Transcriptional Regulation Table of contents

Wingless expression in the wing and haltere

Wingless might be regulated by the Notch pathway. Possible Notch targets are suggested by the results of Notch overexpression in the wing disc. Notch gain-of-function alleles in which Notch activity is not restricted to the dorsoventral boundary cause mis-expression of cut and wingless and overgrowth of the disc, illustrating the importance of localised Notch activation for wing development (Rulifson, 1995 and de Celis, 1996). This work suggests that for the wingless signal to be effective, as it is at the wing margin, Notch must be inactivated. It has been suggested that wingless expression at the dorsal-ventral bounday of the wing disc depends on a signal from dorsal to ventral cells mediated by Serrate and Notch (Diaz-Benjumea, 1995).

Notch-dependent activation of wg, cut and vestigial at the wing margin depends on the activity of Suppressor of Hairless. Su(H)-mutant cells lose expression of the vestigial early enhancer, of wingless and of cut in a cell autonomous manner. Clones of Su(H)-mutant cells cause loss of wing tissue and scalloping of the wing, but only in Notch mutant clones at the D/V boundary. vestigial expression at the D/V boundary does not depend on wingless, since misexpression of wild-type wg cDNA, which results in wing margin bristles, does not cause an expansion of vestigial expression. Likewise, wingless expression does not depend on an early function of vestigial. Both Notch and wingless cooperate to activate cut at the D/V boundary. Later expression of vestigial in the wing pouch is, however, wingless dependent. vestigial is expressed in a broad domain throughout the wing. Removing of Wg activity in late second instar larvae leads to almost complete loss of the secondary expression of vestigial in the wing pouch without affecting expression at the D/V boundary. Taken together with the observation that clones of cells lacking shaggy activity show a cell-autonomous increase of Vestigial expression, these results suggest the vestigial is a direct target of the Wg pathway (Neumann, 1996).

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, 1997).

A study by S. D. Weatherbee (1998) is arguably the best study yet published about how gene regulation differs in homologous structures, and points to future studies for how differential gene regulation will be shown to account for the structural differences between species. The differentiation of the Drosophila haltere from the wing through the action of the Ultrabithorax (Ubx) gene is a classic example of Hox regulation of serial homology. This study reveals several features of the control of haltere development by Ubx which, in principle, are likely to apply to the Hox-regulated differential development of other serially homologous structures in other animals. Specifically, it has been shown that Ubx acts: (1) at many levels of regulatory hierarchies, on long-range signaling proteins and their target genes, as well as genes further downstream; (2) selectively on a subset of downstream target genes of signals common to both wing and haltere, and (3) independently on these diverse targets. This information is presented in terms of the effects of Ubx on gene expression in the three axes of appendage formation, since these axes are to a large extent independently regulated and independent gene regulation in the axes serves to structure the entire wing (Weatherbee, 1998).

In the dorsoventral axis, Ultrabithorax represses Wingless in the posterior compartment and selectively represses genes along the dorsal-ventral boundary. It has long been assumed that the global coordinate systems in homologous appendages are the same and, indeed, the apterous selector gene is expressed in the dorsal compartment of the haltere disc as in the wing. However, it was found that Wg, which is expressed along both the anterior and posterior extent of the DV boundary in the wing disc, is not expressed in the posterior compartment of the haltere disc. Because Wg function along the DV boundary is required for growth and patterning of the wing disc, the absence of Wg in the posterior haltere disc probably contributes to its disproportionately smaller size in comparison to the anterior compartment. In posterior Ubx clones in the haltere disc, Wg is expressed along the DV boundary, suggesting that Ubx represses the posterior portion of the Wg expression pattern. The activation of Wg along the DV boundary occurs via the Notch receptor signaling pathway. This pathway also activates the "boundary" enhancer of the vestigial gene, which is activated along the entire anterior and posterior extent of the DV boundary in the haltere. These results demonstrate that the Notch pathway is active along the entire DV boundary but that Ubx selectively prevents Wg activation by this pathway in the posterior compartment. Wg is expressed in the anterior compartment of the haltere disc, yet its phenotypic effects are markedly different from those in the anterior of the wing disc (Weatherbee, 1998).

Strawberry notch is a nuclear protein that functions downstream of Notch. Subjecting temperature sensitive strawberry notch to heat shock results in a down regulation of wg at the wing margin. Expression of wg in other regions of the wing disc as well as in other imaginal discs is unaffected by the loss of sno function. Likewise sno is required for the expression of vestigial, cut and E(spl)-m8 at the wing margin (Majumdar, 1997).

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, 1998).

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, 1998).

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, 1998).

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, 1998).

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, 1998).

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, 1998).

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, 1998).

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, 1998).

Cell interactions mediated by Notch-family receptors have been implicated in the specification of tissue boundaries in vertebrate and insect development. Although Notch ligands are often widely expressed, tightly localized activation of Notch is critical for the formation of sharp boundaries. Evidence is presented that the POU domain protein Nubbin contributes to the formation of a sharp dorsoventral (DV) boundary in the Drosophila wing. Nubbin represses Notch-dependent target genes and sets a threshold for Notch activity that defines the spatial domain of boundary-specific gene expression (Neumann, 1998).

Certain features of the abnormal wings in flies mutant for nubbin suggest a possible role for Nubbin protein in spatially limiting Notch activity at the DV boundary of the wing. The row of sensory bristles that makes up the wing margin is disorganized in nubbin wing mutants, suggesting a defect in Wingless or Notch activity. In preparations where the wing margin is viewed edge on, this disorganization reflects a broadening of the region where bristles form. Margin bristles are normally specified in cells very close to the DV boundary, reflecting a requirement for high levels of Wingless signaling activity. The broadening of the margin suggests that Wingless might be ectopically expressed in nubbin mutant wing discs. Wingless is normally expressed in a stripe of two to three cells straddling the DV boundary. In nubbin mutant discs, this stripe is widened considerably. Expression of the Notch targets vestigial and cut are similarly expanded at the DV boundary in nubbin mutants (Neumann, 1998).

To determine whether the effect on bristle specification is a direct consequence of removing nubbin activity, clones of nubbin mutant cells were generated in a wild-type background. Ectopic wing margin bristles are found in nubbin mutant clones located near the endogenous wing margins. The nubbin mutant clones show ectopic expression of neuralized, a molecular marker for precursors of the sensory neurons that innervate the bristles. The nubbin mutant clones misexpress wingless and vestigial. The largely autonomous effect of nubbin mutant clones on bristle specification may be due to the relatively low levels of Wg protein expressed in nubbin mutant clones. Together with the results on cut expressioon, these observations suggest that Notch target genes are transcriptionally up-regulated in nubbin mutant cells near the DV boundary (Neumann, 1998).

To test whether ectopic activation of these genes in nubbin mutant clones directly depends on Notch signaling activity, clones of cells were generated that were simultaneously mutant for nubbin and Suppressor of Hairless [Su(H)]. Su(H) encodes a DNA binding protein that mediates transcriptional activity of Notch target genes. Su(H) is autonomously required for the expression of wingless, vestigial, and cut at the DV boundary and binds directly to the vestigial DV boundary enhancer. Clones of cells mutant for both nubbin and Su(H) do not ectopically activate wingless, demonstrating that ectopic expression of wingless in nubbin mutant cells depends on activity of the Notch pathway. To confirm that Nubbin acts downstream of Notch, a test was performed to see whether overexpression of Nubbin could suppress the effects of a ligand-independent form of Notch. When Nubbin is coexpressed with such a constitutively active Notch, ectopic Wingless expression is strongly reduced. Together, these observations suggest that Nubbin may act as a direct repressor of Notch-dependent target gene expression. These findings argue that the effects of Nubbin are unlikely to be mediated by indirect effects on the expression of Notch ligands (Neumann, 1998).

Several different mechanisms have been proposed to account for the activation of Su(H) by Notch. To further investigate how Su(H) activity is regulated, misexpression assays were used with wild-type Su(H) and with modified forms of Su(H) containing either a nuclear localization signal [Su(H)NLS], a transcriptional activation domain [Su(H)VP16], or a deletion of the domain required for interaction with the antagonist Hairless [Su(H)DH]. Only Su(H)VP16 is able to mimic Notch activation effectively in the Drosophila wing, in agreement with the model that Notch activity normally confers coactivator function on Su(H). Neither nuclear localization nor elimination of Hairless binding is sufficient for activation. The phenotypes produced by overexpression of Su(H)wt and Su(H)NLS indicate a mixture of both increased and reduced Notch pathway activity and point to a role for Su(H) in both activation and repression of gene expression, as has been proposed for the mammalian homolog CBF1. Some phenotypes are equivalent to Notch loss-of-function, with wing-nicks and inhibition of a subset of target genes, which is most consistent with the ectopic proteins displacing a Su(H)-coactivator complex. Conversely, other phenotypes are equivalent to Notch gain-of-function, with wing-overgrowths and ectopic target-gene expression. These effects can be explained by the ectopic Su(H)/Su(H)NLS titrating a repressor complex. The wing-overgrowth phenotype is sensitive to the dose of Hairless and the phenotypes produced by coexpressing Su(H) and Hairless suggest that Hairless could form a component of this repressive complex (Furriols, 2000).

The phenotypes produced by misexpressing Su(H)wt and Su(H)NLS appear to combine activation and repression of Notch activity. To confirm whether this interpretation is correct, the effects on genes whose expression at the dorsal/ventral boundary is dependent on Notch were analyzed. Two assays used fragments that are directly responsive to Su(H) and Nicd: mbeta1.5 (a fusion between the E(spl)mbeta regulatory sequences and lacZ) and vgBE-lacZ (a fusion between the vestigial boundary enhancer and lacZ). Expression of wingless and the entire vestigial gene, which may involve indirect as well as direct regulation by the Notch pathway, were also examined. All are ectopically activated by Su(H)VP16, consistent with it mimicking the effects of Nicd. As anticipated, the wing-nick phenotypes produced by the modified Su(H) proteins in combination with ptc-Gal4 correlate with a reduction in the levels of mbeta1.5 and wingless expression at the d/v boundary. mbeta1.5 appears to be more sensitive and is strongly repressed by all three proteins, with Su(H)NLS the most effective as suggested by the wing-nicking phenotypes. The effects on wingless were milder and only Su-(H)NLS strongly represses expression. In contrast, vgBE-lacZ shows a very different response and is ectopically activated by both Su(H)wt and Su-(H)NLS. The activation of vgBE-lacZ is also observed when the levels of misexpressed proteins are lower (using dpp-Gal4) although in this combination Su(H)NLS and Su(H)wt still repress mbeta1.5. The two enhancers therefore appear intrinsically different in the way they respond to Su(H), suggesting that their regulation may involve different thresholds of activating and repressing Su(H) containing complexes (Furriols, 2000).

The mixed loss and gain of Notch function phenotypes produced in the wing by ectopic expression of Su(H)wt and Su(H)NLS suggests that Su(H) has a dual function, acting in some contexts as an activator and in others as a repressor. The simplest model is that Su(H) can exist in at least two complexes -- one where it interacts with a coactivator(s) and the other where it interacts with a corepressor(s). In cells where there is no/low Notch signaling the primary function of Su(H) would be to keep the target genes repressed by interacting with a corepressor complex. One of these corepressors could be Hairless; another could be the HDAC complex described in vertebrates. In contrast, in the cells where Notch is active, Su(H) would be complexed with coactivator(s) (e.g., Nicd so that the transcription of Notch target genes would be initiated). Depending on the relative levels or activity of the components, the equilibrium would shift in favor of one or the other complex (Furriols, 2000).

The development of the Drosophila wing involves progressive patterning events. In the second larval instar, cells of the wing disc are allotted wing or notum fates by a wingless-mediated process and dorsal or ventral fates by the action of apterous and wingless. Notch-mediated signaling is required for the expression of the genes vestigial and scalloped in the presumptive wing blade. Later, wingless, Notch and cut are involved in cell fate specification along the wing margin. The function of scalloped in this process is not well understood and is the focus of this study. Patterning downstream of Notch and wingless pathways is altered in scalloped mutants. Reduction in scalloped expression results in a loss of expression of wing blade- and margin-specific markers. Misexpression of scalloped in the presumptive wing causes misexpression of scalloped, vestigial and wingless reporter genes. However, high levels of scalloped expression have a negative influence on wingless, vestigial and its own expression. sc and vg respond to wg and N signaling in a manner very similar to that characterized for vg in the developing wing. This activation is maintained by auto-regulatory events. It is also suggested that sd and vg serve to regulate and modulate wg expression and to change the pattern observed in the second larval instar to the very different pattern seen in the third instar wing disc (Varadarajan, 1999).

During Drosophila wing development growth and patterning are mediated by signaling from the dorsoventral (D/V) organizer. In the metathorax, wing development is essentially suppressed by the homeotic selector gene Ubx to mediate development of a pair of tiny balancing organs, the halteres. Expression of Ubx in the haltere D/V boundary down-regulates the haltere's D/V organizer signaling compared to that of the wing D/V boundary. Somatic loss of Ubx from the haltere D/V boundary thus results in the formation of a wing-type D/V organizer in the haltere field. Long-distance signaling from this organizer was analyzed by assaying the ability of a Ubx minus clone induced in the haltere D/V boundary to effect homeotic transformation of capitellum cells (the capitellum is the main body of the haltere) away from the boundary. The clonally restored wing D/V organizer in mosaic halteres not only enhances the homeotic transformation of Ubx minus cells in the capitellum but also causes homeotic transformation of even Ubx plus cells in a genetic background known to induce excessive cell proliferation in the imaginal discs. In addition to demonstrating a non-cell-autonomous role for Ubx during haltere development, these results reveal distinct spatial roles for Ubx during maintenance of cell fate and patterning in the halteres. Ubx modulates the expression of wingless and cut in the haltere D/V boundary and represses vestigial in the capitellum, thereby suggesting a mechanism for the Ubx mediated down-regulation of the D/V organizer activity in the haltere. While the repression of wingless and cut expression is cell-autonomous, that of the quadrant vestigial-lacZ is non cell-autonomous: pouch cells farther away from the D/V boundary show more severe reduction in lacZ expression. Given the fact that quadrant vestigial-lacZ repression is dependent on the formation of the D/V boundary, the non-cell-autonomy in quadrant vestigial-lacZ repression by ectopic Ubx would not be surprising if Ubx function is to negatively regulate D/V signaling. Thus all the results provide strong evidence for the negative regulation of D/V organizer signaling by Ubx during haltere specification. It is likely that during haltere development, repression of wing patterning signals results in the specification of cell shape and volume that are unique to the haltere (Shashidhara, 1999).

