome by constitutive activation of Notch. It is suggested that some other Notch-dependent activity may be required to define a region in which joint formation is possible when AP-2 is expressed (Kerber, 2001).

Bowl is required downstream of Notch for elaboration of distal limb patterning

In the Drosophila leg, activation of Notch leads to the establishment of the joints that subdivide the appendage into segments. Mutations in bowl result in phenotypes similar to Notch, causing fusion and truncations of tarsal segments (tarsomeres). Like its close relative Odd-skipped, Bowl is produced in response to Notch signalling at a subset of segment boundaries. However, despite the fact that bowl mutant clones result in fusion of tarsomeres, Bowl protein is only found at the t1/tibial and t5/pretarsal boundaries, not at tarsomere joints. One hypothesis to reconcile these data is that bowl has a role at an earlier stage in tarsal development. Therefore, the effects were investigated of bowl mutations on the expression of leg 'gap' genes that confer regional identity on the developing leg. Several of these genes have altered expression in bowl mutant cells. For example, bric-a-brac2 is normally expressed in the central part of the tarsus domain but expands into distal and proximal regions in bowl clones. Conversely, ectopic bowl leads to a reduction in bric-a-brac2, with a concomitant expansion of proximal (t1) and distal (t5) tarsomere fates. The bowl gene is therefore required for the elaboration of pattern in the tarsus and its effects suggest a progressive model for the determination of P/D identities. This mechanism might be important in the diversification of arthropod limbs, because it explains how segmented tarsomeres could have arisen from an ancestral limb with an unsegmented tarsus (de Celis Ibeas, 2003).

Given the profound effect of bowl mutants on tarsal segmentation and similarities with Notch phenotypes, it was expected that bowl would be expressed at the sites where Notch is active in the tarsus. Therefore, the expression of bowl was compared with E(spl)mß, a known target of Notch signalling in the leg, using an E(spl)mß-lacZ transgene and an antibody that recognizes Bowl. Although Bowl and ß-galactosidase are clearly co-expressed at some positions, including the t5/pretarsus boundary and the tibia/t1 boundary, Bowl was not detected at sites of Notch activity within the tarsus. Indeed, the distribution of Bowl and Odd appears to be identical and neither is detected at tarsomere boundaries (Rauskolb, 1999). Both are present at all the proximal joints (coxa/femur, femur/tibia, tibia/t1) and at a distal site, the t5/pretarsal boundary (the latter has not previously been documented as a site of Notch activity, although it clearly expresses E(spl)mß and gives rise to an articulated joint). In summary, therefore, Bowl and Odd are present at a subset of the segmental boundaries where Notch is active in the developing leg. These correspond to the boundaries between 'true' segments and not to those between tarsomeres (de Celis Ibeas, 2003).

Expression of the Notch ligands is a key step in regulating Notch activity in the developing leg. To investigate the relationship between Bowl and Notch activity, the timing and distribution of Bowl expression was compared with that of Serrate and Delta, which both regulate Notch activity in the leg disc. By monitoring expression from early third instar, it was found that the evolution of Bowl/odd-lacZ expression closely parallels that of the Notch ligands. The only significant discrepancy appears late in the third instar, when Serrate and Delta are detected at intertarsomere boundaries but Bowl and odd-lacZ are not. Before that stage, Bowl/Odd expression occurs distal to each domain of Delta that is established. For example, the central t5/pretarsal ring of Bowl appears at and correlates with the appearance of Delta in the tarsus and a transient expression of Serrate on the distal, pretarsal, side (de Celis Ibeas, 2003).

Whether Bowl accumulation at segment boundaries depends on Notch activity was tested more directly, by generating clones of Notch mutant cells in the disc epithelium. In all cases in which Notch clones crossed between t5 and the pretarsus, the ring of nuclear Bowl protein at the boundary was interrupted. The effects at the t1/tibia and tibia/femur boundaries were less clear cut, with some clones showing absence or reductions in Bowl, whereas others retained apparently wild-type levels. Many of the last group were small clones (seven cells or less). In converse experiments, expression of a constitutively activated form of Notch (Notchicd) resulted in ectopic Bowl accumulation at a subset of locations in the disc. These broadly correspond to the areas where Bowl is normally detected. Taken together, these data indicate that Bowl is responsive to Notch regulation but that the regulation is limited to a specific time window and/or position. Similar results have been obtained with odd, which is only responsive to Notch in selected regions (de Celis Ibeas, 2003).

Notch function in the wing disc

It has been suggested that wingless expression at the dorsal-ventral boundary of the wing disc depends on a signal from dorsal to ventral cells mediated by Serrate and Notch. Wingless expression is lost from the wing margin and the size of the wing is significantly reduced when Notch activity is removed from the third instar larva using a temperature sensitive allele of Notch. In addition, clones of cells mutant for Serrate can cause extensive non-autonomous loss of wing tissue, but only when the clone includes the cells that abut the dorsal compartment boundary. Therefore, it is likely that wingless is regulated by the Notch pathway acting through Suppressor of Hairless (Diaz-Benjumea, 1995).

Notch signalling regulates veinlet expression and establishes boundaries between veins and interveins in the Drosophila wing. The veins in the Drosophila wing have a characteristic width; this is regulated by the activity of the Notch pathway. The expression of the Notch-ligand Delta (Dl) is restricted to the developing veins, and coincides with places where Notch transcription is lower. The regulation of Notch, Dl and E(spl) expression occurs at the transcriptional level, DL mRNA being detected in the vein and Notch in broad stripes that correspond to the interveins in the third instar discs. The expression of Dl is maintained in pupal wings 24 hours after puparium formation (APF) in dorso-ventral stripes 608 cells wide, with those cells at vein-intervein boundaries accumulating maximal levels of DL mRNA. In contrast, the expression of Notch evolves during pupal development; it is gradually lost from intervein territories during the first 12 hours APF, becoming restricted in pupal wings 24 hours APF to stripes of 2-3 cells wide localized at the vein-intervein boundaries. At this stage, the cells that accumulate high levels of Notch mRNA correspond to those in which Dl expression is maximal. This asymmetrical distribution of ligand and receptor leads to activation of Notch on both sides of each vein within a territory of Delta-expressing cells, and to the establishment of boundary cells that separate the vein from adjacent interveins (de Celis, 1997b).

The modulation (upregulation) of Notch expression at the vein/intervein boundaries is independent of the establishment of veins per se. Expression of E(spl)mbeta is severly reduced in the wing pouch of veinlet vein double mutants, demonstrating that Notch, which normally serves to activate E(spl)mbeta is not activated vein/intervein boundaries. There is also a failure to accumulate Notch in vein/intervein boundaries when Notch signaling is strongly reduced, suggesting that this late expression of Notch depends on Notch signaling (de Celis, 1997b).

In the intervein cells, the expression of the Enhancer of split gene mbeta is activated and the transcription of the vein-promoting gene veinlet is repressed, thus restricting vein differentiation. Notch signaling represses veinlet expression, as hyper-activation of Notch signalling results in the complete repression of veinlet in the imaginal disc. Conversely, reductions in Notch signaling result in an increased number of veinlet-expression cells in vein territories. Expression of Delta depends on the previous specification of veins by Egfr activity. Ectopic expression of veinlet in pupal wings, which serves to enhance Egfr activity, leads to ectopic expression of Delta in similar regions (de Celis, 1997b).

It is proposed that the establishment of vein thickness relies on a combination of mechanisms that include:

Cut might be a target of Notch activity. A number of wing scalloping mutations have been examined to determine their effects on the mutant phenotype of cut mutations and on the expression of the Cut protein. The mutations fall into two broad classes, those which interact synergistically with weak cut wing mutations to produce a more extreme wing phenotype than either mutation alone and those that have a simple additive effect with weak cut wing mutations. The synergistically interacting mutations are alleles of the Notch, Serrate and scalloped genes. These mutations affect development of the wing margin in a manner similar to the cut wing mutations. The mutations inactivate the cut transcriptional enhancer for the wing margin mechanoreceptors and noninnervated bristles and prevent differentiation of the organs. Surprisingly, reduction of Notch activity in the wing margin does not have the effect of converting epidermal cells to a neural fate as it does in other tissues of ectodermal origin. Rather, it prevents the differentiation of the wing margin mechanoreceptors and noninnervated bristles (Jack, 1992).

