Interactive Fly, Drosophila

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 patched^Gal4 driver (i.e. crossing the endogenous AP-2 rings). Legs from patched^Gal4/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 patched^Gal4 in a Notch^ts 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, Notch^intra was expressed with the dpp^Gal4 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 (Notch^icd) 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:

independent regulation of Notch and Delta expression in intervein and vein territories,
Notch activation by Delta in cells where Notch and Delta expression overlaps,
positive feedback on Notch transcription in cells where Notch has been activated and
repression of veinlet transcription by E(spl)mbeta and maintenance of Delta expression by Veinlet/Egf-r activity (de Celis, 1997b).

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:

Notch activation in the wing leads to increased production of Delta and Serrate generating a positive feedback loop that maintains signaling.
wingless expression is upregulated by Notch signaling in both dorsal and ventral cells during the early stage of boundary formation. It is proposed that during normal development, Wingless co-operates with Notch to reinforce this positive feedback.
Cut, which is activated by Notch at late stages, acts antagonistically to prevent Delta and Serrate expression in the central D/V cells at the margin. High levels of Delta and Serrate have a dominant negative effect on Notch; at late stages, Notch can only be activated in cells next to the ligand-producing cells that are adjacent to cells of the margin.

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:

ac and sc are expressed in the stg-negative, G-2 arrested cells, but not in G1-arrested cells.
Loss of Wg activity results in loss of expression of ac and sc.
Ectopic Wg or activated Arm expands the domain of stg-negative, Ac-positive cells.
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 (dTCF^DN) 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 N^ts2 was used. When N^ts2 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 rho^ve 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 (N^ts), 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 (N^dn), 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 (N^intra) 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 function in the eye disc

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

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