The scalloped and vestigial genes are both required for the formation of the Drosophila wing, and recent studies have indicated that they can function as a heterodimeric complex to regulate the expression of downstream target genes. The consequences of complete loss of scalloped function, ectopic expression of scalloped, and ectopic expression of vestigial for the development of the Drosophila wing imaginal disc have been analyzed. Clones of cells mutant for a strong allele of scalloped fail to proliferate within the wing pouch, but grow normally in the wing hinge and notum. Cells overexpressing scalloped fail to proliferate in both notal and wing-blade regions of the disc, and this overexpression induces apoptotic cell death. Clones of cells overexpressing vestigial grow smaller or larger than control clones, depending upon their distance from the dorsal-ventral compartment boundary. These studies highlight the importance of correct scalloped and vestigial expression levels to normal wing development. Studies of vestigial-overexpressing clones also reveal two further aspects of wing development. (1) In the hinge region vestigial exerts both a local inhibition and a long-range induction of wingless expression. These and other observations imply that vestigial-expressing cells in the wing blade organize the development of surrounding wing-hinge cells. (2) Clones of cells overexpressing vestigial exhibit altered cell affinities. The analysis of these clones, together with studies of scalloped mutant clones, implies that scalloped- and vestigial-dependent cell adhesion contributes to separation of the wing blade from the wing hinge and to a gradient of cell affinities along the dorsal-ventral axis of the wing (Liu, 2000).

While previous studies have emphasized the autonomous requirement for vg in wing development, these results make clear that this autonomous requirement is restricted to the wing blade and that Sd:Vg has an additional, nonautonomous role in promoting the development of the wing hinge. Null alleles of vg delete the wing blade and most, or sometimes all, of the wing hinge. Even when vg mutant animals retain some hinge tissue, a significant amount of tissue is deleted proximal to the inner Wg expression ring. However, by making clones of cells mutant for sd, it was found that Sd:Vg is autonomously required only distal to the inner Wg expression ring. Similarly, clones of cells that are mutant for a null allele of vg grow normally in the notum, but fail to grow in the wing. The precise border where vg is autonomously required maps to the edge of detectable Vg expression. This places the border distal to the inner Wg expression ring. Altogether, these results suggest that Sd:Vg is required nonautonomously for normal development of the wing hinge. Indeed, clones of cells ectopically expressing Vg frequently reorganize the patterning of surrounding tissue in the wing hinge. This reorganization is visible through changes in the expression of Wg and Nubbin, as well as changes in the folding of the disc epithelia. These studies, along with reports on the function and regulation of hth in the hinge, lead to a model for the regulatory interactions between wing hinge and wing blade (Liu, 2000).

The observation that Sd:Vg is both required nonautonomously for normal hinge development and sufficient to reorganize the normal patterning of surrounding hinge tissue leads to the hypothesis that Sd:Vg-expressing wing blade cells produce a signal (X) that influences gene expression in surrounding wing-hinge cells. Ultimately, one key target of this signal is the inner ring of Wg hinge expression. Wg is essential for wing hinge development; Wg expression is induced non-autonomously by Sd:Vg, and normal Wg hinge expression is reduced or absent in vg mutants. The detection of a spot of Wg expression in some vg mutant discs that appears to correspond to a portion of the inner hinge ring implies that the hypothesized signal X may not be absolutely required for Wg expression. Instead, it may function to maintain and promote Wg hinge expression as the wing pouch grows. Alternatively, it may be, as suggested by the failure of Vg-expressing clones to effectively induce Wg hinge expression near the D-V boundary, that Wg hinge expression near the D-V boundary is regulated by a Vg-independent mechanism, which continues to promote a spot of Wg expression even in vg mutants (Liu, 2000). Although the identity of the signal X is not yet known, nor how direct its regulatory influence on Wg may be, it can be inferred that its action ultimately impinges on enhancers within a 1.2-kb fragment of the wg gene identified as being responsible for the distal ring of Wg hinge expression. Recent studies of Drosophila leg development have implied the existence of signaling from proximal cells to distal cells. Thus, in both legs and wings, normal appendage development appears to rely not just on the direct interpretation of primary signals produced along compartment boundaries, but also on secondary signaling between cells in different domains along the proximal-distal axis (Liu, 2000).

While these studies imply that a Sd:Vg-dependent signal is essential for normal hinge development, hinge cells are uniquely competent to express Wg in response to this signal. This implies that a distinct hinge fate precedes receipt of the signal. In addition, a small amount of wing-hinge tissue, and in some cases Wg expression, remains in vg null mutants. Signaling from the wing blade does not therefore act as an inducer of wing-hinge fate per se, but rather acts to elaborate the patterning and growth of the hinge. hth plays a key role in hinge development, and recent studies have demonstrated that hth is essential for Wg expression in the hinge. Thus Hth, together with its partner protein Extradenticle (Exd), may be at least partially responsible for the distinct responsiveness of hinge cells to Sd:Vg-dependent signaling. Hth expression is itself positively regulated by Wg, and thus the distinct fates of both the wing blade and the wing hinge are maintained in part by positive regulatory loops with Wg. Separate blade and hinge territories are also maintained in part by repressive interactions between Sd:Vg and Exd:Hth. However, while the repression of Hth by Sd:Vg is autonomous, and thus may be direct, Hth does not repress Sd:Vg directly, but instead represses Wg expression along the D-V border, which then indirectly limits Sd:Vg expression (Liu, 2000).

Slimb (Slmb) is an F-box/WD40 protein that potentially participates in the ubiquitin proteolysis machinery. During development, Slmb is required in limb discs to repress Hedgehog (Hh) target genes, i.e. wingless and decapentaplegic, as well as the Wg signal transduction pathway. These repression functions have been proposed from studies using weak slmb alleles. Interestingly, experiments with strong slmb alleles have revealed additional functions in which slmb is required, such as leg dorsal-ventral restriction. New alleles of the slmb gene have been isolated in a screen for new negative regulators of dpp: several amorphs (characterized by genetic and molecular criteria) and a cold-sensitive hypomorph. By performing somatic clone experiments with these new amorphic slmb alleles, it has been determined that regulation of Dpp and Wg by Slmb could be different from what has already been published. In leg discs, lack of slmb function derepresses the transcription of wg, independent of Hh signaling. Ectopic legs resulting from slmb- clone induction come only from wg misexpression in the normal dpp domain, since ectopic proximo-distal axes are induced dorsally, and adult ectopic legs are often perfect with respect to antero-posterior polarity. In wing discs, transcription of wg, which is normally independent of Hh signaling, is also derepressed in the absence of slmb function. In discs bearing amorphic slmb clones and in discs of two other slmb- contexts, a novel pattern of dpp expression is described consisting of an enlargement of the normal dpp domain. Strong evidence indicates that this dpp modification can be linked to imaginal disc regeneration following slmb- cell elimination. The fate of slmb- clones, which disappear before adulthood, has been investigated. It was found that two mechanisms of cell elimination can account for imaginal cell regeneration: an early apoptosis and a mechanism of sorting-out that excludes all slmb- clones from all imaginal discs. This result suggests that Slmb is likely to be involved, in addition to its repression role on Dpp and Wg, in some other essential cellular mechanism, since, in the absence of Slmb, cell affinities are dramatically modified regardless of the deregulated morphogen and of the type of imaginal disc (Miletich, 2000).

These results indicate that slmb is involved in the repression of wg transcription independent of Hh signaling. In the wing pouch, wg transcription depends on communications between dorsal and ventral cells involving the Notch receptor. This finding suggests that slmb is involved in the proteasome-dependent degradation or proteolytic cleavage of a putative regulatory protein of the Notch signaling pathway. This is in good agreement with a proposal that the proteasome is involved in the degradation of an active form of Notch, thus limiting the activation of the Notch targets. In the leg disc, the mechanism responsible for wg transcription in the posterior cells is unknown. In the anterior compartment of imaginal discs, the transcription factor Ci is necessary to activate (or repress) Hh target genes. In the posterior compartment cells, engrailed represses transcription of ci. Thus, a deregulation of wg transcription depending on Hh signaling must be linked to ectopic ci transcription. Ectopic transcription of ci is not observed in the posterior compartment of leg discs bearing slmb- clones; neither is a switching-off of en transcription observed. It is concluded that a repression of wg transcription occurs in leg discs irrespective of the Hh/Ci signaling, and that slmb is involved in this process (Miletich, 2000).

In conclusion, two mechanisms appear to eliminate slmb- cells in imaginal discs: an early apoptosis that only concerns some cells, and a mechanism for sorting-out that excludes all slmb- clones from all imaginal tissues. The early apoptosis is possibly induced by differential cell adhesion: in this case, it would be classified as an early sorting-out. Another possibility is that apoptosis is an alternative path to sorting-out when mutant cells form a 2-4 cell group (the theoretical size of a clone aged 24 h) rather than an organized population of cells (as found 48 h after clone induction). This could be answered by investigating whether early apoptosis is causally linked to a modification of cell affinity. It is also important to investigate whether slmb plays a direct role in the appearance of apoptotic cell death. If so, slmb would act as an anti-apoptotic gene; preliminary results favoring this result have been obtained by generating slmb- clones in a context of apoptosis inhibition (Miletich, 2000).

An important feature of the disappearance of null slmb- clones is that sorting-out occurs regardless of the deregulated signaling pathway and of the type of imaginal disc. Therefore, it seems that this exclusion is not a result of the deregulation of these pathways but rather is the result of the deregulation of some other essential cellular mechanism shared by all imaginal cells. Since slmb encodes an F-box protein that would be involved in targeting degradation of proteins by the proteasome, it is proposed that slmb is necessary in a general process required for the proper functioning of many cellular mechanisms. Alteration of this process would then lead to such dramatic changes in cell affinities that all slmb- cells would be excluded from all types of imaginal discs (Miletich, 2000).

Arthropod and vertebrate limbs develop from secondary embryonic fields. In insects, the wing imaginal disk is subdivided early in development into the wing and notum subfields. The activity of the Wingless protein is fundamental for this subdivision and seems to be the first element of the hierarchy of regulatory genes promoting wing formation. Drosophila epidermal growth factor receptor (Egfr) signaling has many functions in fly development. Antagonizing Egfr signaling during the second larval instar leads to notum to wing transformations and wing mirror-image duplications. Egfr signaling is necessary for confining the wing subregion in the developing wing disk and for the specification of posterior identity. To do so, Egfr signaling acts by restricting the expression of Wingless to the dorsal-posterior quadrant of wing discs, suppressing wing-organizing activities, and by cooperating in the maintenance of Engrailed expression in posterior compartment cells (Baonza, 2000).

To study Egfr function during early wing development, Egfr signaling was reduced at different times by using thermosensitive alleles of Egfr or by overexpression of dominant negative Raf (DNRaf). Hypomorphic vein (vn) and connector enhancer of ksr (cnk) [a regulatory member of the Ras signaling cascade] alleles were also analyzed. Under these conditions, posterior to anterior transformations, proximal (notum) to distal (wing) transformations, and a reduction (or absence) of the notum region were observed with high frequency. When DNRaf is expressed in clones induced during the second instar, different kinds of phenotypes are found. Large clones in the posterior notum/hinge anlage lead to notum to wing transformations, whereas large clones covering the posterior of the wing give rise to posterior to anterior transformations. These phenotypes were found only after inducing a large amount of confluent clones. Clones of cells overexpressing DNRaf in other regions at this age, or anywhere at later stages, give rise to different defects, such as those previously described on cell proliferation and vein cell fates (Baonza, 2000).

Wg is expressed in an anterior-ventral area roughly complementary to vn expression, near the interface between the A/P and D/V compartment boundaries. Reducing Egfr signaling during the second larval instar results in the generation of ectopic wings out from peripheral notal tissue in posterior territories. It is worth pointing out that this ectopic wing tissue does not develop at the expense of the notum, which, in many cases, is not affected. These phenotypes are similar to those observed after early Wg overexpression. They are never observed in the absence of en, which only promotes posterior to anterior notum and wing transformations (Baonza, 2000).

This leads to the proposition that Egfr signaling would restrict the domain of expression of wg and define the boundaries of the field of cells that is going to undergo a wing developmental program. To examine this possibility, the expression of Wg was analyzed in clones ectopically expressing DNRaf. In large clones covering most of the notum/hinge region, Wg expression is up-regulated from the early second larval instar in the posterior of the wing disk, extending progressively. The expansion of Wg expression drives the enlargement of the D/V axis toward posterior territories (manifested in the expanded field of ap-expressing cells in the duplicated areas) giving rise to a fully developed duplicated wing (Baonza, 2000).

To test whether the ectopic expression of Wg, induced by the down-regulation of Egfr activity, is sufficient to promote notum to wing transformations, Wg was interferred with by inducing the expression, in clones, of a dominant-negative form of Wg (DNWg) along with DNRaf. In most cases, double mutant clones covering the whole posterior compartment show a partial reduction in the size of the wing pouch and notum and a suppression of notum to wing transformations or ectopic induction of wing markers (vg expression). It is concluded that the repression of wg expression by Egfr signaling in the notum, under wild-type conditions, is necessary to set a limit for the wing field. This relationship partly resembles the antagonism between the Egfr and wg signaling pathways for the specification of the ventral larval cuticle, although in this case wg and Egfr do not counteract one another, but control epidermal differentiation through the opposite transcriptional regulation of downstream genes (Baonza, 2000).

Interestingly, Wg overexpression induces notum to wing transformations, but never mirror-image duplications, such as those obtained after reducing Egfr activity. This suggests that wg function mediates notum to wing conversion, but its overexpression is not sufficient for posterior to anterior transformation. Indeed, the overexpression of DNWg is not able to rescue the down-regulation of En, the up-regulation of Ci, or wing mirror-image duplications, which result from interfering with Egfr signaling. These data suggest that the effects of Egfr signaling on en and wg functions are independent, although the possibility of regulatory interactions between wg and en cannot be disregarded at this stage (Baonza, 2000).

The reiterative use of Egfr has been demonstrated in the generation of multiple fates in the developing fly eye. A fundamental early function for Egfr in the underlying patterning system of the wing subfield, controlling the activity of two genes, en and wg has been documented. It is significant that fibroblast growth factor [Ras-mitogen-activated protein kinase (MAPK)] activities are implicated in the initiation of the whole program of limb development in vertebrates, a program remarkably similar to that of the Drosophila wing. It remains to be seen whether a similar strategy applies to the activities of Ras-MAPK cascades during vertebrate limb bud development (Baonza, 2000).

In the wing wingless is expressed in a complex and dynamic pattern that is controlled by several different mechanisms. These involve the Hedgehog and Notch pathways and the nuclear proteins Pannier and U-shaped. The mechanisms that drive wingless expression in the wing hinge have been analyzed. Evidence is presented that wingless is initially activated by a secreted signal that requires the genes vestigial, rotund and nubbin. Later in development, wingless expression in the wing hinge is maintained by a different mechanism, which involves an autoregulatory loop and requires the genes homothorax and rotund. The role of wingless in patterning the wing hinge is discussed (Rodriguez, 2002).