The role of the Notch and Wingless signaling pathways has been investigated in the maintenance of wing margin identity through the study of cut, a homeobox-containing transcription factor and a late-arising margin-specific marker. By late third instar, a tripartite domain of gene expression can be identified in the area of the dorsoventral compartment boundary, which marks the presumptive wing margin. A central domain of cut- and wingless-expressing cells is flanked on the dorsal and ventral side by domains of cells expressing elevated levels of the Notch ligands Delta and Serrate. Cut acts to maintain margin wingless expression, providing a potential explanation for the cut mutant phenotype. Notch, but not Wingless signaling, is autonomously required for cut expression. Rather, Wingless is required indirectly for cut expression; the results suggest this requirement is due to the regulation by wingless of Delta and Serrate expression in cells flanking the cut and wingless expression domains. Delta and Serrate play a dual role in the regulation of cut and wingless expression. Normal, high levels of Delta and Serrate can trigger cut and wingless expression in adjacent cells lacking Delta and Serrate. However, high levels of Delta and Serrate also act in a dominant negative fashion, since cells expressing such levels cannot themselves express cut or wingless. It is proposed that the boundary of Notch ligand along the normal margin plays a similar role as part of a dynamic feedback loop that maintains the tripartite pattern of margin gene expression (Micchelli, 1997).

Notch function is required at the dorsoventral boundary of the developing Drosophila wing for its normal growth and patterning. Clones of cells expressing either Notch or its ligands Delta and Serrate in the wing mimic Notch activation at the dorsoventral boundary, producing non-autonomous effects on proliferation and activating expression of the target genes E(spl), wingless and cut. The analysis of these clones reveals several mechanisms important for maintaining and delimiting Notch function at the dorsoventral boundary:

Thus the combined effects of Notch and its target genes cut and wingless regulate the expression of Notch ligands, which restricts Notch activity to the dorsoventral boundary (de Celis, 1997c).

Different signals activate separate enhancers to control vestigial expression: initially, in the dorsal/ventral organizer through the Notch pathway, and subsequently, in the developing wing blade by Decapentaplegic, and by a signal from the dorsal/ventral organizer. Signal integration must be a general feature of genes like vestigial, that regulate growth or patterning along more than one axis (Kim, 1996).

The Notch receptor mediates cell interactions controlling the developmental fate of a broad spectrum of undifferentiated cells. By modulating Notch signaling in specific precursor cells during Drosophila imaginal disc development, it has been demonstrated that Notch activity can influence cell proliferation. The activation of the Notch receptor in the wing disc induces the expression of the wing margin patterning genes vestigial and wingless, and strong mitotic activity. However, the effect of Notch signaling on cell proliferation is not the simple consequence of the upregulation of either vestigial or wingless. On the contrary, Vestigial and Wingless display synergistic effects with Notch signaling, resulting in the stimulation of cell proliferation in imaginal discs (Go, 1998a).

To explore the consequences of Notch signaling modulation during Drosophila development, the UAS-GAL4 system was used. Loss-of-function phenotypes were elicited through the expression of either a truncated, dominant negative form of the Notch receptor (d.n.N) lacking the intracellular domain, or the Hairless (H) protein, a negative regulator of Notch signaling. Gain-of-function phenotypes were induced by expressing a constitutively activated form of the Notch receptor (act.N) (Go, 1998a).

To examine the link between the H misexpression phenotypes and Su(H)-dependent Notch activity, transgenic animals were generated carrying a lacZ reporter construct driven by the fusion between multimerized Su(H)-binding sites and an E(spl)m g promoter, a known Su(H) target. This construct consists almost exclusively of engineered Su(H)-binding sites. In a cell culture based reporter, the expression from the reporter construct is induced by the simultaneous expression of Su(H) and act.N, while the expression of any one construct alone fails to induce transcription. Strong lacZ expression is detected in the posterior part of the eye disc of late third instar transgenic larva. This expression is effectively suppressed by misexpression of H using the GAL4 line T113 and results in small eye discs, indicating that H overexpression can suppress Su(H)-dependent Notch signaling in vivo. The size of the eye is significantly affected and, in extreme cases, the eye is missing. In addition to small eyes, small wings and halteres are observed as well as more typical Notch loss-of-function phenotypes, such as extra thoracic bristles. The 'small eye' phenotype induced by H expression is not associated with severe eye roughness. This 'small eye' phenotype, together with the wing and haltere abnormalities, is reminiscent of Serrate loss-of-function mutations. To further explore the possibility that the observed eye phenotype reflects Ser-dependent Notch signaling, the genetic interactions were examined with Beaded of Goldschmidt (BdG), a dominant negative mutation of Ser known to affect wing margin development. In combination with BdG, strong synergistic effects are observed displaying phenotypes characteristic of Ser, such as small eyes, wings and halteres. Therefore, H misexpression can mimic Ser loss-of-function mutations, raising the possibility that Ser/Notch signaling may control eye morphogenesis (Go, 1998a).

To further investigate the role Notch signaling plays in morphogenesis, the H and d.n.N transgenes were expressed at the d/v compartment boundary of the wing disc using the vestigial-GAL4 driver. Misexpression of either H or d.n.N results in similar phenotypes, which range from wing margin notches to rudimentary wings. The effect of H misexpression can be suppressed by expressing act.N and vice versa. For example, the lethality associated with misexpression of act.N is suppressed by simultaneous expression of H. Conversely, the phenotypes elicited by H misexpression are largely suppressed by act.N. This mutual suppression is observed with other GAL4 lines as well. Given that the actions of act.N and H seem to be manifested through Su(H), it is likely that the mutual suppression of act.N and H is also mediated by Su(H). It is noteworthy that, even though both H and d.n.N act as antagonists of Notch signaling and the phenotypes associated with their expression are similar, their interactions with act.N are different. While act.N is an effective suppressor of the phenotypes induced by H misexpression, it fails to suppress the effects of d.n.N (Go, 1998a).

The relationship between Notch signaling and the expression of vg and wg was examined, since the induction of both genes is considered to be essential for wing morphogenesis. When either d.n.N or H is misexpressed along the anterior/posterior (a/p) boundary using the ptc-GAL4 line, expression from the vg d/v boundary enhancer, as well as the wg enhancer, is effectively repressed near the intersection between the a/p and d/v boundaries. In contrast, the vg quadrant enhancer, which is normally silent at the intersection between a/p and d/v boundaries, is induced by the identical constructs. Essentially the opposite effect is observed when act.N is misexpressed, demonstrating that Notch signaling has opposite effects on two distinct enhancers of vg (Go, 1998a).

The wing phenotypes elicited by misexpression of act.N are similar to those induced by Abruptex (Ax) mutations, which are Notch gain-of-function alleles associated with point mutations in the extracellular domain of the protein. A heteroallelic Ax combination results in the activation of Notch signaling and the expression of Notch downstream genes is induced. For instance, ectopic wg expression is found around the d/v boundary. Induction of the vg d/v boundary enhancer and repression of the vg quadrant enhancer around the d/v boundary are found, similar to the effect of expression of act.N. Activation of Notch signaling around the d/v boundary of the wing disc through either misexpression of act.N or the Ax mutations results in a substantial enlargement of the disc. BrdU incorporation experiments indicate that these phenotypes are associated with an elevated mitotic activity. BrdU incorporation is stimulated and is particularly obvious in the peripheral region of the wing pouch, suggesting that the periphery Is more responsive than other regions. Misexpression of act.N in other parts of the wing disc also results in the stimulation of mitotic activity. When act.N is expressed in the wing pouch, the disc grows in such a way that the characteristic folded structures of the wing pouch are pushed to the periphery. Conversely, the same structures are 'pushed' toward the d/v boundary when act.N is expressed in the periphery. When act.N is misexpressed in a discrete pattern in the periphery using the GAL4 line 766, a regional correspondence is observed between Notch signaling activation and high mitotic activity, demonstrating a local effect of Notch activity on cell proliferation in the periphery. However, as is particularly evident when the Notch receptor is activated around the d/v boundary, the region of Notch signaling activation does not coincide with the region of the highest mitotic activity. It is therefore concluded that the effect of Notch signaling on cell proliferation must be indirect (Go, 1998a).

The effect of Notch activity on cell proliferation is not the simple consequence of vg induction Since vg is a direct target of Su(H)-dependent Notch signaling, it is possible that the mitogenic effect of Notch is mediated by the upregulation of vg. In this case, misexpression of Vg would be expected to result in phenotypes similar to those elicited by act.N. Misexpression of act.N in the dorsal side of the wing pouch, using the GAL4 line A9, induces expression from the vg d/v boundary enhancer as well as the wg enhancer The dorsal side of the wing pouch region appears enlarged. In contrast, when Vg is misexpressed in the same region, the dorsal side of the wing pouch becomes much smaller than the ventral side, while wg expression in the periphery of the dorsal side was suppressed. The loss of dorsal wing pouch induced by Vg misexpression is significantly rescued by expressing Wg simultaneously. This is consistent with the notion that the observed phenotype caused by misexpression of Vg is due to the repression of wg, whose expression in the wing pouch is more uniform at earlier stages. Misexpression of Wg alone in the dorsal side, unlike the misexpression of act.N, does not have a significant effect on cell proliferation in the wing pouch. These results indicate that the effect of act.N expression on mitosis is separable from vg induction. In addition, they indicate that Vg is capable of repressing wg expression in the wing pouch, but not at the d/v boundary (Go, 1998a).

Misexpression of Vg compared to act.N has opposite effects in the wing disc. Thus, Vg misexpression in the wing disc induces wg downregulation and small discs. In contrast, misexpression of Vg in the eye discs upregulates wg and results in a clear enlargement of the discs, demonstrating that Vg can either repress or induce wg expression in a context-dependent manner. The observed context-dependent effect of Vg on wg expression raises the possibility that Notch signaling may be capable of modulating the way Vg affects wg expression. This is of particular interest in view of the possibility that Vg does not suppress wg expression at the d/v boundary because of the existing high level of Notch signaling activity. In fact, the simultaneous expression of act.N and Vg reveals a striking synergistic effect on cell proliferation. The most notable effects are in the eye discs, where tissue expressing the two proteins shows striking overgrowth associated with strong wg induction. The other discs are also clearly affected, displaying cellular overgrowth, but the effects are far less dramatic than the eye discs. This overgrowth phenotype is also evident when act.N and Vg misexpression are driven by dpp-GAL4, even though the synergistic effect is less dramatic. In contrast, the effect of misexpression of Vg with dpp-GAL4 on wg induction and cell proliferation in the eye discs is, in some cases, significantly suppressed by simultaneous expression of H. These experiments demonstrate that the proliferative potential of certain tissues can be modulated by the synergistic action of Notch with other genes. Moreover, they identify Notch signaling as an important factor in the way Vg affects wg expression and cell proliferation at the d/v boundary during wing morphogenesis (Go, 1998a).

The expression of vestigial during wing development is regulated through two enhancers: the second intron or boundary enhancer (vgBE), and the fourth intron quadrant enhancer (vgQE). These names reflect the patterns of expression directed by these regulatory regions: vgBE produces a thin stripe over the prospective wing margin, and vgQE produces a pattern in four quadrants that are complementary to the vgBE and which fill in the developing wing blade. Both, vgBE and vgQE, act as integrators of signaling systems that drive wing development and, in this manner, these regulatory regions determine the tempo and the mode of wing development (Klein, 1999 and references).

The vgBE is activated first during the second instar. Its expression pattern is very similar to that of the Vestigial protein at this stage suggesting that, in these early stages, the vgBE is responsible for the complete pattern of vg expression. Deletion analysis of the enhancer reveals two regions essential for its activity: a binding site for Suppressor of Hairless and sequences contained in the first 80 base pairs of the enhancer. In an attempt to map the nature and timing of the inputs into this enhancer, the activity of the wild-type vgBE and of deletions of the two essential regions have been compared during wing development. These results suggest that the cells in which the expression of the vgBE is upregulated at the end of the second instar represent the anlage of the wing and require Notch/Su(H) signaling. These cells are located at the DV interface, on the domain of wg expression and overlap the expression of nubbin. The suggestion that these cells represent the primordium of the wing pouch can explain why a deletion of the vgBE results in the abolition of the development of the wing pouch; in such a mutant, the anlage would never be defined (Klein, 1999).

Notch signaling is also required for the initial activity of the Quadrant Enhancer (vgQE). The activity of the vgQE can be detected first at the beginning of the third instar, several hours after the upregulation of the vgBE, when it closely outlines the realm of the growing wing. This enhancer is only expressed in the growing wing blade and thus provides a unique and most specific marker for wing blade tissue. A variety of experiments have shown that the vgQE receives a negative input from Notch signaling and a positive one from Dpp. The presence of an E(spl)-binding site in the sequence of the vgQE has led to the suggestion that this suppression by Notch is mediated by the E(spl) protein. However, no strong suppression of the activity of the vgQE is found if E(spl)-m8 is ectopically expressed, suggesting that the effect of Notch requires other mediators. Although the vgQE is suppressed in the domain of Notch activity, Notch signaling plays a non-autonomous role in its activation. For example, the vgQE is never active in Serrate (Ser) mutants in which wing development initiates normally but is aborted very early. Ectopic expression of Delta rescues the wing pouch and leads to the activation of the vgQE. Interestingly, this activity arises in regions devoid of Notch signaling. This result suggests that Notch signaling influences the activity of the vgQE in two ways: it represses the activity of the vgQE autonomously but it is also required for its activity in a non-autonomous way (Klein, 1999).

In developing organs, the regulation of cell proliferation and patterning of cell fates is coordinated. How this coordination is achieved, however, is unknown. In the developing Drosophila wing, both cell proliferation and patterning require the secreted morphogen Wingless (Wg) at the dorsoventral compartment boundary. Late in wing development, Wg also induces a zone of non-proliferating cells at the dorsoventral boundary. This zone gives rise to sensory bristles of the adult wing margin. How Wg coordinates the cell cycle with patterning has been investigated by studying the regulation of this growth arrest. Wg, in conjunction with Notch, induces arrest in both the G1 and G2 phases of the cell cycle in separate subdomains of the zone of non-proliferating cells (ZNC). The ZNC is composed of three subdomains, each about four cells wide. Cells in the central domain express wg. This domain is flanked by dorsal and ventral domains, which, in the anterior compartment, express Achaete and Scute. Cells in the ZNC stop proliferating 30 h before most of the other cells in the disc but re-enter the cell cycle for two or three divisions after pupariation. This arrest is seen by an absence of cells in the S phase of mitosis. The domain architecture of the ZNC is suggested by the expression of string and the G2 cyclins A and B. In the anterior compartment, cells in the dorsal and ventral domains do not express STG messenger RNA but accumulate high leves of G2 cyclins in the cytoplasm. Since Stg is required for mitosis and Stg and the G2 cyclins are degraded at cell division, these patterns are indicative of arrest in G2. In contrast, in the central domain CycA and CycB proteins are undetectable, but STG mRNA is expressed. This indicates that these cells may be arrested in G1. G1 arrest may be due to inactivation of dE2F, a factor required to activate the transcription of genes needed for DNA replication (Johnston, 1998).

Loss of wingless function during disc development abolishes both the G1 and G2 arrests and allows string expression in the anterior dorsal and ventral domains. Four observations suggest that the proneural genes achaete and scute regulate the G2 arrest of the ZNC:

  1. ac and sc are expressed in the stg-negative, G-2 arrested cells, but not in G1-arrested cells.
  2. Loss of Wg activity results in loss of expression of ac and sc.
  3. Ectopic Wg or activated Arm expands the domain of stg-negative, Ac-positive cells.
  4. Expression of ac and sc in the ZNC is extinguished just before re-entry into the cell cycle, after pupariation.

Together, these results indicate that Wg induces G2 arrest in two subdomains by inducing the proneural genes achaete and scute, which downregulate the mitosis-inducing phosphatase String (Cdc25). Notch activity creates a third domain by preventing arrest at G2 in wg-expressing cells, resulting in their arrest in G1. To test whether Notch directly regulates the G1 arrest, discs were constructed lacking Wg activity, but expressing activated Notch in the ZNC. These discs do not form a ZNC at all. Thus, in the absence of Wg activity, Notch is not sufficient to induce a G1 arrest. It is noted that the string promoter contains putative Ac/Sc-binding sites, indicating that these basic helix-loop-helix proteins can repress string expression directly (Johnston, 1998).

Pattern formation during animal development is often induced by extracellular signaling molecules, known as morphogens, which are secreted from localized sources. During wing development in Drosophila, Wingless (Wg) is activated by Notch signaling along the dorsal-ventral boundary of the wing imaginal disc and acts as a morphogen to organize gene expression and cell growth. Expression of wg is restricted to a narrow stripe by Wg itself, repressing its own expression in adjacent cells. This refinement of wg expression is essential for specification of the wing margin. A homeodomain protein, Defective proventriculus (Dve), mediates the refinement of wg expression in both the wing disc and embryonic proventriculus, where dve expression requires Wg signaling. These results provide evidence for a feedback mechanism that establishes the wg-expressing domain through the action of a Wg-induced gene product (Nakagoshi, 2002).