The adult wing is formed by a continuous monolayer of epidermal cells that folds to form the dorsal and ventral surfaces of the wing pouch. The two surfaces contact at the margin of the wing and extend proximally through the wing hinge to the dorsal notum and the ventral pleura. In the presumptive wing region of the wing disc, wg is expressed in a narrow stripe of cells that runs all along the wing margin and in two rings that surround the wing pouch. The phenotypes and wg expression have been examined in several mutants in which the wing hinge is deleted (Rodriguez, 2002).

The effects of removing wg expression in the inner ring (IR) can be observed in spade (spd) mutants. spd mutations are a type of wg allele that specifically removes wg expression from the IR, with no effects on other expression domains. In spdfg wings, the hinge region is deleted, and the wing pouch appears directly joined to more proximal cells. In these wings, both wg-expressing cells and surrounding cells are missing. It has been shown that this phenotype is not caused by cell death but is a consequence of underproliferation in this region, suggesting that one of the functions of Wg in the IR is to promote local cell proliferation (Rodriguez, 2002).

The rotund (rn) gene is a member of the Krüppel family of zinc-finger encoding genes. Among other phenotypes, rn mutations delete the wing hinge and remove wg expression from the IR. nubbin encodes a member of the POU family of transcription factors. In strong nub mutations wings are vestigial, but phenotypic analysis of weaker alleles shows that the wing hinge is deleted and the expression of wg in the IR is missing. The hinge phenotype of the triple mutant spdfg nub2; rnDelta2-2 was examined, and it is similar to the phenotype of each of them, suggesting that the main cause of the phenotype is the lack of wg expression in the IR (Rodriguez, 2002).

vestigial encodes a nuclear protein with no homology with other identified families of nuclear proteins. Based on its interaction with scalloped (sd) it has been suggested that the function of Vg is to mediate transcriptional activation by Sd. vg expression in the wing is regulated by two separate enhancers: the boundary enhancer (BE) and the quadrant enhancer (QE). The BE is activated by the Notch signaling pathway and drives vg expression at the dorsal/ventral boundary in middle/late second instar larval stage. The QE is activated by the combined action of Wg and Dpp, and drives vg expression in the rest of the wing pouch from early third instar larval stage (Rodriguez, 2002).

The expression patterns of vg, rn and nub were examined. In mature wing discs vg, rn and nub are expressed in three concentric domains, the Vg domain being the smallest one. At this stage the wing hinge is lined with several anterior/posterior folds. The boundary of vg expression coincides with the distal-most fold of the disc. The Rn domain is slightly broader and its boundary coincides with a second fold in the disc. The Nub domain contains the Rn domain and coincides with the third fold in the disc. The IR domain corresponds to the proximal-most area of the Rn domain (Rodriguez, 2002).

The expression of these genes was examined in early larval development. In middle/late second instar larvae the expression domains of vg, rn and nub in the presumptive wing pouch are slightly broader than the vg domain. The rest of the cells of the disc, those that do not express nub, express the gene teashirt (tsh). wg is expressed only in a stripe of cells that corresponds to the presumptive wing margin. In early third instar larvae, wg starts to be expressed in the IR. This expression domain corresponds to cells that express rn and nub but do not express vg. wg expression in the IR promotes the growth of the hinge and, in third instar larvae, gives rise to the expression patterns described above for vg, rn and nub. At this stage, the cells that express the wg IR enhancer are located at the limit of the domain 3 (Rn + Nub), and are several cells away from the boundary of vg expression (Rodriguez, 2002).

The results indicate that Vg is required to activate the expression of rn and nub genes in the wing disc. This activation is restricted to the cells that will take wing fate and takes place in the cell that express vg, and also in the surrounding cells, suggesting that a Vg-dependent short-range signal activates rn and nub expression. At this time, the expression of nub and tsh in the wing disc are complementary and cover the whole disc (Rodriguez, 2002).

The expression of these genes in a domain broader than the Vg domain creates a ring of cells that express rn and nub but not vg. Evidence is presented indicating that a signal from vg-expressing cells activates the wg IR enhancer in adjacent rn/nub-expressing cells. Unlike the activation of rn and nub, the activation of wg expression by the IR enhancer is repressed in cells that also express vg. So, the IR enhancer is activated only in cells that surround the Vg domain. During the development of the disc, the position of the IR moves several cells away from the Vg domain. This implies either that the Vg-dependent signaling is able to activate the IR over a long range, or that a different, Vg-independent, mechanism maintains the IR (Rodriguez, 2002).

When artificial Vg/Rn-Nub interfaces are generated experimentally, the IR enhancer is activated in rn-nub-expressing cells that abut the Vg domain. This ectopic IR is around four cells wide, indicating the active range of the signal that activates wg expression. The results indicate that at distances greater than this, a Vg-independent mechanism maintains wg expression in the IR (Rodriguez, 2002).

Several results presented here indicate that Wg signaling activates hth expression, which is in turn required to maintain wg expression. wg and hth are co-expressed in the IR and OR, and wg expression precedes hth expression. Furthermore, hth expression is missing in spdfg discs, and wg expression is lost in hth mutant clones. Nevertheless, spdfg discs show activation of the IR enhancer, as revealed by the spd-lacZ construct and wg expression is not affected in hth mutant clones when observed in early third instar larvae. This indicates that Hth, while required to maintain IR activation, is not required to initiate wg expression (Rodriguez, 2002).

The rn clonal analysis indicates that Rn is also required for wg expression. One interesting observation is that when the IR moves away from the Vg domain, wg-expressing cells are always maintained at the limit of rn expression. rn is activated by a Vg-dependent signal. This implies that the activity range of the signal and the lifetime of the Rn protein together limit the domain of rn expression. So one explanation for why the IR is always maintained in the limit of rn expression is that, as a consequence of cell proliferation, cells drop out of the range of the Vg-dependent signaling. Thus, cells simultaneously lose the expression of both rn and wg. The result of the rn lineage-tracing experiment supports this prediction. Taken together, these results suggest that an autoregulatory loop involving Hth and Rn maintains wg expression. Although hth expression depends on Wg, rn expression depends on Vg, so wg expression in the IR is not maintained by lineage. wg autoregulation has been reported in embryo development, and a negative mechanism of 'self-refinement' has been suggested in wing margin specification. However, in neither of these cases has a role been reported for Hth or Rn (Rodriguez, 2002).

Wg-promoted cell proliferation generates a new domain between the IR and the Vg domain. This indicates that at this stage Vg-dependent signaling is unable to activate wg expression in adjacent cells. Otherwise the IR would be expressed in the whole Rn domain. One explanation for this could be that there is a temporal window for the activation of wg, but vg-expressing clones induced in mid/late third instar larvae are able to activate wg. Another explanation could be that a repressor is expressed in this domain. Clones of vg-expressing cells placed in this domain do not activate wg, which supports this explanation. In the experiment in which vg expression was presented in the Dpp domain, the new stripe of ectopic Wg does not recognize this domain, suggesting that the proposed repressor may be a target of Vg signaling. One alternative explanation is that wg refines its own expression domain by repressing the Vg-dependent activation. This has been proposed for the expression in the wing margin, but does not seem to be the case here. In experiments in which third instar larvae carrying a thermosensitive allele of wg (wgts/wgcx4) were reared at the restrictive temperature (16 hours at 30°C) and stained with Wg antibody, no changes were detected in the pattern of wg expression in the IR. However, it was observed that the expression in the wing margin was widened (Rodriguez, 2002).

Thus, the proximal and distal limits of the IR would be defined respectively by the limit of rn expression and by the limit of the expression of the proposed repressor. In summary, these results suggest that at least four different target genes are independently activated by one or more signals that emanate from vg-expressing cells: rn and nub are activated in second instar larvae; wg is activated in early third instar larvae (this activation requires the function of Rn and Nub and is repressed by Vg); and finally the repressor, which would be activated in middle third instar larvae (Rodriguez, 2002).

One interesting observation that can be made from these results relates to how the hinge is patterned. As a result both of local cell interactions and Wg-promoted cell proliferation, several domains, which are defined by different combinations of gene expression, are established. The generation of these domains is, in part, a consequence of the fact that the expression of these genes are not maintained by lineage, but also because there is not evidence of lineage restrictions. Thus, cells at the borders of both the IR domain and the Vg domain lose wg and vg expression, and fall into adjacent domains. However the expression in cells within a given domain, away from the border, must be more efficiently maintained by a phenomena similar to the reported community effect, because no holes are detected in the pattern of expression (Rodriguez, 2002).

In Drosophila, Suppressor of deltex [Su(dx)] mutations display a wing vein gap phenotype resembling that of Notch gain of function alleles. The Su(dx) protein may therefore act as a negative regulator of Notch but its activity on actual Notch signalling levels has not been previously demonstrated. Su(dx) is shown to regulate the level of Notch signalling in vivo, upstream of Notch target genes and in different developmental contexts, including a previously unknown role in leg joint formation. Overexpression of Su(dx) is capable of blocking both the endogenous activity of Notch and the ectopic Notch signalling induced by the overexpression of Deltex, an intracellular Notch binding protein. In addition, using the conditional phenotype of the Su(dx)sp allele, it has been shown that loss of Su(dx) activity is rapidly followed by an up-regulation of E(spl)mß expression, the immediate target of Notch signal activation during wing vein development. While Su(dx) adult wing vein phenotypes are quite mild, only affecting the distal tips of the veins, the initial consequence of loss of Su(dx) activity is more severe than previously thought. Using a time-course experiment it has been shown that the phenotype is buffered by feedback regulation illustrating how signalling networks can make development robust to perturbation (Mazaleyrat, 2003).

To begin to unravel the mechanism of action of Su(dx), it is an important prerequisite to establish whether Su(dx) acts on the Notch pathway itself, or whether the genetic interactions observed reflect an indirect, parallel, or downstream activity. The data argue that Su(dx) can indeed negatively regulate Notch signalling, upstream of the immediate Notch target genes. (1) It has been shown, using the temperature sensitivity of the Su(dx)sp wing vein gap phenotype, that Su(dx) loss of function is rapidly followed by the up-regulation of E(spl)mß expression in the pupal wing. (2) In third instar wing imaginal discs, it has been shown that in two enhancing genetic backgrounds, Su(dx) loss of function results in the up-regulation of wingless, another Notch target gene at the D-V boundary. (3) Su(dx) overexpression in the wing imaginal disc is capable of down-regulating the Notch-dependent expression of three genes, wingless and cut at the D-V boundary, and the vgBE-LacZ element at both the D-V and the A-P boundaries. These data show that Su(dx) is capable of downregulating Notch in different developmental contexts and that its activity on Notch is not limited to the particular situation of wing vein development (Mazaleyrat, 2003).

Su(dx) is capable of blocking the stimulation of Notch signalling, which is induced by the overexpression of Deltex, a regulatory protein which binds to the Notch intracellular domain. Thus these data suggest that the activity of Su(dx) lies upstream of the regulation of Notch target gene expression but downstream of, or at the level of, Deltex. This, together with the rapidity of the response of increased Notch signalling that is observed following Su(dx) loss of function, supports the hypothesis that Su(dx) acts directly on the Notch pathway. In vivo data are thus consistent with the in vitro observation that a related mammalian Nedd4 family protein, Itch, can promote the ubiquitination of the Notch1 intracellular domain (Mazaleyrat, 2003).

The phenotype of Su(dx)sp was examined in two different enhancing genetic backgrounds and different consequences on the spatial distribution of ectopic Notch activation were obtained at the wing disc D-V boundary, as monitored by wingless expression. Su(dx) mutations alone have no wing margin phenotype. wingless expression was investigated in the background of Notch alleles that enhance the Su(dx) wing vein phenotype, i.e., notchoid1 (nd1) and AbruptexE2 (AxE2). Ectopic wingless expression in nd1;Su(dx)sp discs is restricted to the ventral side of the D-V boundary, but is found on both sides of this boundary in AxE2;Su(dx)sp discs. A similar ventral compartment-specific Notch activation is observed when Serrate is expressed along the anterior-posterior axis, while expression of constitutively active Notch intracellular domain does not show such a restriction (Mazaleyrat, 2003).

In the third thoracic segment of Drosophila, wing development is suppressed by the homeotic selector gene Ultrabithorax (Ubx) in order to mediate haltere development. Ubx represses dorsoventral (DV) signaling to specify haltere fate. The mechanism of Ubx-mediated downregulation of DV signaling has been studied. Wingless (Wg) and Vestigial (Vg) are differentially regulated in wing and haltere discs. In wing discs, although Vg expression in non-DV cells is dependent on DV boundary function of Wg, it maintains its expression by autoregulation. Thus, overexpression of Vg in non-DV cells can bypass the requirement for Wg signaling from the DV boundary. Ubx functions, at least, at two levels to repress Vestigial expression in non-DV cells of haltere discs. At the DV boundary, it functions downstream of Shaggy/GSK3ß to enhance the degradation of Armadillo (Arm), which causes downregulation of Wg signaling. In non-DV cells, Ubx inhibits event(s) downstream of Arm, but upstream of Vg autoregulation. Repression of Vg at multiple levels appears to be crucial for Ubx-mediated specification of the haltere fate. Overexpression of Vg in haltere discs is enough to override Ubx function and cause haltere-to-wing homeotic transformations (Prasad, 2003).

Several experiments were designed to test the current model of Wg and Vg regulation (which is essentially based on studies on wing imaginal discs) in haltere discs. In wing discs, both Wg and Vg are subjected to an elaborate regulatory circuit. Wg and Vg interact to maintain each other's expression at the DV boundary. Vg-mediated activation of Wg is independent of Arm and TCF/pan function, which suggests that Vg may activate Wg either directly or through the N signaling pathway. Vg is capable of specifying wing development, even in the absence of Wg signaling. Overexpression of Vg in a vg1/vg1 background (in which no Wg or Vg is expressed) is sufficient to rescue wing phenotypes. This is particularly significant because Vg was expressed in this experiment only in non-DV cells. These results also suggest that Vg cell-autonomously regulates its own expression through its quadrant enhancer. Clonal analysis of arm suggests that Wg is required to activate vg-QE and Arm is not able to activate this enhancer in vg1 background. Wg signaling might activate Vg either indirectly or by activating some other enhancer of Vg. Once activated, Vg might maintain its expression by autoregulation, which is mediated through its quadrant enhancer. This could ensure the maintenance of Vg expression in non-DV cells, once it is activated by Wg signaling. It might also explain how the Wg gradient is translated into uniformly higher levels of Vg in non-DV cells (Prasad, 2003).