The expression pattern of dve raises the possibility that repression of dve along the D-V boundary is mediated by N signaling, which plays a pivotal role in the activation of margin-patterning genes, such as wg and cut. To examine this possibility, the N signal was ectopically activated along the A-P boundary by expressing N ligands Delta (Dl) and Serrate (Ser) under the control of patched (ptc)-Gal4. Dl and Ser trigger N signaling in the dorsal half and the ventral half of wing discs, respectively. In ptc-Gal4/UAS-Ser discs, N signaling is ectopically activated along the ventral A-P boundary and results in the ventral expression of the margin-patterning genes wg and cut. In these discs, dve expression was found to be repressed along the ventral A-P boundary. In ptc-Gal4/UAS-Dl discs, N signaling is ectopically activated along the dorsal A-P boundary, leading to dorsal dve suppression. Thus, the ectopic N signal can repress dve expression. In order to determine whether or not N indeed suppresses Dve expression along the D-V boundary of wing discs, mosaic clones were generated lacking N activity. The loss of N activity results in the ectopic activation of Dve in the region where Dve expression is normally suppressed along the D-V boundary. These results indicate that N signaling represses dve along the D-V boundary of wing discs to create a domain in which Dve is absent and wg is activated (Nakagoshi, 2002).

Wg signaling appears to be necessary for dve repression along the D-V boundary. The action of Wg, which is up-regulated by N at the D-V boundary, might explain N-mediated repression of dve. To examine the cell autonomy for N-mediated repression of dve, dve expression was examined in N mutant clones at later stages. N mutant clones crossing the D-V boundary cause the derepression of Dve, with varying levels of dve expression within clones. This suggests that there is some nonautonomous effect on dve repression. Mutant mosaic clones were generated for zeste-white 3 (zw3), in which Wg signaling is constitutively active. Partial repression of Dve was observed in zw3 mutant clones at early to mid-third instar. At later stages, when N signaling is strongly activated along the D-V boundary, ectopic Dve repression in zw3 mutant clones is more evident outside the D-V boundary. N-mediated dve repression thus depends largely on Wg signaling that is activated by N. At the late third instar, expansion of Dve repression at the distal region also depends on Wg and Dpp signaling. Inhibition of these signals by expressing a dominant-negative form of Wg signaling molecule (dTCFDN) or a negative regulator for Dpp signaling (dad) along the A-P boundary results in elevated expression of Dve. These results support the notion that N-mediated repression of Dve has a nonautonomous effect, although Wg signaling alone is insufficient for complete repression at early stage. It is inferred that N-mediated events along the D-V boundary modulate the Wg and Dpp signaling, or another secreted signaling molecule, such as Spitz, might be involved in dve repression because the Spitz ligand is up-regulated along the D-V boundary. Indeed, inhibition of EGF signaling by expressing a dominant-negative form of Drosophila EGF receptor along the A-P boundary results in derepression of Dve (Nakagoshi, 2002).

N-mediated activation of wg together with Vg function is important for disc growth. In addition, repression of dve at the D-V boundary largely depends on N-mediated Wg signaling and is also crucial for disc outgrowth and patterning of wing discs. Complementary pattern of dve and wg expression at mid- to late third instar appears to be important for wing patterning. How do these events organize growth and patterning? Studies involving flip-out Nact clones might provide an insight in this issue. These experiments suggested that the two types of Nact clones arise from a difference in the level of N signaling within clones: lower N signaling-clones express dve but not wg and cut, and higher N signaling-clones express wg and cut but not dve. The second type of clone appears to mimic the situation at the D-V boundary. It is remarkable that the on and off states of dve expression within the clones are tightly correlated with the induction of N-target gene expression. Considering the ability of Dve to repress wg, this observation makes it possible to hypothesize a threshold of N-mediated signaling that defines both wg activation and subsequent dve repression; N-mediated signaling over this threshold can repress dve and results in the sustained expression of wg. Thus, it establishes a complementary pattern of dve and wg expression at the D-V boundary of wing discs at mid- to late third instar. This threshold also appears to define nonautonomous induction of cell growth. By utilizing the cold-sensitivity of Gal4 to drive gene expression, different levels of N signaling were induced. These experiments also suggest the notion that the level of N signaling that represses dve is important for disc outgrowth. Thus, the model assuming a threshold of the N-mediated signal repressing dve might provide a clue for understanding the coordination between cell growth and patterning through shaping of the Wg stripe (Nakagoshi, 2002).

During Drosophila wing development, Hedgehog (Hh) signaling is required to pattern the imaginal disc epithelium along the anterior-posterior (AP) axis. The Notch (N) and Wingless (Wg) signaling pathways organise the dorsal-ventral (DV) axis, including patterning along the presumptive wing margin. A functional hierarchy of these signaling pathways is described that highlights the importance of the competing influences of Hh, N, and Wg in establishing gene expression domains. Investigation of the modulation of Hh target gene expression along the DV axis of the wing disc has revealed that collier/knot (col/kn), patched, and decapentaplegic are repressed at the DV boundary by N signaling. Attenuation of Hh signaling activity caused by loss of fused function results in a striking down-regulation of col, ptc, and engrailed (en) symmetrically about the DV boundary. This down-regulation depends on activity of the canonical Wg signaling pathway. It is proposed that modulation of the response of cells to Hh along the future proximodistal (PD) axis is necessary for generation of the correctly patterned three-dimensional adult wing. These findings suggest a paradigm of repression of the Hh response by N and/or Wnt signaling that may be applicable to signal integration in vertebrate appendages (Glise, 2002).

Short-range Hh signaling, partly through activation of Col function, is essential for correct AP patterning and differentiation of L3-L4 intervein tissue. N and Wg first define the DV boundary and later subdivide the region near this boundary into a number of distinct subregions that will eventually differentiate into wing margin bristles and vein tissue. These signals overlap spatially and temporally and lead to opposite fates. It is proposed that in and close to the DV boundary, N, Wg, and Hh signaling exist in a delicate balance to allow vein tissue, bristle, and sensory organ differentiation along the adult wing margin (Glise, 2002).

The Hh target genes col/kn and ptc, in contrast to en, are repressed in a wild type wing in cells corresponding to the presumptive wing margin. It has been demonstrated, using both gain- and loss-of-function experiments, that this repression is mediated by N signaling and that its inhibition results in aberrant morphogenesis of the wing. Hh signaling, achieved either by overexpression of Hh or loss of Ptc activity, is not sufficient to give maximum activation of Hh targets in cells of the prospective wing margin, suggesting that a finely tuned balance of activation and repression is required to achieve the appropriate biological output. However, overexpression of a stabilized form of Ci under the ptc-Gal4 driver results in the activation of Col in the prospective wing margin and defects in wing margin differentiation, indicating that N repression can be overcome by hyperactivity of the Hh signaling pathway. N signaling may lead to the repression of col, ptc, and dpp directly or it may act indirectly by affecting the ability of Ci to act as a transcriptional activator. Since expression of en, which requires the highest level of Hh signaling and Ci activity, appears immune to N repression, the former possibility is favored (Glise, 2002).

The Drosophila wing is a classical model for studying the generation of developmental patterns. Previous studies have suggested that vein primordia form at boundaries between discrete sectors of gene expression along the antero-posterior (A/P) axis in the larval wing imaginal disc. Observation that the vein marker rhomboid (rho) is expressed at the center of wider vein-competent domains led to the proposal that narrow vein primordia form first, and produce secondary short-range signals activating provein genes in neighboring cells. This study examined how the central L3 and L4 veins are positioned relative to the limits of expression of Collier (Col), a dose-dependent Hedgehog (Hh) target activated in the wing A/P organizer. rho expression is first activated in broad domains adjacent to Col-expressing cells and secondarily restricted to the center of these domains. This restriction, which depends upon Notch (N) signaling, sets the L3 and L4 vein primordia off the boundaries of Col expression. N activity is also required to fix the anterior limit of Col expression by locally antagonizing Hh activation, thus precisely positioning the L3 vein primordium relative to the A/P compartment boundary. Experiments using Nts mutants further indicate that these two activities of N can be temporally uncoupled. Together, these observations highlight new roles of N in topologically linking the position of veins to prepattern gene expression (Crozatier, 2003).

In the Drosophila wing, L3 vein is decorated with campaniform sensory organs (CS). Formation of the L3 vein and sensory organs is generally thought to be topologically linked through expression of rho and the proneural genes ac and sc in overlapping A/P positions in third instar wing discs. In wild-type wings, CS overlap the posterior-most row of L3 vein cells. In heterozygous N55 mutant wings, veins are wider than in wild-type, due to defective partitioning of the provein into vein and intervein cells, but selection of sensory organ precursors (SOPs) is not affected. It was observed, however, that CS still overlap the posterior-most L3 vein cells, unlike what would be predicted if widening of L3 vein due to defective vein resolution were centered over its initial coordinate position. The position of SOP relative to the A/P border is not changed in N mutant discs, indicating that in the adult, the position of L3 vein is shifted anteriorly by one or two rows of cells. In order to determine whether this shift and the defect in vein resolution are coupled, the temperature-sensitive allele Nts2 was used. When Nts2 mutants are shifted to restrictive temperature between 104 and 128 h AEL [8-32 h after puparium formation (APF)], the L3 vein is broader, indicative of abnormal vein resolution, but, unlike N55/+ wings, the CS are now centered over the broader vein. Reciprocally, when a transient temperature shift is applied at 60-80 h AEL (mid-second/mid-third larval stages), the L3 vein retains a wild-type width but, as in N55/+, its position is shifted anteriorly relative to the CS. Nts mutant analysis thus reveals a new role of N in positioning the L3 vein relative to the A/P border, temporally uncoupled from its known role in partitioning provein into vein and intervein cells at the pupal stage. This has led to a detailed investigation of the molecular mechanisms involved in positioning L3 vein (Crozatier, 2003).

Longitudinal vein primordia can be visualized in third instar larval wing discs as a series of stripes of cells expressing provein genes, alternating with domains of D-SRF expression. col activates D-SRF expression in A/P organizer cells and positions L3 vein by limiting L3 vein competence to cells expressing iro-C but not col. Therefore col transcription was examined in N55/+ third instar wing discs; it is expanded toward the anterior by one to two rows of cells. The position of the SOPs were examined, using a neuralised (neu)-lacZ reporter gene (transgenic line A101). Whereas in wild-type, one row of cells separates SOPs from the anterior limit of Col expression, SOPs are found immediately adjacent to cells expressing high levels of Col protein in N55/+ discs. Counterstaining of discs with propidium iodide (which labels all nuclei) confirms that the position of SOPs relative the A/P border (anterior limit of hh/posterior limit of Col expression) is unchanged, leading to the conclusion that reducing N activity in third instar larvae specifically results in anterior expansion of Col expression. Col expression was then examined in clones of N mutant cells generated in a heterozygous N55/+ background and spanning the A border of Col expression; it was found not to be expanded further anteriorly. col expression is established in response to Hh in a dose-dependent manner. The present data indicate that: (1) only one or two rows of cell activate col in response to Hh in the absence of Notch signaling, and (2) the same expansion on col expression results from complete absence of N or 2-fold reduction of N signaling suggesting that col expression is very dose sensitive. Thus, the expansion of col expression observed in N55/+ discs indicates that N signaling locally antagonizes Hh activation of col transcription, to precisely position the posterior limit of the L3 vein primordium. Repression of col transcription by Notch signaling has already been reported in formation of an embryonic muscle and at the wing margin but the molecular mechanisms underlying this expression remain to be determined. iro-C expression is also expanded anteriorly in late 3rd instar larval discs in N55/+ mutants, indicating that the entire L3 vein-competence domain is shifted anteriorly. The opposite, posterior shift of iro-C expression (and consequently L3 vein position) observed in col mutant discs (this correlates with the posterior shift of L3 vein observed in these mutants) is linked to the modified range of Dpp signaling resulting from lack of col activity. Similarly, it is proposed that the anterior expansion of iro-C expression in N55/+ mutant discs reflects a modified range of Dpp signaling induced by anterior extension of Col expression. Thus, in wild-type discs the cross-regulation between Hh, N and Dpp signalling allows the positioning of the L3 vein primordium in register with CS. Next, the question of the relation between the A and P boundaries of Col expression and positions of L3 and L4 veins versus proveins was addressed (Crozatier, 2003).

The current view is that vein primordia form at borders between adjacent A/P sectors of gene expression. According to this view, and based on col expression and requirement in cells along the A/P compartment boundary, the L4 and L3 vein primordia are predicted to edge the Col-expressing domain. It was observed, however, that in late 3rd instar wing discs, the col expression domain is not directly flanked by rho-expressing cells, but is separated from them by one to two rows of cells expressing neither gene and expressing Dl. This led to an examination at an earlier stage. In mid-third instar larvae, the col and rho expressing domains are immediately adjacent to one another, suggesting that the vein-centered-over-provein pattern is established secondarily as the disc continues to grow in size due to cell proliferation. Contrary to wild-type, in N55/+ mutant discs, col and rho expression domains remain juxtaposed, correlating with an increased number of rows of rho-expressing cells. These observations indicate that Notch signaling is involved in restricting rho expression and EGFR signaling to single rows of cells at the center of provein domains, probably via lateral inhibition. This process therefore operates already in mid-third instar larvae and results in a displacement of one to two cells between the positions of L3 and L4 vein primordia and the boundaries of Col expression. This displacement offers an explanation for the observation that the adult L4 vein is separated by several rows of intervein cells from the A/P compartment boundary. Although rho expression at the center of proveins in late third instar larvae likely prefigures the position of adult veins, the provein into vein and intervein resolution process can be initiated or, conversely abort later during pupal development, as shown by analysis of various mutants including Nts mutants. While consonant with the view that different A/P boundaries of prepattern gene expression in the wing primordium define the positions where provein domains are specified, the data do not support the suggestion that secondary short-range signals organize proveins around vein primordia. They rather support a sequential induction mechanism in which activation of the EGFR pathway defines vein-competent groups of cells in early 3rd instar larvae as well as promote the expression of Dl; in turn, Dl activation initiates lateral inhibitory signaling and restricts EGFR signaling to cells at the center of vein-competent domains, through a feed-back regulatory loop requiring Notch. This mechanism is consistent with the loss of the L3 and L4 stripes of Dl expression in rhove vn1 (vn, an EGFR ligand) mutants, indicating that Dl expression is dependent on EGF-R signaling. A similar EGFR-->Dl sequential induction model has recently been proposed to operate in differentiating photoreceptor cells in the developing eye of Drosophila. In conclusion, these observations highlight the importance of cross-talk between the Hh and N signaling pathways in assigning overlapping A/P positions to the L3 vein and associated sensory organs and the role of N in precisely positioning vein primordia, thus intimately linking prepattern to the vein resolution process (Crozatier, 2003).

In Drosophila, muscles attach to epidermal tendon cells are specified by the gene stripe (sr). Flight muscle attachment sites are prefigured on the wing imaginal disc by sr expression in discrete domains. The mechanisms underlying the specification of these domains of sr expression have been examined. The concerted activities of the wingless (wg), decapentaplegic (dpp) and Notch (N) signaling pathways, and the prepattern genes pannier (pnr) and u-shaped (ush) establish domains of sr expression. N is required for initiation of sr expression. pnr is a positive regulator of sr, and is inhibited by ush in this function. The Wg signal differentially influences the formation of different sr domains. These results identify the multiple regulatory elements involved in the positioning of Drosophila flight muscle attachment sites (Ghazi, 2003).

The role of N as a potential regulator of sr was examined, since it is known to influence multiple events in wing disc morphogenesis from proliferation to bristle patterning. Using a temperature sensitive allele (Nts), the protein function was inactivated by growing animals at non-permissive temperatures during the third larval instar. sr expression was examined at 0 h APF. Loss of sr expression is observed in these animals. In hemizygous males, this effect is most severe and sr expression is completely abolished. Females, with one normal copy of N, showed faint sr expression. This suggested that N may be required for initiation of sr expression. A dominant negative form of N (Ndn), was expressed in the pnr domain; abolition of sr expression was found. This was observed most clearly in the anterior medial domain covered by pnr. The lateral domains showed some reduction in sr as well. In a gain of function experiment, a constitutively active form of N (Nintra) was expressed in the same region and results in an increase in sr-lacZ. The N ligand Ser is known to regulate sr expression in the embryonic segment border cells. Mis-expression of Ser in the presumptive notum region resulted in loss of sr expression. Together, these results show that the initiation of sr expression relies on N, which is antagonized by Ser in this activity (Ghazi, 2003).