However, the above-mentioned model does not reconcile the observation that Vg, and not Wg, is capable of activating vg-QE in Ser background. Since the vg gene is intact in Ser background, ectopic expression of Wg using dpp-GAL4 should have activated one of the enhancers to induce Vg expression, which in turn would activate vg-QE. A model that reconciles all the results would, therefore, include a third component, which may act either parallel to or downstream of Wg and Vg at the DV boundary. Although there is no direct evidence for the existence of such a molecule, the fact that N23-GAL4 expression in non-DV cells is dependent on N function and independent of Vg and Wg function suggests such a possibility (Prasad, 2003).

The downregulation of Wg signaling by Ubx occurs at the level of Arm stabilization. Ubx inhibits stabilization of Arm by acting on event(s) downstream of Sgg. Normally, the Arm degradation machinery is very efficient and can degrade even overexpressed Arm. This is evident from the fact that embryos overexpressing Arm (from armS2) secrete normal denticle belts. If a downstream component functions with enhanced efficiency (either by direct enhancement of its expression by Ubx or owing to repression of a positive component of Wg signaling), residual activity of Sgg may be sufficient to cause enhanced degradation of Arm. Thus, enhanced degradation of Arm in haltere discs provides a new assay system to identify additional components of Wg signaling. For example, in microarray experiments to identify genes that are differentially expressed in wing and haltere discs, several transcripts of known (e.g., Casein kinase) and putative (e.g., Ubiquitin ligase) negative regulators of Wg signaling are upregulated in haltere discs (Prasad, 2003).

The results suggest that Wg and Vg regulation in haltere discs is different from that in wing discs. Wg is not autoregulated in haltere discs. In addition, Vg expression at the haltere DV boundary is independent of Wg function. However, in both wing and haltere discs, Wg expression at the DV boundary is dependent on Vg. Wg expression at the anterior DV boundary of haltere discs could be redundant because overexpression of DN-TCF at the haltere DV boundary shows no phenotype. However, Vg at the DV boundary appears to have an independent function. vg1 flies exhibit much smaller halteres than do wild-type flies. Since Wg function (and expression in the posterior compartment) is already repressed in haltere discs, reduction in haltere size in vg1 flies suggests Wg-independent long-range effects of Vg from the DV boundary. This could be one of the reasons why Ubx does not affect Vg expression at the DV boundary but represses Vg expression in non-DV cells. In wing discs too, Vg may have such a function on cells at a distance (Prasad, 2003).

One way to test the requirement of Ubx in DV and non-DV cells directly is by removing Ubx only from the haltere DV boundary or from non-DV cells. Clonal removal of Ubx solely from the haltere DV boundary does not induce cuticle phenotype in the capitellum. However, it was not possible to ascertain the effect on vg-QE because of haploinsufficiency: Ubx-heterozygous haltere discs themselves show activation of lacZ in the entire haltere pouch. The activation of vg-QE in Ubx/+ haltere discs could be a result of reduced Ubx function at the DV boundary, or in non-DV cells, or in both. Misexpression of Ubx at the wing disc DV boundary causes non-cell-autonomous reduction in vg-QE expression. The current results suggest that Ubx represses additional event(s) in non-DV cells to downregulate Vg expression. This is consistent with the recent report on cell-autonomous repression of vg-QE by ectopic Ubx in wing discs. It is proposed that Ubx inhibits the activation of Vg in non-DV cells at three different levels: (1) Wg in the posterior compartment; (2) event(s) downstream of Sgg that inhibit the stabilization of Arm, and (3) additional event(s) downstream of Arm in non-DV cells. In wing discs, Wg and a hitherto unknown DV component may function together to activate Vg in non-DV cells. Since Vg-autoregulation is not inhibited in haltere discs, it is possible that Ubx represses Vg activation in non-DV cells by interfering with the Wg-mediated activation of Vg and/or by repressing the activity of the unknown DV-signal molecule in the haltere (Prasad, 2003).

The Drosophila homolog of the human TEF-1 gene, scalloped (sd), is required for wing development. The Sd protein forms part of a transcriptional activation complex with the protein encoded by vestigial (vg) that, in turn, activates target genes important for wing formation. One sd function involves a regulatory feedback loop with vg and wingless (wg) that is essential in this process. The dorsal-ventral (D/V) margin-specific expression of wg is lost in sd mutant wing discs while the hinge-specific expression appears normal. In the context of wing development, a vg::sdTEA domain fusion produces a protein that mimics the wild-type SD/VG complex and restores the D/V boundary-specific expression of wg in a sd mutant background. Further, targeted expression of wg at the D/V boundary in the wing disc is able to partially rescue the sd mutant phenotype. It is inferred from this that sd could function in either the maintenance or induction of wg at the D/V border. Another functional role for sd is the establishment of sensory organ precursors (SOP) of the peripheral nervous system at the wing margin. Thus, the relationship between sd and senseless (sens) in the development of these cells was also examined, and it appears that sd must be functional for proper sens expression, and ultimately, for sensory organ precursor development (Srivastiva, 2003).

Because Ly mutations are gain of function alleles of sens and because Ly interacts genetically with sd, it is possible that this could result in alterations of Sens protein levels in sd mutant wing discs. Wing discs derived from wild-type flies and from flies harboring sd58 were stained with an anti-Sens antibody. In wild-type discs, Sens is localized to the region fated to become the wing margin with higher levels at the anterior margin in SOP cells. In addition, sens is also expressed in other SOPs distributed throughout the wing disc. In sd58 discs, the wing margin-specific expression of sens is completely lost, but expression in other SOPs is unaffected. Substantial margin-specific expression is restored when the vg::sdTEA fusion construct is expressed in sd58 discs using a vg-Gal4 driver. That this restoration of Sens is not complete could be attributed to the amount of the fusion VG::SD TEA protein being produced from the transgene. However, this level of restoration is consistent with the notion that the fusion construct can restore the margin-specific expression of wg, and emphasizes the involvement of wg in specifying the formation of SOPs. The mutual enhancement of mutant wing phenotypes by sd and Ly mutations can also be explained based on the role of wg in SOP formation. Because sd mutations affect the margin-specific expression of wg, and in Ly mutations there is a repression of wg expression, it is predictable that in transheterozygotes the overall Wg signal is further reduced at the margin, resulting in the phenotypic enhancement of wing margin loss (Srivastiva, 2003).

In conclusion, a further characterization of the functions of the SD/VG complex during wing development is reported by analyzing the roles of sd, via the vg::sdTEA fusion during patterning by wg, during growth and during SOP development. In the narrow context of the D/V specific expression of wg, the SD/VG complex appears to act upstream of wg as evidenced by the rescue of the D/V WG stripe by the fusion construct and the rescue of sd wing mutations by the expression of exogenous WG. In addition, the relationship between sd and sens in the development of margin-specific bristles is clarified and the results show that sens needs sd function for proper development of the PNS organs. The current model for actions of the SD/VG complex during wing development, incorporating the new data herein, is that the SD/VG complex either induces or maintains the expression of Wg. This, in turn, causes expression of Sd and Vg to promote cell proliferation in the wing pouch. At the D/V boundary Wg also mediates the expression of sens via its actions on the achaete scute (AS-C) complex that, in the presence of Sd, helps to specify the SOP fate (Srivastiva, 2003).

Koshikawa, S., Giorgianni, M. W., Vaccaro, K., Kassner, V. A., Yoder, J. H., Werner, T. and Carroll, S. B. (2015). Gain of cis-regulatory activities underlies novel domains of wingless gene expression in Drosophila. Proc Natl Acad Sci U S A 112: 7524-7529. PubMed ID: 26034272

Gain of cis-regulatory activities underlies novel domains of wingless gene expression in Drosophila

Changes in gene expression during animal development are largely responsible for the evolution of morphological diversity. However, the genetic and molecular mechanisms responsible for the origins of new gene-expression domains have been difficult to elucidate. This study sought to identify molecular events underlying the origins of three novel features of wingless (wg) gene expression that are associated with distinct pigmentation patterns in Drosophila guttifera. The activity of cis-regulatory sequences (enhancers) across the wg locus in D. guttifera and Drosophila melanogaster were compared, and strong functional conservation was found among the enhancers that control similar patterns of wg expression in larval imaginal discs that are essential for appendage development. For pupal tissues, however, three novel wg enhancer activities were found in D. guttifera associated with novel domains of wg expression, including two enhancers located surprisingly far away in an intron of the distant Wnt10 gene. Detailed analysis of one enhancer (the vein-tip enhancer) revealed that it overlapped with a region controlling wg expression in wing crossveins (crossvein enhancer) in D. guttifera and other species. These results indicate that one novel domain of wg expression in D. guttifera wings evolved by co-opting pre-existing regulatory sequences governing gene activity in the developing wing. It is suggested that the modification of existing enhancers is a common path to the evolution of new gene-expression domains and enhancers (Koshikawa, 2015).

A large body of comparative studies has shown that changes in the spatiotemporal expression of toolkit genes and the target genes they regulate correlate with the evolution of morphological traits. In a considerable number of instances, these spatiotemporal changes in gene expression have been demonstrated to involve the modification of enhancers. However, there are relatively few cases in which the origins of new enhancers have been elucidated, and none involving regulatory genes themselves (Koshikawa, 2015).

This study has shown that three novel domains of wg expression in D. guttifera are governed by three novel enhancers, respectively. The evolution of wg cis-regulatory sequences within the D. guttifera lineage played a role in the gain of each enhancer activity, and the evolution of trans-acting regulatory factors was also necessary for the activity of two elements (gutCS and gutTS). Detailed analysis of the D. guttifera vein-tip enhancer revealed that it evolved within another conserved enhancer, whereas two other enhancers (the campaniform sensilla and thoracic stripe enhancers) arose within in an intron of the distant Wnt10 locus. These results bear on the understanding of the mechanisms underlying the evolution of new enhancers and domains of gene expression (Koshikawa, 2015).

The D. guttifera vein-tip enhancer activity was localized within a 756-bp DNA segment that was also active in the developing pupal crossveins. This DNA segment is orthologous to segments of DNA in D. melanogaster and D. deflecta that were only active in the crossveins. The segments are all collinear, and contain numerous blocks of identical sequence, which suggests that the vein-tip enhancer activity evolved within the pre-existing crossvein enhancer (Koshikawa, 2015).

One explanation for the presence of two activities in this one fragment is that they share functional sites: that is, binding sites for common transcription factors. Because both activities appear in the pupal wing, it is likely that they use common tissue-specific (wing) and temporal (pupal) inputs. The evolution of a new activity in the vein tips could have arisen through the addition of DNA-binding sites for transcription factors that were already present active in cells at vein tips. In this scenario, the novel enhancer activity would have resulted from the evolutionary co-option of an existing enhancer (Koshikawa, 2015).

There is precedent for multifunctional enhancers and for this mechanism of co-option. For example, one enhancer of the D. melanogaster even-skipped gene governs two domains of gene expression that are controlled by shared inputs. In addition, it has been demonstrated that a novel optic lobe enhancer of the Drosophila santomea Neprilysin-1 gene arose via co-option of an existing enhancer. Moreover, it was shown that co-option had occurred in just a few mutational steps. The co-option of existing elements is an attractive explanation for the evolution of novel enhancers because it requires a relatively short mutational path (Koshikawa, 2015).

One surprising property of enhancers is their ability to control gene transcription at promoters located at considerable linear distances away in the genome. For example, the enhancer that drives Sonic hedgehog (Shh) expression in the developing amniote limb bud is located in the intron of another gene ~1 Mb from the Shh locus. A growing body of evidence indicates that long segments of DNA are looped out in accommodating long-range enhancer-promoter interactions. The ability of enhancers to act over such long ranges suggests that new enhancers could evolve at considerable distances from the promoters that they regulate (Koshikawa, 2015).

This study identified two enhancers in an intron of the D. guttifera Wnt10 gene that control transcription of the wg gene from a distance of ~70 kb, and separated by the Wnt6 locus. The data suggest that the gutTS enhancer preferentially regulates wg transcription and not Wnt10 or Wnt6 transcription, although the authors cannot explain this preference. The origins of the gutCS and gutTS enhancers are not as clear as the vein-tip enhancer. No pupal enhancer activity was detected in the orthologous DNA segments of D. melanogaster, there was no evidence of enhancer co-option. Nor were any obvious insertions found in these DNA segments such as a transposon. Nevertheless, the discovery of these novel, distant elements reflects the functional flexibility of cis-regulatory elements and their contribution to the evolution of gene regulation and morphological diversity (Koshikawa, 2015).

Refinement of wingless expression Defective proventriculus

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 dve gene is essential for proventriculus development and is initially triggered by the Wg signal in its primordium. The expression of wg and dve initially overlaps, but then segregates into adjacent but exclusive domains. The function of dve in regulating wg expression was examined. In the absence of dve, wg expression expands posteriorly to the region that normally expresses dve. This indicates that dve defines the posterior border of wg expression by repressing its expression. Thus, Wg refines its expression domain via dve gene activation in the embryonic proventriculus (Nakagoshi, 2002).

Wg refines its own expression along the D-V boundary in wing imaginal discs. The above result of the dve function in the embryonic proventriculus led to an examination of the possibility that dve acts in the refinement of wg expression at the D-V boundary. The expression of dve in wing discs was examined. At early to mid-third larval instar (48-24 h before pupariation, BP), dve expression begins throughout the prospective wing pouch, which overlaps partly with wg expression. The coexpression of dve and wg is obvious until mid-third instar, and subsequently, dve is excluded from the D-V boundary at mid- to late third instar (24-12 h BP). At this stage, dve is expressed complementary to wg. The activation of wg and repression of dve are both mediated by Notch (N) signaling at the D-V boundary. At late third instar (12-0 h BP), dve expression is reduced in the distal region, whereas it remains strong at the proximal region of the wing pouch. The significance of this repression is unclear (Nakagoshi, 2002).

The embryonic expression of dve depends on the Wg signal in the proventriculus and on the Dpp signal in the middle midgut. Whether or not dve expression in wing discs depends on these signals was examined. When the Wg signal is ectopically activated in a ring pattern around a wing pouch under the control of the 30A-Gal4 driver, dve is ectopically activated only in cells at the intersection of the ring with the A-P boundary, which normally expresses Dpp. In contrast, activation of the Dpp signal in the same ring pattern results in the ectopic expression of dve only at the D-V boundary, where Wg is normally expressed. Thus, the combined Wg and Dpp signals appear to induce dve expression in wing discs. To examine this possibility, flip-out recombination clones were generated that simultaneously express an activated form of the Dpp receptor (TkvQ253D) and that of a Wg signaling molecule (DeltaArm). Some of these clones induced ectopic Dve expression autonomously outside the compartment boundaries. These results strongly support the above notion that the combined activities of Wg and Dpp signals induce dve expression rather than other signals generated at compartment boundaries (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).