Notch-mediated repression of bantam miRNA contributes to boundary formation in the Drosophila wing

Subdivision of proliferating tissues into adjacent compartments that do not mix plays a key role in animal development. The Actin cytoskeleton has recently been shown to mediate cell sorting at compartment boundaries, and reduced cell proliferation in boundary cells has been proposed as a way of stabilizing compartment boundaries. Cell interactions mediated by the receptor Notch have been implicated in the specification of compartment boundaries in vertebrates and in Drosophila, but the molecular effectors remain largely unidentified. This study presents evidence that Notch mediates boundary formation in the Drosophila wing in part through repression of bantam miRNA. bantam induces cell proliferation and the Actin regulator Enabled was identified as a new target of bantam. Increased levels of Enabled and reduced proliferation rates contribute to the maintenance of the dorsal-ventral affinity boundary. The activity of Notch also defines, through the homeobox-containing gene cut, a distinct population of boundary cells at the dorsal-ventral (DV) interface that helps to segregate boundary from non-boundary cells and contributes to the maintenance of the DV affinity boundary (Becam, 2011).

Cell divisions lead to cell rearrangements that may challenge straight and sharp compartment boundaries. The DV boundary of mid- and late third instar wing primordia is characterized by a reduced rate of cell proliferation which defines the zone of non-proliferating cells (ZNC). The contribution of the ZNC to the maintenance of the DV affinity boundary was proposed many years ago but this notion was subsequently questioned. This study provides evidence that the ZNC does indeed play a role in boundary formation. bantam miRNA positively modulates the activity of the E2F transcription factor and drives G1-S transition in Drosophila tissues. Notch-mediated downregulation of bantam miRNA defines the ZNC and contributes to maintain a stable DV affinity boundary. Induction of proliferation in boundary cells by the ectopic expression of bantam, the cell cycle regulators Cyclin E and String, or the proto-oncogene dMyc, which is known to drive G1-S transition, compromises the formation of a smooth DV affinity boundary. A similar reduction in proliferation rates is observed at the rhombomere boundaries in the developing hindbrain, suggesting that reduced rate of cell proliferation might often be used in compartment boundary formation (Becam, 2011).

Notch-mediated downregulation of bantam activity is not only required to define the ZNC but also to establish the actomyosin cables observed at the interface between boundary and non-boundary cells. Ena, a regulator of Actin elongation, was identified as a direct target of bantam that is involved in DV boundary formation. The multiple roles of bantam in promoting G1-S transition and tissue growth, blocking apoptosis and regulating Actin dynamics unveil a new molecular connection between these three processes that might have relevance in growth control and tumorigenesis (Becam, 2011).

Intriguingly, bantam miRNA has no major role in the maintenance of the anterior-posterior compartment boundary of the developing wing and this boundary is not affected upon depletion of Ena protein levels. Thus, different regulators of actin elongation might be at work to regulate the actomyosin cytoskeleton and direct cell sorting in diverse developmental contexts. Whether reduced levels of bantam miRNA and increased levels of Ena protein are required to maintain differential cell sorting in the embryonic ectoderm or other imaginal tissues remains to be elucidated (Becam, 2011).

Cut is a late target of Notch that is expressed in boundary cells and is required to induce a stable Notch signaling center. This study demonstrate that Cut activity has also a specific function in reducing Ena mRNA and protein levels in boundary cells. Although depletion of Cut compromises the formation of the actomyosin cables at the interface of boundary and non-boundary cells and the maintenance of a stable DV affinity boundary, cell lineage and clonal analysis of wild-type and cut mutant cells reveal that Cut plays a major role in sorting boundary from non-boundary cells. The finding that the Notch signaling pathway defines, through Cut, a distinct population of boundary cells at the DV interface reinforces the mechanistic similarities in the maintenance of compartment boundaries within the vertebrate hindbrain and the Drosophila wing. In both developmental contexts, Notch defines a distinct population of boundary cells and contributes to segregating boundary from non-boundary cells. Although Cut mediates the role of Notch in the Drosophila wing, the molecular effectors mediating the role of vertebrate Notch in boundary formation remain uncharacterized. The data indicate that the later subdivision into boundary and non-boundary cells contributes to the maintenance of a stable DV affinity barrier in the mature wing primordium (Becam, 2011).

Regulation of cell growth by Notch signaling and its differential requirement in normal vs. tumor-forming stem cells in Drosophila

Cancer stem cells (CSCs) are postulated to be a small subset of tumor cells with tumor-initiating ability that shares features with normal tissue-specific stem cells. The origin of CSCs and the mechanisms underlying their genesis are poorly understood, and it is uncertain whether it is possible to obliterate CSCs without inadvertently damaging normal stem cells. This study shows that a functional reduction of eukaryotic translation initiation factor 4E (eIF4E) in Drosophila specifically eliminates CSC-like cells in the brain and ovary without having discernable effects on normal stem cells. Brain CSC-like cells can arise from dedifferentiation of transit-amplifying progenitors upon Notch hyperactivation. eIF4E is up-regulated in these dedifferentiating progenitors, where it forms a feedback regulatory loop with the growth regulator dMyc to promote cell growth, particularly nucleolar growth, and subsequent ectopic neural stem cell (NSC) formation. Cell growth regulation is also a critical component of the mechanism by which Notch signaling regulates the self-renewal of normal NSCs. These findings highlight the importance of Notch-regulated cell growth in stem cell maintenance and reveal a stronger dependence on eIF4E function and cell growth by CSCs, which might be exploited therapeutically (Song, 2011).

The differential cell growth rates observed between ectopic NBs and normal or primary NBs and the correlation between cell growth defects and NB fate loss prompted a test of whether slowing down cell growth might selectively affect the formation of ectopic NBs. Attenuation of TOR signaling, a primary mechanism of cell growth regulation, through NB-specific overexpression of TSC1/2, a strong allele of eIF4E antagonist 4EBP [4EBP(LL)s], or a dominant-negative form of TOR (TOR.TED) all partially suppressed ectopic NB formation in α-adaptin (ada) mutants without affecting normal or primary NBs. Interestingly, RNAi-mediated knockdown of eIF4E, a stimulator of oncogenic transformation and a downstream effector of TOR signaling, showed a better suppression than manipulating other TOR pathway components, suggesting that eIF4E might play a more important role in ectopic NB formation. Strikingly, the brain tumor phenotypes caused by overactivation of N signaling - as in lethal giant larvae (lgl) mutant, aPKCCAAX overexpression, or N overexpression conditions - were also fully suppressed by eIF4E knockdown. Furthermore, the brain tumor phenotypes of brat mutants were also completely rescued by eIF4E RNAi (Song, 2011).

In contrast, normal NB formation or maintenance was not affected by eIF4E knockdown. NBs with eIF4E knockdown remained highly proliferative, as evidenced by the mitotic figures, and displayed relatively normal apical basal cell polarity. There are several other eIF4E-like genes in the fly genome (Hernandez, 2005), which may play partially redundant roles in normal NB maintenance. eIF4E knockdown appeared to specifically block ectopic NB formation caused by the dedifferentiation of IPs in type II NB lineages, since it did not affect ectopic type I NB formation in cnn or polo mutants that are presumably caused by symmetric divisions of type I NBs. In addition, cell fate transformation induced by N overactivation in the SOP lineage was not affected by eIF4E RNAi, supporting the idea that eIF4E is particularly required for type II NB homeostasis. Supporting the specificity of the observed eIF4E RNAi effect, another eIF4E RNAi transgene (eIF4E-RNAi-s) also prevented ectopic NB formation. Moreover, a strong loss-of-function mutation of eIF4E also selectively eliminated ectopic NBs induced by N overactivation without affecting normal NBs, reinforcing the hypothesis that ectopic NBs exhibit higher dependence on eIF4E (Song, 2011).

To further support the notion that the ectopic NBs are particularly vulnerable to eIF4E depletion, a conditional expression experiment was carried out in which eIF4E-RNAi-s was turned on in brat mutants using the 1407ts system, after ectopic NBs had been generated. Whereas the brain tumor phenotype exacerbated over time in the brat mutants, 1407-GAL4-driven eIF4E-RNAi-s expression in brat mutants effectively eliminated ectopic NBs, leaving normal NBs largely unaffected (Song, 2011).

In normal type II NB lineage, eIF4E protein was enriched in the NBs. Ectopic NBs induced by N overactivation in ada mutants also expressed eIF4E at high levels, whereas spdo mutant NBs exhibited reduced eIF4E expression. Thus, eIF4E up-regulation correlates with N-induced ectopic NB formation in a dedifferentiation process that likely involves elevated cell growth (Song, 2011).

Given the coincidence of nucleolar size change with ectopic NB formation, the involvement of the growth regulator dMyc was tested. dMyc protein levels were up-regulated in normal or N overactivation-induced ectopic NBs, but were down-regulated in spdo mutant NBs. Furthermore, dMyc transcription, as detected with a dMyc-lacZ transcriptional fusion reporter, was also up-regulated in both normal and ectopic NBs in ada mutants. A previous study in Drosophila S2 cells identified dMyc as a putative N target. In vivo chromatin immunoprecipitation (ChIP) experiments were carried out to assess whether dmyc transcription is directly regulated by N signaling in NBs. Using chromatin isolated from wild-type larval brains and a ChIP-quality antibody against the N coactivator Suppressor of Hairless [Su(H)], specific binding was demonstrated of Su(H) to its putative binding sites within the second intron of dmyc (dmyc-A). No binding to an internal negative control region proximal to the first exon of dmyc (dmyc-B) or to the promoter region of the rp49 gene was detected. N signaling thus directly activates dMyc transcription in the NBs. Similar to eIF4E RNAi, knockdown of dMyc strongly suppressed ectopic NB formation induced by Brat or Ada inactivation or N overactivation. Intriguingly, the strong tumor suppression effect of eIF4E knockdown was partially abolished by dMyc overexpression. Furthermore, dMyc function, as reflected by its promotion of nucleolar growth in IPs, was attenuated by eIF4E RNAi, although eIF4E RNAi alone had no obvious effect. Different from the reported eIF4E regulation of Myc expression in mammalian cells (Lin, 2008), dMyc promoter activity or protein levels remained unaltered under eIF4E RNAi conditions, suggesting that eIF4E may modulate dMyc activity without altering its expression. One possibility is that eIF4E may enter the nucleus to interact with Myc and promote its transcriptional activity. To test this hypothesis, HEK293T cells were transfected with Flag-tagged human eIF4E alone or in combination with HA-tagged dMyc. Indeed, both Drosophila dMyc and endogenous human c-Myc specifically coimmunoprecipitated with human eIF4E from nuclear extracts, indicating a conserved interaction between eIF4E and Myc within the nuclei of proliferating cells. Consistent with these biochemical data, dMyc transcriptional activity within NBs, which could be monitored with an eIF4E-lacZ reporter, was drastically reduced upon eIF4E knockdown (Song, 2011).

In contrast, eIF4E transcription, as detected with an eIF4E-lacZ transcriptional fusion reporter, as well as eIF4E protein levels detected by immunostaining were up-regulated upon dMyc overexpression and down-regulated by dMyc RNAi. It is unlikely that the changes in eIF4E-lacZ activity were due to global increases or decreases in β-galactosidase (β-gal) translation caused by altered dMyc levels, since lacZ expression from a dMyc-lacZ reporter was unaffected under similar conditions. Furthermore, like dMyc protein, eIF4E-lacZ reporter expression was up-regulated in normal NBs or ectopic NBs in ada mutants, further supporting the notion that dMyc may up-regulate eIF4E transcription. Moreover, ChIP experiments using chromatins isolated from wild-type larval brains and a ChIP-quality antibody against dMyc demonstrated specific binding of dMyc to an eIF4E promoter region harboring a cluster of adjacent noncanonical E boxes, supporting a direct regulation of eIF4E transcription by dMyc. dMyc and eIF4E thus appeared to form a regulatory feedback loop that promoted NB growth and renewal. Consistent with this model, while knocking down either dMyc or eIF4E had no noticeable effect on type II NB maintenance and only a mild effect on NB nucleolar size in the case of dMyc RNAi, their simultaneous knockdown led to a significant reduction in nucleolar size, premature neuronal differentiation, and loss of NBs (Song, 2011).

If the dMyc-eIF4E axis of cell growth control is a crucial downstream effector of N signaling in regulating NB maintenance, its up-regulation might be able to rescue the type II NB depletion phenotype resulting from reduced N signaling. Indeed, the loss of NBs associated with reduced Notch signaling was preventable when cell growth was boosted by dMyc overexpression. Thus, while N-IR directed by 1407-GAL4 led to complete elimination of type II NBs, the coexpression of dMyc, but not CD8-GFP or Rheb, an upstream component of the TOR pathway, resulted in the preservation of approximately half of type II NBs with apparently normal cell sizes, cell fate marker expression, and lineage composition. A similar effect was observed when dMyc was coexpressed with N-IR using the conditional 1407ts system, with transgene expression induced at the larval stage. While both dMyc and Rheb promote cell growth, they do so through distinct mechanisms, with the former increasing nucleolar size and the latter expanding cytoplasmic volume. These results thus provide compelling evidence that control of cell growth, particularly nucleolar growth, is a critical component in the maintenance of NB identity by N signaling (Song, 2011).

The differential responses of normal and tumor-initiating stem cells to functional reduction of eIF4E prompted a test of whether chemicals that specifically inhibit eIF4E function might have therapeutic potential in preventing CSC-induced tumorigenesis. Indeed, the brain tumor phenotypes induced by N overactivation or ada loss of function were effectively suppressed by feeding animals with fly food containing Ribavirin, an eIF4E inhibitor that interferes with eIF4E binding to mRNA 5' caps and promotes the relocalization of eIF4E from the nucleus to the cytoplasm (Kentsis, 2004; Assouline, 2009) (Song, 2011).

The CSC hypothesis was initially developed based on studies in mammalian systems. Various studies have supported the notion that CSCs share many functional features with normal stem cells, such as signaling molecules, pathways, and mechanisms governing their self-renewal versus differentiation choice. However, the cellular origin of CSCs and the molecular and cellular mechanisms underlying their development or genesis remain poorly understood. It has been proposed that CSCs could arise from (1) an expansion of normal stem cell niches, (2) normal stem cells adapting to different niches, (3) normal stem cells becoming niche-independent, or (4) differentiated progenitor cells gaining stem cell properties. This study has showen that in the Drosophila larval brain, CSCs can arise from the dedifferentiation of transit-amplifying progenitor cells back to a stem cell-like state. Importantly, eIF4E was identified as a critical factor involved in this dedifferentiation process. More significantly, it was shown that reduction of eIF4E function can effectively prevent the formation of CSCs without affecting the development or maintenance of normal stem cells. This particular dependence on eIF4E function by CSCs appears to be a general theme, as reduction of eIF4E function also effectively prevented the formation of CSCs, but not normal GSCs, in the fly ovary. These findings may have important implications for stem cell biology and cancer biology, in terms of both mechanistic understanding and therapeutic intervention (Song, 2011).

This study also offers mechanistic insights into the cellular processes leading to the dedifferentiation of progenitors back to stem cells. In Drosophila type II NB clones with overactivated N signaling, ribosome biogenesis within ectopic NBs appears to be faster than in normal NBs, as shown by the fact that the ratio of nucleolar to cellular volume of the ectopic NBs is approximately fivefold higher than that of normal NBs. The faster growth rate is accompanied by the up-regulation of dMyc and eIF4E and appears to be essential for transit-amplifying progenitors to undergo complete dedifferentiation back to a stem cell-like state. When the function of cell growth-promoting factors such as eIF4E is attenuated, the faster cell growth of ectopic NBs can no longer be sustained and the dedifferentiation process stalls. As a result, brain tumor formation caused by uncontrolled production of ectopic NBs is suppressed. In contrast, normal NBs, which presumably have relatively lower requirements for cell growth and hence eIF4E function, maintain their stem cell fate and development under similar conditions. Therefore, a potential key to a successful elimination of CSC-induced tumors would be to find the right level of functional reduction in eIF4E, which causes minimal effects on normal stem cells but effectively obliterates CSCs. An ongoing clinical trial with Ribavirin in treating acute myeloid leukemia (AML) (Assouline, 2009), a well-characterized CSC-based cancer, demonstrated exciting proof of principle that such a strategy is feasible. The current version of Ribavirin, however, has certain limitations, such as its poor specificity and the high dosage (micromolar range) required for effective treatment. Thus, more specific and effective eIF4E inhibitors are urgently needed. The drug treatment experiments with Ribavirin validated Drosophila NBs as an excellent CSC model for searching further improved drugs. More importantly, the nuclear interaction between eIF4E and Myc unraveled by this biochemical analysis not only provides a new mechanistic explanation for the synergistic effects of eIF4E and Myc in tumorigenesis (Ruggero, 2004; Wendel, 2007), but also sheds new light on how to rationally optimize drug design and therapy for treating CSC-based cancer (Song, 2011).