In both wing discs and the proventriculus, the initially overlapped expression of wg and dve becomes segregated into complementary patterns via the ability of Dve to suppress wg gene expression, which leads to refinement of the border of the wg-expressing domain. In wing discs, several different mechanisms limit wg expression to the D-V boundary: (1) restriction of N activation to the D-V boundary, which requires the Fringe function, and a positive feedback loop between Ser and Dl expression; (2) the inhibition of N signaling in Ser- and Dl-expressing cells in a dominant-negative manner; (3) the local suppression by the Wg signal itself near the D-V boundary. This study has revealed a feedback mechanism by which a Wg-induced gene product refines the source of wg expression to shape a morphogen gradient. Refinement of wg expression is important to specify the structure of the wing margin. In dsh mutant clones that abut the D-V boundary, wg expression expands. The dve mutant clone encompassing the D-V boundary also allows the expansion of wg expression, as observed in dsh mutant clones. The action of Dve in the refinement of wg expression appears to attenuate N-mediated gene expression. How is the Dve function related to the Wg signaling cascade in this process? When dsh mutant clones are created so as to abut the D-V boundary, dve expression is still observed in such clones. These observations for dsh clones suggest that Dve activity in the absence of the Wg signal input is insufficient to refine wg expression. However, when dve mutant clones are created adjacent to the D-V boundary, wg expression is expanded within the clones, but Wg-dependent accumulation of Ser seems to be normal. Thus, the Dsh-mediated Wg signal also appears to be insufficient to refine wg expression in the absence of Dve. Taken together, both Wg signaling and Dve appear to be necessary for the refinement of wg. There might be some interaction between Dve and Wg signaling downstream of N (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).

Nab controls the activity of the zinc-finger transcription factors Squeeze and Rotund in Drosophila development. Rotund in turn regulates wingless

Nab proteins form an evolutionarily conserved family of transcriptional co-regulators implicated in multiple developmental events in various organisms. They lack DNA-binding domains and act by associating with other transcription factors, but their precise roles in development are not known. This study analyzed the role of nab in Drosophila development. By employing genetic approaches it was found that nab is required for proximodistal patterning of the wing imaginal disc and also for determining specific neuronal fates in the embryonic CNS. Two partners of Nab were identified: the zinc-finger transcription factors Rotund and Squeeze. Nab is co-expressed with squeeze in a subset of neurons in the embryonic ventral nerve cord and with rotund in a circular domain of the distal-most area of the wing disc. These results indicate that Nab is a co-activator of Squeeze and is required to limit the number of neurons that express the LIM-homeodomain gene apterous and to specify Tv neuronal fate. Conversely, Nab is a co-repressor of Rotund in wing development and is required to limit the expression of wingless (wg) in the wing hinge, where wg plays a mitogenic role. Pull-down assays show that Nab binds directly to Rotund and Squeeze via its conserved C-terminal domain. Two mechanisms are described by which the activation of wg expression by Rotund in the wing hinge is repressed in the distal wing (Félix, 2007).

Precise temporal and spatial control of gene transcription is crucial for development. Sequence-specific DNA-binding factors and their association with a variety of modulator proteins, the co-factors, achieve this control. Co-factors do not bind DNA but act as adaptors between DNA-binding factors and other proteins. A number of transcription factors have been characterized, many of which act by recruiting multiprotein complexes with chromatin-modifying activities. By recruiting co-factors, a DNA-binding protein can act as co-activator or as co-repressor depending on the context. An example of a co-repressor is the retinoblastoma protein that converts the E2F transcription factor into a repressor of cell-cycle genes. The identification of co-factors and the determination of their precise roles are crucial for understanding the mechanisms that govern development (Félix, 2007).

Nab (NGFI-A-binding protein) proteins form an evolutionarily conserved family of transcriptional regulators. Nab was originally identified in mouse as a strong co-repressor by virtue of its capacity to interact directly with the Cys2-His2 zinc-finger transcription factor Egr1 (Krox24; NGFI-A) and inhibit its activity. Two Nab genes, Nab1 and Nab2, have been identified in vertebrates. Nab proteins do not bind DNA but they can repress (Svaren, 1998) or activate (Sevetson, 2000) gene expression by interacting with Egr transcription factors. Nab proteins have two regions of strong homology: NCD1 and NCD2. The NCD1 domain interacts with the R1 domain of Egr1 (Svaren, 1998). The NCD2 domain is required for transcriptional regulation (Swirnoff, 1998). Mice harboring targeted deletions of Nab1 and Nab2 have phenotypes very similar to Egr2 (Krox20)-deficient mice, suggesting that they act as co-activators of this gene (Le, 2005). In zebrafish, egr2 controls expression of the Nab gene homologs in the r3 and r5 rhombomeres of the developing hindbrain (Mechta-Grigoriou, 2000). Egr2 has been implicated in determining the segmental identities of r3 and r5 by controlling the expression of several target genes as well as cell proliferation. Misexpression experiments suggest that Nab1/Nab2 antagonize Egr2 transcriptional activity by a negative-feedback regulatory loop. Nevertheless, Nab proteins might have additional functions as these experiments also led to alterations of the neural tube not found in Egr2-deficient embryos (Mechta-Grigoriou, 2000). Conversely, Egr2-deficient mice have a severe hindbrain segmentation defect that is not found in mice deficient in Nab1 and Nab2. Nab might also have Egr-independent functions in mice because, although epidermal hyperplasia has been observed in Nab1 Nab2 double mutant mice, this phenotype has not been observed in mice lacking any of the Egr proteins (Le, 2005; Félix, 2007 and references therein).

In Drosophila, only one Nab gene has been identified; it is highly homologous to vertebrate Nab genes in the NCD1 and NCD2 domains. Drosophila nab mutants are early larval lethal. Detection of nab transcripts by in situ hybridization indicates expression in a subset of neuroblasts of the embryonic and larval CNS and weak expression in imaginal discs (Clements, 2003). The role of Nab in Drosophila development is not known and so far no binding partner has been identified. This report shows that nab is a component of the combinatorial code that determines the number of neurons that express the gene apterous (ap) in embryonic neural development, and that nab specifies the Tv neuronal fate in the ap thoracic cluster of neurons (Félix, 2007).

In early larval development, the wing fate is established in the distal-most region of the wing disc by a combination of two factors: activation of the gene vestigial (vg) and repression of the gene teashirt (tsh). Later, in early third instar larvae, wingless (wg) is activated in a ring of cells (the inner ring, IR) that borders the vg expression domain in the presumptive wing region. It has been suggested that activation of the IR involves a signal from the vg-expressing cells to the adjacent cells. Interpretation of this signal by the adjacent cells requires the transcription factors encoded by rotund (rn) and nubbin (nub). Expression of wg in the IR plays a mitogenic role; hence, as a consequence of wg expression, cells proliferate and the IR moves away from the vg border. At a distance from the source of the signal that drives the initial activation, wg IR expression is maintained by an autoregulatory loop that involves homothorax (hth). It is thought that an additional mechanism distally represses wg IR expression and, in so doing, controls cell proliferation in the wing hinge. In this report, it is shown that during imaginal disc development, nab is strongly expressed in the wing presumptive domain under the control of vg, and that nab is required in proximodistal axis development to control the expression of wg in the wing hinge (Félix, 2007).

Two putative partners of Nab have been identified: Rn and Squeeze (Sqz). These proteins are members of the Krüppel family of zinc-finger proteins. Pull-down assays show that that Nab interacts with both proteins via a conserved C-terminal domain, and evidence is presented that Nab acts as co-activator of Sqz in embryo development and as co-repressor of Rn in wing development. Finally, it is proposed that there are two mechanisms to repress the activation of wg expression by Rn in the wing pouch: the first involves Nab as a co-repressor of Rn; the second involves Sqz as a competitor of Rn for binding to specific DNA target sites (Félix, 2007).

Antibody against Nab revealed a low level of expression in all imaginal discs. In late third instar wing discs, Nab was strongly expressed in a circular domain that delimits the expression of wg in the inner ring. Nab expression was first detected in early third instar larvae, in a group of cells of the distal-most wing, and was maintained throughout the remainder of the larval and pupal stages. There was a low level of expression in the rest of the wing disc, except in the hinge where there was no detectable expression. In the eye disc, Nab was detected in a stripe corresponding to the morphogenetic furrow (Félix, 2007).

It was asked whether, as with other genes involved in proximodistal patterning, nab expression in the wing is dependent upon vg. No expression of nab was detected in the distal wing of vg83b27r wing discs. However, nab is ectopically expressed in clones of vg-expressing cells. Together, these results indicate that the expression of nab in the wing depends on vg. In wild-type discs and vg ectopic-expressing clones, the domain of nab expression is broader than that of vg, pointing to the nonautonomous control of nab expression. A similar mechanism has been proposed for other genes, such as rn and nub, whose expression depends on vg. Expression of vg in the wing starts in second instar larvae, whereas nab expression is first detected at early third instar. This suggests that some other mechanism controls the initiation of nab expression (Félix, 2007).

The nabSH143 allele is a P(lacW) insertion in the first exon. Most larvae homozygous for this allele die in first instar. Thus, to analyze the role of nab in development of the wing nabSH143 homozygous mutant clones were generated by mitotic recombination using the FLP/FRT mitotic recombination system. In the wing, these clones activated wg ectopically. However, it was noted that not all the clones activated wg. It is therefore possible that there is functional redundancy between Nab and other proteins (Félix, 2007).

Two enhancers drive the expression of wg in the wing: the wing margin enhancer, which is activated by the Notch signaling pathway, and the spade (spd) enhancer, which drives wg expression in the inner ring. Previous results suggest that activation by the latter depends on a nonautonomous signal coming from the vg-expressing cells. nab co-expresses with wg in the wing margin and abuts on wg expression in the inner ring. It was therefore assumed that Nab should repress activation of the inner ring enhancer derepressed in nab clones. To obtain independent evidence that the inner ring enhancer is being activated, tests were performed to see whether other genes activated in the wing margin were activated in the nab clones. To this end, cut (ct) was analyzed, and no ectopic expression was detected. It has been reported that wg expression can be detected in the wing after induction of cell death. To detect cell death in the nab clones, use was maed of an antibody that recognizes the activated form of Caspase 3, but no cell death was detected. These results, together with the pattern of expression, strongly suggest that the inner ring enhancer is being activated in the nab clones and, therefore, that in normal development Nab acts as a repressor of the wg inner ring enhancer in the distal wing. To confirm this hypothesis, nab was expressed ectopically in the inner ring domain using the nubGAL4 driver, which is expressed in a circular domain that includes the inner ring. In nubGAL4>UASnab larvae, expression of wg in the inner ring was lost, whereas its expression in the wing margin was not affected. Clones of nab-expressing cells were generated, and it was found that wg expression was cell-autonomously lost in these clones, whereas wg expression in the wing margin was not affected. In the light of these results, it is proposed that the function of nab in wing development is to delimit, distally, the domain of wg expression in the inner ring by inhibiting the mechanism of inner ring activation (Félix, 2007).

The mammalian Nab partner Egr1 contains an inhibitory domain called R1. When this domain is deleted the transcriptional activity of Egr1 increases 15-fold. It has been shown that the R1 domain mediates a functional interaction between Nab and Egr1. Since no R1 domain has been identified in the fly genome and all the previously identified partners of Nab are Krüppel-type zinc-finger transcription factors, transcription factors of the Krüppel family expressed in the wing were examined as potential Nab partners in the fly. The gene rn encodes a Krüppel-like zinc-finger protein that in the wing is expressed in a circular domain slightly broader than the nab domain. The wg inner ring enhancer is active only in the cells that express rn and that do not express nab. Previous studies have shown that Rn is required for activation of the wingless spd enhancer. The results so far suggest that Rn could be a partner of Nab in the wing: first, nab is expressed in the rn-expressing cells that do not express wg; second, nab loss-of-function clones contain ectopic Wg; and third, nab misexpression represses the wg inner ring enhancer (Félix, 2007).

rn was also expressed in leg discs in a broad ring that corresponded to three tarsal segments (T2-4). In rn mutant legs, the T2-4 tarsal segments were deleted. It would therefore be expected that if Rn were a partner of Nab, ectopic expression of nab in the leg would generate the same phenotype as the lack of Rn. This proved to be the case when nab was misexpressed in the rn expression domain under the control of the rnGal4 driver. The phenotype of these flies was indistinguishable from the rn mutant phenotype in both legs and wings. The specificity of this interaction was examined by rescuing the phenotype caused by nab misexpression by co-expressing rn (rnGal4>UASrn+UASnab), as well as by misexpressing nab in a broader domain using Distal-less Gal4 (DllGal4), which is expressed from mid-tibia to distal leg (DllGal4>UASrn). In the first experiment, the phenotype was markedly reduced in both wing and leg, indicating that adding more rn antagonizes the inhibitory effect of nab misexpression. In the second experiment, although nab was misexpressed in a broader domain of the leg, the phenotype was unaltered and was restricted to the area where rn was expressed. Taken together, these results support a role for Rn as a potential partner of Nab and that Nab acts as co-repressor of Rn function in the cells where both are expressed. The rn mutant phenotype in the wing is caused by the loss of wg expression in the inner ring. Whether wg expression was affected in rnGal4 UASnab and rnGal4 UASnab UASrn wings was examined. In the first case, the inner ring was found to be absent, whereas in the second it was partially restored. In summary, these results indicate that Nab functions in wing development by antagonizing the transcriptional activation function of Rn (Félix, 2007).

In order to analyze the molecular role of Nab as a co-factor of Sqz and Rn GST pull-down assays were performed. The complete nab cDNA was cloned in a glutathione S-transferase (GST) vector and incubated with radioactively labeled Rn or Sqz. Nab-GST, but not GST alone, readily retained [35S]methionine-labeled Rn or Sqz. Rn and Sqz share a C-terminal domain of 32 amino acids with a homology greater than 80%. To further test whether this domain mediates the interaction with Nab, the pull-down assays were repeated with an [35S]Rn in which the C-terminal domain was deleted. This deletion removes the region from amino acid 894 to the C-terminus (943) of the protein (RnΔ894). The ability of Nab-GST to retain the [35S]RnΔ894 was notably reduced. It is concluded that this conserved domain mediates the direct interaction of Nab with Rn and Sqz. To further test whether the C-terminal domain is sufficient to mediate this interaction, the Nab-GST was incubated with a 32 amino acid peptide containing just the sequence of the C-terminal domain. Nab-GST did not retain the peptide, indicating that the C-terminal domain is not sufficient to mediate Nab-Rn interaction. Since no other conserved domains have been identified between Rn and Sqz besides the zinc-finger and C-terminal domains, it is considered that either secondary structure or an additional modification of the protein is required for binding Nab. In order to provide an in vivo functional test of this hypothesis, the rnΔ894 fragment was cloned into the pUAST vector and clones of cells misexpressing UASrnΔ894 were generated (Act>Gal4>UASrnΔ894). These clones activated the expression of wg throughout the wing pouch. As a control experiment, the wild-type version of rn (Act>Gal4>UASrn) was misexpressed. These clones only activated wg expression in the wing hinge, outside of the nab expression domain (Félix, 2007).

sqz expression was examined in the wing disc. Because of the high degree of sequence homology between rn and sqz and to avoid interference with the rn mRNA present in the wing, in situ hybridization assay was performed in rn mutant discs. sqz expression was detected by in situ hybridization in rn20 wing discs in a circular pattern that faded off laterally and whose proximal limit coincided with the limit of vg expression; this corresponded to the distal-most wing fold. To determine whether sqz plays a role in wing development the phenotype was analyzed of sqz mutant clones induced by mitotic recombination. These clones had no adult phenotype, nor did they alter the expression of wg. Since Sqz and Rn share zinc-finger and the C-terminal domains and differ in their N-terminal domains, it was wondered whether the roles of Sqz and Nab might be functionally redundant, both repressing Rn activity but by different mechanisms: Nab would repress Rn activity by direct binding to Rn protein as a co-repressor, whereas Sqz would compete for binding to the same DNA targets. To test this hypothesis, the effect was analyzed of misexpressing sqz in the rn expression domain. rnGal4/UASsqz UASGFP flies had small deletions of the wing hinge and shortened legs, a phenotype that resembles the nab misexpression and rn mutant phenotypes. In agreement with these results, wg expression in the inner ring was downregulated in rnGal4/UASsqz wing discs. An alternative explanation for these results is that sqz activates nab expression, but no nab misexpression was seen in this experiment. It is suggested that there must be some functional redundancy, irrespective of whether Nab and Sqz play similar roles in the wing by repressing Rn activity, and this would account for the low penetrance of the nab mutant clones. Because nab and sqz map on different chromosome arms it was not possible to generate double-mutant clones. Therefore nabSH143 homozygous clones were generated in a sqzlacZ/+ background. In this situation, the frequency of clones misexpressing wg increased by 38%). It was also noted that the clones that showed wg misexpression were preferentially located in the lateral-most regions of the wing, which correspond to the regions with the lowest levels of sqz expression. Taken together, these observations support the hypothesis that Nab and Sqz play similar roles in wing development: Nab as a co-repressor of Rn via its conserved C-terminal domain, and Sqz by competing with Rn for binding to its DNA targets. This function of Sqz would differ from its above-proposed role as a transcriptional activator in CNS development, and would not require Nab (Félix, 2007).

This study presented evidence that Nab is a co-activator of Sqz. This protein has been implicated in two aspects of embryonic ventral nerve cord development: first, in a Notch-dependent lateral inhibition mechanism that specifies the number of cells that express ap in the ap thoracic neuronal cluster; and second, in the specification of the Tv neuronal fate. nab and sqz are co-expressed in a subset of neurons, including several of the ap cluster, as well as the Tv neuron. nab loss-of-function embryos reproduce all the phenotypes of sqz loss-of-function embryos: additional cells express ap in the cluster and the Tv neuronal fate is lost. In addition, in both nab and sqz mutants an increased number of cells in the clusters express dimm. These findings indicate that Nab is required for all identified Sqz functions in embryonic development. Although this analysis focused on the ap thoracic cluster of neurons, both sqz and nab are co-expressed in many cells in the ventral nerve cord and others expressed either sqz or nab. But no other functions have been identified for sqz and it is not known how the expression of sqz is controlled. It has been reported that the expression of nab in the ventral nerve cord depends on the gene castor (Clements, 2003). Thus, the results presented in this study reveal greater complexity in the mechanisms of neuronal fate specification. The combined expression of genes, whose expression is individually activated by different mechanisms, is required to determine specific neuronal fates (Félix, 2007).

Sqz and Rn share two regions of strong homology: the zinc finger and a stretch of 32 amino acids in the C-terminal domain. By contrast, only rn has a long N-terminal domain. The results indicate that the C-terminal domain mediates the interaction with Nab. By GST pull-down assays, it was shown that Nab binds to the full-length Rn protein but not to the RnΔ894 version, and clones of cells misexpressing rnΔ894 activate wg expression in the nab expression domain. The similarity between sqz misexpression and rn loss-of-function phenotypes in leg and wing suggests that Sqz acts like a dominant-negative form of Rn in the rn domain: both proteins would bind to the same target sites but have opposite effects, and the results indicate that this role of Sqz would not require interaction with Nab. It is possible that the long N-terminal region of Rn is involved in interaction with other partners specifically required for Rn function (Félix, 2007).

Thus, these results indicate that Nab has a dual role as co-repressor of Rn and co-activator of Sqz. Previous studies in vertebrates also suggest that Nab is involved in both repression and activation of transcription. Co-repressors are proteins that bridge the interaction of the repressor with its target. Two main co-repressors have been identified in Drosophila: Groucho and CtBP. CtBP binds to a specific sequence motif (P-DLS-K) that has been found in the sequence of three repressors present in the early embryo: Snail, Knirps and Krüppel. All three are zinc-finger transcription factors, and genetic evidence suggests that they all require CtBP to repress their targets. Neither Rn nor Sqz have a CtBP-binding motif but one has been in Nab (P-DLS--K). Although the functional significance of this motif remains to be confirmed, itis suggested that Nab is acting as a bridge between Rn and CtBP (Félix, 2007).

Control of Drosophila wing growth by the vestigial quadrant enhancer: A Wg-dependent feed-forward circuit

Following segregation of the Drosophila wing imaginal disc into dorsal (D) and ventral (V) compartments, the wing primordium is specified by activity of the selector gene vestigial (vg). Evidence is presented that vg expression is itself driven by three distinct inputs: (1) short-range DSL (Delta/Serrate/LAG-2)-Notch signaling across the D-V compartment boundary; (2) long-range Wg signaling from cells abutting the D-V compartment boundary; and (3) a short-range signal sent by vg-expressing cells that entrains neighboring cells to upregulate vg in response to Wg. These inputs define a feed-forward mechanism of vg autoregulation that initiates in D-V border cells and propagates from cell to cell by reiterative cycles of vg upregulation. Evidence is provided that this feed-forward mechanism is required for normal wing growth and is mediated by two distinct enhancers in the vg gene. The first is a newly defined 'priming' enhancer (PE), that provides cryptic, low levels of Vg in most or all cells of the wing disc. The second is the previously defined quadrant enhancer (QE), which activates by the combined action of Wg and the short-range vg-dependent entraining signal, but only if the responding cells are already primed by low-level Vg activity. Thus, entrainment and priming constitute distinct signaling and responding events in the Wg-dependent feed-forward circuit of vg autoregulation mediated by the QE. It is posited that Wg controls the expansion of the wing primordium following D-V segregation by fueling this autoregulatory mechanism (Zecca, 2007b).

The dramatic expansion of the Drosophila wing primordium following the D-V compartmental segregation provides a valuable paradigm of organ growth. Growth in this context is manifest as a rapid ~200-fold expansion of the population of cells expressing the wing selector gene vg, under the control of the long-range morphogens Wg and Dpp. This system thus poses the fundamental question of how morphogens organize the increase in the mass and number of cells that express a given selector gene, to yield an adult appendage of appropriate size and shape (Zecca, 2007b).

A novel autoregulatory property of vg has been defined that appears crucial for this process. Evidence is presented that vg-expressing cells send a short-range feed-forward signal that neighboring cells must receive in order to express vg in response to Wg. This led to a hypothesis that Wg controls wing development by fueling this non-autonomous autoregulatory mechanism. This study establishes that the vg quadrant enhancer (QE) can mediate vg autoregulation in response to Wg and then uses a transgene that expresses Vg under QE control to provide a proof-in-principle that wing growth normally depends on the operation of the autoregulatory circuit (Zecca, 2007b).

Wing growth following D-V segregation is envisioned as an outcome of vg autoregulation, primed by cryptic, low-level Vg in all cells that is seeded by DSL-Notch-mediated induction of specialized D-V border cells that express high levels of vg and wg, and then propagated by the capacity of vg-expressing cells to induce and sustain vg expression in neighboring cells in response to Wg. In support, it has been possible to restore wing growth in vg0 discs in a step-wise manner by the sequential addition of transgenes that provide, first priming (rp49-vg), then initiation (BE-vgGFP), and finally feed-forward propagation (5XQE>vg). Priming is necessary but not sufficient for wing development, initiation provides local rescue of wing tissue, and propagation is responsible for the dramatic expansion in the size of the prospective wing (Zecca, 2007b).

Importantly, priming and feed-forward signaling are linked in a self-reinforcing autoregulatory circuit in which a gain in either input leads to an amplification of both. It is envisaged that the QE normally integrates both the priming and feed-forward inputs together with Wg in a way that is sensitive to the initial strength of each input and subject to autoamplification. For example, in the 'resting' state, cells have a low level of priming that falls beneath the minimal threshold necessary to specify the wing state or generate appreciable feed-forward signal. Upon receipt of sufficient Wg and feed-forward signal, the level of Vg expression rises, crossing the threshold defining the wing state and enhancing the capacity of the responding cell both to send and to receive the feed-forward signal. Amplification of this circuit then leads to the maximum output of Vg expression and feed-forward signaling that can be supported by the strength of the Wg signal received (Zecca, 2007b).

The self-reinforcing nature of this autoregulatory circuit, both between and within cells, helps explain how Wg spreading from D-V border cells normally fuels the expansion of the population of vg-expressing cells. It also helps account for the unexpected responses observed in experiments using the rp49-vg, BE-vgGFP and 5XQE>vg transgenes to mimic the normal priming, initiation and feed-forward inputs. All of these transgenes depend on heterologous promoters and potentially complex enhancer elements operating outside of their normal genomic contexts. Consequently, weak, inappropriate activities of any of these transgenes (e.g. cryptic priming by BE-vgGFP and 5XQE>vg transgenes, or faint QE activity of the BE-vgGFP transgene) could be amplified by the autoregulatory circuitry, yielding spatially inappropriate responses. Nevertheless, despite these experimental limitations, the results indicate that the major factor governing the expansion of the wing primordium is feed-forward autoregulation mediated by the QE (Zecca, 2007b).

Wing growth does not depend solely on the capacity of Wg to recruit and maintain cells in the wing primordium by fueling vg autoregulation. Instead, even when wing pouch cells are supplied constitutively with exogenous Vg (thus bypassing the requirement for vg autoregulation), they still depend on continuous Wg input to survive and grow within the context of the wing primordium. This is in contrast to cells in the more proximal hinge and notum primordia, which survive and grow without Wg input. Thus, Wg appears to promote wing growth via two distinct mechanisms: by continuously 'selecting' which cells enter and remain within the wing primordium, and by allowing the survival and growth of cells so selected. The relative contributions of these two mechanisms cannot be distinguished. However both appear essential, as cells fail to enter, or stay, within the wing primordium when either one is eliminated (Zecca, 2007b).

Wing growth depends not only on Wg emanating from D-V border cells, but also on Dpp secreted by A compartment cells along the A-P compartment boundary, suggesting that the QE might mediate feed-forward autoregulation in response to Dpp, as well as Wg. In support, the QE contains binding sites for the Dpp transducer Mad, and there is evidence that these sites, as well as Mad itself, contribute to QE activity. Moreover, clones of cells that cannot transduce Dpp behave like those that cannot transduce Wg: they cease to express Vg and are lost specifically from the wing primordium, in contrast to clones located in the more proximal hinge and notum primordia. Hence, it is likely that Dpp and Wg act together to fuel the feed-forward autoregulatory circuit, and by so doing, regulate the size and shape of the developing wing (Zecca, 2007b).

The ability of Wg, and potentially Dpp, to promote wing growth by fueling a non-autonomous autoregulatory circuit of vg expression is, to our knowledge, novel, and has implications for the control of organ growth by morphogens. As epitomized by the developing wing, a long-standing enigma is that gradient morphogens drive relatively uniform growth and proliferation across a tissue at the same time that they function in a concentration-dependent manner to organize complex patterns of gene expression and cell differentiation. It is suggested that a minimum threshold level of morphogen might be sufficient to fuel both feed-forward autoregulation of organ selector genes and the growth and proliferation of cells so selected. Accordingly, organ growth would be governed primarily by the progressive expansion in the range of morphogen (a process that might itself depend on the ability of morphogen to regulate expression of its receptors and other binding proteins) and by any boundary conditions that limit the availability and capacity of surrounding cells to respond (Zecca, 2007b).

SoxF is part of a novel negative-feedback loop in the wingless pathway that controls proliferation in the Drosophila wing disc

Wnt molecules act as mitogenic signals during the development of multiple organs, and the aberrant activity of their pathway is often associated with cancer. Therefore, the production of Wnts and the activity of their signaling pathway must be tightly regulated. This study has investigated the mechanisms of this regulation in the Drosophila hinge, a domain within the wing imaginal disc that depends on the fly Wnt1 ortholog wingless (wg) for its proliferation. The results uncover a new feedback loop in the wg pathway in which the spatially restricted activation of the Sox gene SoxF (Sox15) by wg represses its own transcription, thus ensuring tight regulation of growth control. rotund, a wing proximodistal patterning gene, excludes SoxF from a thin rim of cells. These cells are thus allowed to express wg and act as the source of mitogenic signal. This novel mode of action of a Sox gene on the Wnt pathway -- through transcriptional repression of a Wnt gene -- might be relevant to human disease, as loss of human SoxF genes has been implicated in colon carcinoma (Dichtel-Danjoy, 2009).

One of the long-standing questions in biology is how organ growth is coordinated with tissue patterning. Research during recent decades has shown that a limited set of signals and signaling pathways control this coordination. Some of these signals are mitogenic, and their production at specific sites, called signaling centers, links spatial information to cell proliferation within developing organs. Normal organ growth not only needs mitogens, but also mechanisms to control their production, transport, reception and/or transduction to ensure that proliferation is limited in space and time. Alterations in these control mechanisms often lead to disease (Dichtel-Danjoy, 2009).