The results offer new information on how N signaling helps specify and maintain NSC fate. N signaling regulates stem cell behavior in various tissues of diverse species. However, it remains unclear how differential N signaling determines distinct cell fate within the stem cell hierarchy. This study demonstrates that N signaling maintains Drosophila NSC fate at least in part through promoting cell growth. The following evidence supports that cell growth, but not cell fate, change is the early and primary effect of N signaling inhibition in type II NBs: (1) Pros expression is not immediately turned on in spdo mutant NBs with reduced cell sizes. Instead, it gradually increases during the course of spdo mutant NB divisions. (2) Up-regulation of Pros is not the cause of stem cell fate loss in spdo mutant NBs, as shown by spdo pros double-mutant analysis. (3) Cell growth defects precede the up-regulation of Ase expression in aph-1 mutant NBs. (4) Promotion of cell growth, and particularly nucleolar growth, by dMyc is sufficient to prevent NB loss caused by N inhibition. At the molecular level, N signaling appears to regulate the transcription of dMyc, which in turn up-regulates the transcription of eIF4E. Such a transcriptional cascade and feedback regulation of dMyc activity by eIF4E may help to sustain and amplify the activity of the Notch-dMyc-eIF4E molecular circuitry. Hence, differential N signaling within the lineage can lead to different cell growth rates, which partially determine differential cell fates. Consistent with this notion, knockdown of both eIF4E and dMyc results in defects of NB cell growth and loss of stem cell fate (Song, 2011).

While many signaling pathways and molecules have been implicated in the maintenance of stem cell identity, the question of how a stem cell loses its 'stemness' at the cellular level remains poorly understood. A stem cell may lose its stem cell fate by undergoing a symmetric division to yield two daughter cells that are both committed to differentiation or through cell death. Earlier studies provided intriguing hints that cell growth and translational regulation could influence stem cell maintenance in the Drosophila ovary. This study usded detailed clonal analyses of NSCs over multiple time points to provide direct evidence that a NSC with impaired N signaling will gradually lose its identity due to a gradual slowing down of cell growth and loss of cell mass. Remarkably, such loss of stem cell fate can be prevented when cell growth is restored by dMyc, but not Rheb, overexpression, demonstrating the functional significance of regulated cell growth, particularly nucleolar growth, in stem cell maintenance. More importantly, this information offers clues on how to specifically eliminate tumor-initiating stem cells. These studies suggest that a stem cell, normal or malignant, has to reach a certain growth rate in order to acquire and maintain its stemness, presumably because when the stem cell grows below such a threshold, its proliferative capacity becomes too low, whereas the concentration of differentiation-promoting factors becomes too high to be compatible with the maintenance of stem cell fate. Consistent with this notion are the strong correlation between the expression of ribosomal proteins and cellular proliferation (van Riggelen, 2010) as well as the correlation between the reduction of NB sizes and the up-regulation of differentiation-promoting factor Pros or Ase in different developmental contexts (Song, 2011).

The results also provide new insights into how the evolutionarily conserved tripartite motif and Ncl-1, HT2A, and Lin-41 (TRIM-NHL) domain proteins regulate stem cell homeostasis. The TRIM-NHL protein family, to which Brat and Mei-P26 belong, include evolutionarily conserved stem cell regulators that prevent ectopic stem cell self-renewal by inhibiting Myc. However, the downstream effectors of the TRIM-NHL proteins remain largely unknown. This study identified eIF4E as such a factor. NB-specific knockdown of eIF4E completely suppresses the drastic brain tumor phenotype caused by loss of Brat. Interestingly, eIF4E knockdown is even more effective than dMyc knockdown in this regard. N signaling and Brat have been proposed to act in parallel in regulating Drosophila type II NB homeostasis. However, at the molecular level, how deregulation of these two rather distinct pathways causes similar brain tumor phenotypes remain largely unknown. The current results suggest that these two pathways eventually converge on the dMyc-eIF4E regulatory loop to promote cell growth and stem cell fate. N overactivation and loss of Brat both result in up-regulation of eIF4E and dMyc in transit-amplifying progenitors, accelerating their growth rates and helping them acquire stem cell fate. Consistent with a general role of eIF4E and dMyc in stem cell regulation, it was shown that partial reduction of eIF4E or dMyc function in the Drosophila ovary effectively rescues the ovarian tumor phenotype due to the loss of Mei-P26. The vertebrate member of the TRIM-NHL family, TRIM32, is shown to suppress the stem cell fate of mouse neural progenitor cells, partially through degrading Myc. Whether eIF4E acts as a downstream effector of TRIM32 in balancing stem cell self-renewal versus differentiation in mammalian tissues awaits future investigation (Song, 2011).

Notch signaling activates Yorkie non-cell autonomously in Drosophila

In Drosophila imaginal epithelia, cells mutant for the endocytic neoplastic tumor suppressor gene vps25 stimulate nearby untransformed cells to express Drosophila Inhibitor-of-Apoptosis-Protein-1 (DIAP-1), conferring resistance to apoptosis non-cell autonomously. This study shows that the non-cell autonomous induction of DIAP-1 is mediated by Yorkie, the conserved downstream effector of Hippo signaling. The non-cell autonomous induction of Yorkie is due to Notch signaling from vps25 mutant cells. Moreover, activated Notch in normal cells is sufficient to induce non-cell autonomous Yorkie activity in wing imaginal discs. These data identify a novel mechanism by which Notch promotes cell survival non-cell autonomously and by which neoplastic tumor cells generate a supportive microenvironment for tumor growth (Graves, 2012).

This study identifies a novel role of Notch signaling for non-cell autonomous control of apoptosis via induction of Yki activity in neighboring cells. It has previously been shown that Notch signaling controls cell proliferation both autonomously and non-cell autonomously in the developing eye. The non-cell autonomous component of proliferation control was attributed to Notch-dependent activation of Jak/Stat signaling although that recently came into question. Nevertheless, Jak/Stat activation is not sufficient to mediate the effect of Notch on non-cell autonomous control of apoptosis. This study identified the Hpo/Wts/Yki pathway as a target of Notch signaling for the non-cell autonomous control of apoptosis both in eye and wing imaginal discs. Because the Hpo/Wts/Yki pathway also controls proliferation, it is likely that Notch promotes non-cell autonomous proliferation through both Jak/Stat and Hpo/Wts/Yki activities (Graves, 2012).

It is also interesting to note that this non-autonomous control of the Hpo/Wts/Yki pathway by Notch occurs in a position-dependent manner. For example, vps25 mutant clones or NICD-expressing clones located in the hinge and notum of wing discs triggered non-cell autonomous up-regulation of ex-lacZ, while clones in the wing pouch did not. Additionally, vps25 mutant clones located anterior to the morphogenetic furrow triggered non-cell autonomous up-regulation of ex-lacZ, while clones in the posterior of the eye disc did not. The reason for this position-dependence is unknown. However, the regions which do not induce Hpo/Wts/Yki signaling non-autonomously correspond to the zone of non-proliferating (ZNP) cells in the wing disc and post-mitotic, differentiating cells in the eye disc. Therefore, one potential reason for the position-dependence may be that the post-mitotic nature of the cells in the ZNP of the wing pouch and in the posterior of the eye disc render them inert to growth-promoting signals that trigger the Hpo/Wts/Yki pathway. However, while this is one possibility, there may also be additional mechanisms that influence the response to growth-promoting signals (Graves, 2012).

How Notch exerts this non-autonomous effect is an important and interesting question. Based on its function as a transcriptional regulator, it is possible that increased Notch signaling in vps25 mutant cells could lead to transcription of a secreted or transmembrane protein that communicates to surrounding tissue and induces Yki activity. Expression of proteins known to non-cell autonomously activate Yki signaling such as Fat, Dachsous (Ds) and Four-Jointed as well as ds-lacZ, however, are not altered in vps25 mosaic discs. Identification of this non-cell autonomous signaling mechanism may also be critical for understanding tumorigenesis, as mutations in the Notch pathway, the Hippo pathway and in ESCRT components have been implicated in many different types of human cancer. In conclusion, this study provides a mechanism by which neoplastic cells influence the behavior of neighboring wild-type cells, which may be critical for generating a supportive microenvironment for tumor growth by preventing cell death and promoting the proliferation of wild-type cells (Graves, 2012).

Notch function in the eye disc

Continued: Notch Targets of Activity part 3/3 | return to part 1/3

Notch: Biological Overview | Evolutionary Homologs | Protein Interactions | Post-transcriptional regulation of Notch mRNA | Developmental Biology | Effects of Mutation | References

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