The Wnt/β-catenin signaling pathway promotes cell proliferation during normal development and disease. Wnts are lipid-modified glycosylated signaling molecules that can reach distant cells. Binding of Wnts to the receptor complex [composed of a Frizzled family receptor and an Arrow (LRP) co-receptor] results in the stabilization of the transcriptional co-factor β-catenin [armadillo (arm) in Drosophila]. Thereby, β-catenin/Arm accumulates in the nucleus, where it associates with Tcf/LEF DNA-binding transcription factors to regulate the expression of Wnt target genes. Research in a number of model organisms has demonstrated that the Wnt/β-catenin pathway controls cell proliferation in a variety of tissues, including the nervous system and the progenitors of the intestine and hematopoietic systems in mammals, and during imaginal disc development in Drosophila. It is also known that most colorectal tumors, and a number of other tumor types, are caused by aberrant Wnt/β-catenin signaling, which underlines the necessity of tight regulation of this pathway (Dichtel-Danjoy, 2009).

The range and intensity of the signaling elicited by Wnt molecules have been shown to be regulated by many different mechanisms, including negative-feedback loops. These have been particularly well studied for the main Drosophila Wnt gene, wingless (wg). wg is required in the imaginal discs for the growth and patterning of the adult body structures. wg signaling results in the downregulation of its two receptors, Dfz-2 (fz2 - FlyBase) and fz and in the upregulation of Dfz-3 (fz3 - FlyBase), a non-productive low-affinity receptor, and of the extracellular Wg inhibitor Notum (wingful). Intracellularly, high levels of wg/Wnt signaling induce the expression of two inhibitors of the pathway: naked cuticle and nemo. All these feedback loops result in an attenuation of the signal at the sites of maximal wg production and are generally implicated in all processes in which wg is required (Dichtel-Danjoy, 2009).

The Drosophila wing disc gives rise to the wing blade, the notum (body wall) and the hinge, which joins the wing blade to the body wall and articulates its movements. wg is expressed in two concentric rings in the hinge domain and has been shown to be required for the proliferation of hinge cells. Moreover, wg overexpression is sufficient to drive hinge overgrowths without causing major repatterning. Therefore, the precise regulation of the wg pathway is crucial to control the growth of the hinge. The mitogenic effect of wg on hinge cells contrasts with its effect on the neighboring wing pouch cells which, upon similar wg overexpression, are mostly driven into sensory organ differentiation. One prediction from these results is that the hinge-specific proliferative function of wg needs dedicated control mechanisms to ensure normal hinge size and shape. To identify these mechanisms, genes were sought that are differentially expressed in the hinge territory for a role in wg-mediated proliferation. SoxF (Sox15) belongs to the family of sequence-specific HMG Sox transcription factors and has been shown to be expressed in the prospective hinge of third larval stage (L3) wing discs). The functions of Sox genes have been extensively studied in mammals, in which they play essential roles during development. In addition, misregulation of Sox genes is often associated with cancer (Dichtel-Danjoy, 2009).

Only two of the eight Sox family genes present in the Drosophila genome have been studied in detail: Dichaete (D) and SoxNeuro (SoxN). They belong to the SoxB group and have prominent roles in embryonic segmentation and nervous system development. In addition, it has recently been shown that both genes negatively regulate the activity of the wg/Wnt pathway during cell fate specification in the embryonic epidermis (Dichtel-Danjoy, 2009 and references therein).

This paper reports that SoxF, which is the sole member of this Sox group in Drosophila, is also required to restrain wg signaling, but using a novel mechanism: the transcriptional repression of wg. In the absence of SoxF, wg transcription spreads through the hinge causing its overproliferation. SoxF is itself under the control of the canonical wg/Wnt pathway such that wg and SoxF regulate each other's transcription through a feedback loop. Moreover, the expression of rotund (rn), which is part of the proximodistal patterning mechanism of the wing disc, allows the exclusion of SoxF from a thin rim of cells, allowing them to express wg. Thereby, this rim becomes a spatially well-defined mitogen-producing center necessary to ensure normal hinge growth. This novel mode of action of a Sox gene on the Wnt pathway -- the transcriptional repression of a Wnt gene -- might be relevant to human disease, as loss of human SoxF genes has been implicated in colon carcinoma (Dichtel-Danjoy, 2009).

In order to determine the role played by SoxF during hinge development, a SoxF allele, Sox15KG09145 (now renamed SoxFKG09145) was characterized. The SoxFKG09145 allele carries an insertion of the P[SUPor-P] transposon in an intronic region of the gene, which also harbors the CG30071 transcript. Most homozygous SoxFKG09145 flies die as pharate adults, and escapers are weak with held-out wings. This latter phenotype is indicative of hinge defects. In fact, these flies show abnormal proximal hinge structures: the sclerites, the alula and the costa are affected. Although the insertion does not affect SoxF coding sequence, it was observed by RT-PCR and in situ hybridization that SoxF expression is completely lost in the wing disc of mutant L3 larvae. Sice this P-element carries insulator sequences, it was also checked by RT-PCR that expression of CG30071 and of the 5' neighboring gene, RpS23, was not affected by the insertion, which was indeed the case. This study has also generated new alleles by imprecise excision of the P transposon from the original allele. In addition to full revertants, more than ten mutant lines were isolated in which different lengths of intron sequences were deleted, without affecting the coding region, and which showed a range of phenotypic severity. These results suggest that this intronic region carries crucial elements for the regulation of SoxF expression. Some alleles were isolated that disrupt the coding sequence. Among them, SoxF26 is specific to the SoxF gene and deletes the first exon and part of the first large intron, and is therefore likely to be a null allele. This allele has the same phenotype as the initial insertion. In addition, the phenotype and escaper rates of individuals carrying SoxFKG09145 over a deficiency uncovering the SoxF locus, Df(2R)Exel7130, are the same as for homozygous SoxFKG09145 flies. Therefore, SoxFKG09145 behaves as a genetic null allele. It has been reported that SoxF is expressed in the embryonic Peripheral nervous system (PNS). Adult escapers of the molecular null allele SoxF26 exhibit, in addition to their abnormally folded wings, are also weak and die shortly after eclosion. Other hinge mutants, such as wg spd-fg, are much healthier. Therefore, it is possible that the larval lethality and weakness of adult escapers is due to abnormal PNS development (Dichtel-Danjoy, 2009).

This study describes a novel negative-feedback mechanism in the wg pathway that is required to restrain the expression of wg itself, and which is essential to control organ growth. During Drosophila development, the wg pathway often leads to the activation of genes that attenuate its signaling pathway. This is the case, for example, for Notum and Dfz-3, which are expressed in the wing disc in response to peak levels of signaling to reduce ligand availability for the Wg receptors, and for nemo, which acts intracellularly to block the signal transduction pathway. In all cases described, these negative-feedback components act in all domains of wg expression and none regulates wg expression at the transcriptional level. However, in the case investigated in this study, the putative transcription factor SoxF is activated non-autonomously by wg in a hinge-specific manner. SoxF in turn represses wg transcription driven by the wg spd-fg enhancer, thus restricting the production of wg to the thin inner ring (IR) domain. Interestingly, the SoxF phenotype is similar to those of dominant Dichaete (D) mutations. D is a SoxB gene not normally expressed in the wing disc. However, flies carrying dominant D mutations show reduced hinge structures. This phenotype is caused by ectopic D expression in the prospective hinge region of the disc. One of the salient features of D discs is the repression of the wg IR, which is reminiscent of the wg repression by SoxF described in this study. Therefore, and taking into account the similarity between Sox proteins in their HMG DNA-binding domain, the ectopic D might be mimicking the repression of wg that is normally exerted by SoxF (Dichtel-Danjoy, 2009).

The tight regulation of the growth of the hinge depends critically on the wg-induced activation of SoxF in the growing territory. Nevertheless, this activation is 'polarized' along the PD axis, taking place only in cells adjacent and proximal to the IR. It is proposed that this directionality in SoxF activation results from the mechanisms that pattern the wing disc along its PD axis. It has been suggested that wg is activated non-autonomously by a signal produced by the vg-expressing wing pouch cells, but excluded from them. This would generate a circular domain of wg expression surrounding the wing pouch. However, in the absence of SoxF, the domain of wg is abnormally broad and causes hinge overgrowth. This ectopic wg expression does not seem to result from a misregulation of hinge-specific genes: the expression of nub, tsh, hth and rn and their relative positioning in the hinge are unaffected in SoxF mutant discs. Therefore, it seems that in the absence of SoxF, hinge cells cannot respond to the wg activating signals with enough precision to give rise to a thin ring of wg expression. The results show that this precision is achieved through a double repression mechanism. First, wg activates its own transcriptional repressor, SoxF. This would lead to the extinction of wg expression if it were not for rn, which acts as a repressor of SoxF. Second, rn, by repressing SoxF, permits wg transcription. The result is that wg expression becomes restricted to a narrow circular stripe at the edge of the rn domain that provides a highly localized source of Wg. This signal activates, simultaneously and in the same cells, proliferation and the upregulation of SoxF, which restricts the production of the signal. Therefore, SoxF joins SoxN and SoxD (Sox102F - FlyBase)as the third Drosophila Sox known to antagonize the wg pathway. The vertebrate Sox proteins Sox9, XSox3 and XSox17 have also been shown to downregulate the Wnt/β-catenin pathway. Therefore, this antagonism seems evolutionarily conserved (Dichtel-Danjoy, 2009).

The relationship between SoxF genes, the wg/Wnt pathway and the control of tissue proliferation seems to extend to disease. The SoxF Sox17 is normally expressed in the gut epithelium where it downregulates Wnt signaling via degradation of β-catenin and TCF. In colon carcinomas, the expression of the SoxB gene Sox17 is often reduced, and this is associated with tissue overproliferation. Moreover, inactivation of the SoxE gene Sox9 leads to increased cell proliferation and hyperplasia in the mouse intestine. The authors concluded that Sox9 is essential for the fine-tuning of the transcriptional activity of the Wnt pathway. Interestingly, the expression of Sox9 is regulated by the Wnt pathway itself. These results in Drosophila point to the possibility that the transcriptional regulation of Wnt expression by Sox genes might be a common feature of this proliferation-associated feedback loop (Dichtel-Danjoy, 2009).

The ecdysone receptor controls the post-critical weight switch to nutrition-independent differentiation in Drosophila wing imaginal discs

In holometabolous insects, a species-specific size, known as critical weight, needs to be reached for metamorphosis to be initiated in the absence of further nutritional input. Reaching critical weight depends on the insulin-dependent growth of the prothoracic glands (PGs) in Drosophila larvae. Because the PGs produce the molting hormone ecdysone, it is hypothesized that ecdysone signaling switches the larva to a nutrition-independent mode of development post-critical weight. Wing discs from pre-critical weight larvae [5 hours after third instar ecdysis (AL3E)] fed on sucrose alone showed suppressed Wingless (Wg), Cut (Ct) and Senseless (Sens) expression. Post-critical weight, a sucrose-only diet no longer suppresses the expression of these proteins. Feeding larvae that exhibit enhanced insulin signaling in their PGs at 5 hours AL3E on sucrose alone produced wing discs with precocious Wg, Ct and Sens expression. In addition, knocking down the Ecdysone receptor (EcR) selectively in the discs also promotes premature Wg, Cut and Sens expression in the wing discs of sucrose-fed pre-critical weight larvae. EcR is involved in gene activation when ecdysone is present, and gene repression in its absence. Thus, knocking down EcR derepresses genes that are normally repressed by unliganded EcR, thereby allowing wing patterning to progress. In addition, knocking down EcR in the wing discs causes precocious expression of the ecdysone-responsive gene broad. These results suggest that post-critical weight, EcR signaling switches wing discs to a nutrition-independent mode of development via derepression (Mirth, 2009).

The PGs involved in regulating the critical weight transition in Drosophila. This involvement has led to the hypothesis that levels of ecdysone might be important in determining when critical weight has been reached. Demonstrated here, at least in the imaginal discs, ecdysone signaling is involved in mediating the switch in the developmental response to starvation that occurs at critical weight (Mirth, 2009).

The finding that knocking down EcR in the wing discs allows them to pattern in the absence of nutrition offers evidence that ecdysone is acting via EcR to regulate the critical weight transition. Either enhancing insulin signaling in the PGs, which upregulates the ecdysone biosynthetic genes phm and dib (Caldwell, 2005; Colombani, 2005), or knocking down EcR in the imaginal discs, results in premature differentiation of WG and SENS expression patterns under sucrose-only conditions. Furthermore, because knocking down EcR in the wing discs, but not overexpressing EcRDN, results in premature differentiation, it is concluded that the release of repression mediated by unliganded EcR-USP underlies the switch to nutrition-independent differentiation of the wing disc post-critical weight (Mirth, 2009).

In pre-critical weight larvae fed on sucrose alone, Wg expression was not only developmentally delayed but also showed suppressed levels of expression. This suppression was not due to an overall reduction in translation because Actin, Armadillo, En, Ptc and Tubulin were all expressed at the same levels in pre-critical weight larvae fed on sucrose alone and in larvae fed on a standard diet. Instead, the fact that Wg expression was reduced in P0206>Pten and C765>EcRDN larvae fed on standard medium implies that ecdysone might be important in maintaining Wg expression in pre-critical weight larvae (Mirth, 2009).

Classic studies of disc overgrowth mutants and of disc fragmentation have led many authors to speculate that critical weight is modified by the growth status of the imaginal discs. Injecting fragmented imaginal discs into prewandering larvae causes a substantial delay to metamorphosis. Furthermore, the disc overgrowth mutant lethal (2) giant larvae also exhibits a significant delay to metamorphosis. More recently, Stieper (2008) has shown that slowing disc growth alters critical weight. Either damaging discs using X-rays, or reducing their growth rate by knocking down the ribosomal RNA gene Minute specifically in the discs, delays critical weight and produces overgrowth phenotypes. Clearly, imaginal disc growth has at least an inhibitory effect on critical weight (Mirth, 2009).

Although slow growth in the discs can delay critical weight, these results show that premature differentiation in the disc does not cause critical weight to be reached prematurely. C765>EcRi larvae showed precocious differentiation in their discs when starved, yet their critical weight was not altered. Furthermore, when discs are completely ablated, metamorphosis occurs on time. Thus, although the discs are able to modulate critical weight, this modulation is only inhibitory (Mirth, 2009).

Although the ecdysone response pathway has been well described for late-stage larvae and prepupae, less is known about the regulation of ecdysone-responsive genes in the early third instar. broad seemed a likely candidate for a gene that could act downstream of EcR to promote post-critical weight development. Br is a zinc-finger transcription factor that is expressed early in the third instar, at least in the fat body. In addition, Schubiger (2005) has found that in wandering-stage wing discs, Br is both upregulated in usp mutant clones and necessary for the premature differentiation of wing sensilla in these clones (Mirth, 2009).

In the current studies it was found that although Br is upregulated in post-critical weight discs, in C765>EcRi discs, and in the dorsal wing pouch of MS1096>EcRi discs, it was insufficient for stimulating premature differentiation of the Wg, Ct and Sens expression patterns. Thus, it seems that Br, although necessary for the later stages of imaginal disc patterning, does not function in promoting earlier phases of differentiation during the critical weight transition (Mirth, 2009).

One principal difference between the studies conducted by Schubiger (Schubiger, 2005) and the present study is that premature differentiation was observed only in discs from larvae that were malnourished. This implies that pre-critical weight, nutrition modulates ecdysone biosynthesis and that this in turn controls the patterning of gene expression (Mirth, 2009).

It has been shown that insulin-dependent growth in the PGs is involved in regulating the critical weight transition. This study has found that attaining critical weight signifies a developmentally important event that allows tissues to continue patterning even in starved or severely malnourished larvae. Furthermore, this study has shown that ecdysone signaling is the link between the PG-mediated regulation of critical weight and the developmental consequences of reaching critical weight. Ecdysone signaling is important in switching imaginal discs to a post-critical weight developmental program, and this switch is mediated through the derepression of genes repressed by unliganded EcR (Mirth, 2009).

Defective proventriculus specifies the ocellar region in the Drosophila head: Dve represses wg and cut in the wing

A pair of the Drosophila eye-antennal disc gives rise to four distinct organs (eyes, antennae, maxillary palps, and ocelli) and surrounding head cuticle. Developmental processes of this imaginal disc provide an excellent model system to study the mechanism of regional specification and subsequent organogenesis. The dorsal head capsule (vertex) of adult Drosophila is divided into three morphologically distinct subdomains: ocellar, frons, and orbital. The homeobox gene orthodenticle (otd) is required for head vertex development, and mutations that reduce or abolish otd expression in the vertex primordium lead to ocelliless flies. The homeodomain-containing transcriptional repressor Engrailed (En) is also involved in ocellar specification, and the En expression is completely lost in otd mutants. However, the molecular mechanism of ocellar specification remains elusive. This study provides evidence that the homeobox gene defective proventriculus (dve) is a downstream effector of Otd, and also that the repressor activity of Dve is required for en activation through a relief-of-repression mechanism. Furthermore, the Dve activity is involved in repression of the frons identity in an incoherent feedforward loop of Otd and Dve (Yorimitsu, 2011).

This study presents evidence that Dve is a new member involved in ocellar specification and acts as a downstream effector of Otd. The results also revealed a complicated pathway of transcriptional regulators, Otd-Dve-Ara-Ci-En, for ocellar specification (Yorimitsu, 2011).

Transcription networks contain a small set of recurring regulation patterns called network motifs. A feedforward loop (FFL) consists of three genes, two input transcription factors and a target gene, and their regulatory interactions generate eight possible structures of feedforward loop (FFL). When a target gene is suppressed by a repressor 1 (Rep1), relief of this repression by another repressor 2 (Rep2) can induce the target gene expression. When Rep2 also acts as an activator of the target gene, this relief of repression mechanism is classified as a coherent type-4 feedforward loop (c-FFL). During vertex development, Ara is involved in hh repression, and the Dve-mediated ara repression is crucial for hh expression and subsequent ocellar specification. However, the cascade of dve-ara-hh seems to be a relief of repression rather than a cFFL, because Dve is not a direct activator of the hh gene. Furthermore, dve RNAi phenotypes were rescued in the ara mutant background, suggesting that a linear relief of repression mechanism is crucial for hh maintenance (Yorimitsu, 2011).

In photoreceptor R7, Dve acts as a key molecule in a cFFL. Dve (as a Rep1) represses rh3, and the transcription factor Spalt (Sal) (as a Rep2) represses dve and also activates rh3 in parallel to induce rh3 expression. Interestingly, Notch signaling is closely associated with the relief of Dve-mediated transcriptional repression in wing and leg disks. These regulatory networks may also be cFFLs in which Dve acts as a Rep1, although repressors involved in dve repression are not yet identified. In wing disks, expression of wg and ct are repressed by Dve, and Notch signaling represses dve to induce these genes at the dorso-ventral boundary. The Dve activity adjacent to the dorso-ventral boundary still represses wg to refine the source of morphogen. In leg disks, Dve represses expression of dAP-2, and Notch signaling represses dve to induce dAP-2 at the presumptive joint region. The Dve activity distal to the segment boundary still represses dAP-2 to prevent ectopic joint formation. Taken together, these results suggest that Dve plays a critical role as a Rep1 in cFFLs in different tissues. In the head vertex region, it is likely that the repressor activity of Dve is repressed in a cFFL to induce frons identity (Yorimitsu, 2011).

The homeodomain protein Otd is the most upstream transcription factor required for establishment of the head vertex. During second larval instar, Otd is ubiquitously expressed in the eye-antennal disk and it is gradually restricted in the vertex primordium until early third larval instar. Expression of an Otd-target gene, dve, is also detected in the same vertex region at early third larval instar. Otd is required for Dve expression, and the Otd-induced Dve is required for repression of frons identity through the Hh signaling pathway in the medial region. However, Otd is also required for the frons identity in both the medial and mediolateral regions (Yorimitsu, 2011).

This regulatory network is quite similar to the incoherent type-1 feedforward loop (iFFL) in photoreceptor R7. Otd-induced Dve is involved in rh3 repression, whereas Otd is also required for rh3 activation. iFFLs have been known to generate pulse-like dynamics and response acceleration if Rep1 does not completely represses its target gene expression. However, the repressor activity of Dve supersedes the Otd-dependent rh3 activation, resulting in complete rh3 repression in yR7. In pR7, Dve is repressed by Sal, resulting in rh3 expression through the Otd- and Sal-dependent rh3 activation. Thus, Dve serves as a common node that integrates the two loops, the Otd-Dve-Rh3 iFFL and the Sal-Dve-Rh3 cFFL (Yorimitsu, 2011).

In the head vertex region, Otd and Dve are expressed in a graded fashion along the mediolateral axis with highest concentration in the medial region. It is assumed that Otd determines the default state for frons development through restricting the source of morphogens Hh and Wg, and also that high level of Dve expression in the medial ocellar region represses the frons identity through an iFFL. It is likely that repression of dve by an unknown repressor X occurs in a cFFL and induces the frons identity in the mediolateral region (Yorimitsu, 2011).

Interlocked FFLs including Otd and Dve appear to be a common feature in the eye and the head vertex. However, other factors are not shared between two tissues. In R7, a default state is the Otd-dependent Rh3 activation, an acquired state is (1) Rh3 repression through the Otd-Dve iFFL and (2) Spineless-dependent Rh4 expression. In the vertex, a default state is Otd-dependent frons formation, an acquired state is (1) frons repression through the Otd-Dve iFFL and (2) Hh-dependent ocellar specification associated with En and Eya activation (Yorimitsu, 2011).

Both Otd and Dve are K50-type homeodomain transcription factors, and they bind to the rh3 promoter via canonical K50 binding sites (TAATCC). The Otd-Dve iFFL in the eye depends on direct binding activities to these K50 binding sites, but the iFFL in the vertex seems to be more complex. Although target genes for frons determination are not identified, the iFFL in the vertex includes some additional network motifs. For instance, in the downstream of Dve, Hh signaling is critically required for repression of the frons identity (Yorimitsu, 2011).

Since iFFLs also act as fold-change detection to normalize noise in inputs, interlocked FFLs of Dve-mediated transcriptional repression may contribute to robustness of gene expression by preventing aberrant activation. It is an intriguing possibility that, in wing and leg disks, Dve also serves as a common node that integrates the two loops as observed in the eye and the vertex. Further characterization of regulatory networks including Dve will clarify molecular mechanisms of cell specification (Yorimitsu, 2011).

Requirements for mediator complex subunits distinguish three classes of notch target genes at the Drosophila wing margin

Spatial and temporal gene regulation relies on a combinatorial code of sequence-specific transcription factors that must be integrated by the general transcriptional machinery. A key link between the two is the mediator complex, which consists of a core complex that reversibly associates with the accessory kinase module. Genes activated by Notch signaling at the dorsal-ventral boundary of the Drosophila wing disc fall into three classes that are affected differently by the loss of kinase module subunits. One class requires all four kinase module subunits for activation, while the others require only Med12 and Med13, either for activation or for repression. These distinctions do not result from different requirements for the Notch coactivator Mastermind or the corepressors Hairless and Groucho. It is proposed that interactions with the kinase module through distinct cofactors allow the DNA-binding protein Suppressor of Hairless to carry out both its activator and repressor functions (Janody, 2011).

Intercellular signaling pathways drive many processes during development. Their activation results in changes in transcription factor activity that lead to the activation or repression of specific target genes. An important goal is to understand the transcriptional regulatory codes that allow the combinations of proteins bound to enhancer elements to direct precise patterns of gene expression. One well-characterized developmental paradigm is the specification of the Drosophila wing margin by Notch signaling. The Notch receptor is specifically activated at the dorsal-ventral boundary of the larval wing imaginal disc, due to the restricted expression of its ligands Delta and Serrate and of the glycosyltransferase Fringe. Notch activation results in expression of the target genes Enhancer of split m8 (E(spl)m8), cut, wingless (wg), and vestigial (vg), the last through a specific enhancer element known as the boundary enhancer (vgBE). Wg signaling then leads to the differentiation of characteristic sensory bristles adjacent to the margin of the adult wing (Janody, 2011).

Upon ligand binding, Notch is cleaved by the γ-secretase complex, and its intracellular domain (Nintra) enters the nucleus, where it interacts with the DNA-binding protein Suppressor of Hairless (Su(H)). In the absence of Notch activation, Su(H) represses target gene expression through interactions with the corepressor Hairless (H), which binds to Groucho (Gro) and C-terminal binding protein (CtBP). Nintra displaces these corepressors from Su(H) and recruits coactivators such as Mastermind (Mam). It has been proposed that only a subset of Notch target genes require Su(H) to recruit coactivators, while others require Notch signaling only to relieve Su(H)-mediated repression, allowing transcription to be activated by other factors. However, the mechanisms by which Su(H) directs both activation and repression are not fully understood (Janody, 2011).

The mediator complex is thought to promote transcriptional activation by recruiting RNA polymerase II (Pol II), the general transcriptional machinery, and the histone acetyltransferase p300 to promoters, and by stimulating transcriptional elongation by Pol II molecules paused downstream of the promoter. The 'head' and 'middle' modules of the core complex bind to Pol II and general transcription factors, while the 'tail' module consists largely of adaptor subunits that bind to sequence-specific transcription factors. This core complex reversibly associates with a fourth 'kinase' module that consists of the four subunits Med12, Med13, Cdk8, and Cyclin C (CycC). Several studies have implicated the kinase module in transcriptional repression, which can be mediated by phosphorylation of Pol II and other factors by Cdk8, by histone methyltransferase recruitment, and by occlusion of the Pol II binding site. However, this module also appears to function in activation in some contexts; for example, it promotes Wnt target gene expression during Drosophila and mouse development, in mammalian cells, and in colon cancer. Although all four subunits have very similar mutant phenotypes in yeast, loss of Med12 or Med13 has more severe effects on Drosophila development than loss of Cdk8 or CycC, suggesting that Med12 and Med13 have evolved additional functions in higher eukaryotes (Janody, 2011).

This study shows that Notch target genes at the wing margin can be divided into three classes based on their requirements for kinase module subunits. An E(spl)m8 reporter requires all four subunits for its activation, cut requires only Med12 and Med13 (known as Kohtalo [Kto] and Skuld [Skd], respectively, in Drosophila) for its activation, and wg and the vgBE enhancer require Med12 and Med13 for their repression in cells close to the wing margin. Because Med12 and Med13 coimmunoprecipitate with Su(H), regulate an artificial reporter driven by Su(H) binding sites, and can be replaced by a VP16 activation domain or a WRPW repression signal fused to Su(H), it is proposed that the kinase module directly regulates Notch target genes. All four Notch target genes fail to be expressed in the absence of Mam and are similarly affected by the loss of Hairless or Gro, suggesting that other more specific cofactors might recruit kinase module subunits to these genes (Janody, 2011).

The kinase module of the mediator complex is conserved throughout eukaryotes, yet its functions in transcription remain poorly understood. In yeast, loss of any of the four subunits has a very similar effect. In Drosophila, however, loss of Med12 or Med13 has more dramatic effects than loss of Cdk8 or CycC. The kinase module was originally thought to be primarily important for transcriptional repression, mediated by the kinase activity of Cdk8. However, Med12 and Med13 appear to directly activate genes regulated by Wnt signaling in Drosophila and mammalian systems, and also play a positive role in gene activation by the Gli3 and Nanog transcription factors. The data presented in this study confirm that Med12 and Med13 have functions distinct from Cdk8 and CycC. In addition, evidence is provided that all four kinase module subunits contribute to the activation of E(spl)m8 (Janody, 2011).

The human Mastermind homologue MAM has been shown to recruit Cdk8 and CycC to promoters of Notch target genes, where Cdk8 phosphorylates the intracellular domain of Notch, leading to its ubiquitination by the Fbw7 ligase and degradation (Fryer, 2004). This mechanism would be expected to reduce Notch target gene expression, consistent with the increase in E(spl)mβ expression seen in clones lacking the Drosophila Fbw7 homologue Archipelago (Nicholson, 2011); thus it cannot explain the positive effects of Cdk8 and CycC on E(spl)m8. A function for Cdk8 and CycC in Notch-mediated activation would be analogous to recent findings showing that Cdk8 phosphorylation of Smad transcription factors and of histone H3 promotes activation. Cdk8 phosphorylation of RNA polymerase II (Pol II) is also important for transcriptional elongation (Janody, 2011).

Of interest, the current data also suggest that Med12 and Med13 are involved in the repression of wg and the vgBE enhancer in the absence of Notch signaling. The kinase module has been proposed to inhibit transcription through steric hindrance of Pol II binding, independently of Cdk8 kinase activity. Removal of this module on the C/EBP promoter is thought to convert the mediator complex to its active form. In contrast, this study find that wg and vgBE require Med12 and Med13 for their repression but not their activation, while cut and E(spl)m8 require Med12 and Med13 only for their activation, arguing that the two functions occur on different promoters. It cannot be ruled out that Med12 and Med13 have only indirect effects on some of the genes examined; however, their physical association with Su(H) and the requirement for Su(H) binding sites for misexpression of an artificial reporter in skd and kto mutant clones are consistent with a direct effect of Med12 and Med13 on the Su(H) complex (Janody, 2011).

Med12 and Med13 are found associated with both active and inactive promoters in genome-wide chromatin immunoprecipitation studies, suggesting that they can have different effects on transcription when bound to distinct interaction partners. Although both are very large proteins, they contain no domains predicted to have enzymatic activity, and may instead act as scaffolds for the assembly of transcriptional complexes (Janody, 2011).

It has been proposed that Notch target genes could be categorized into two classes: permissive genes, for which the primary function of Notch is to relieve repression by the Su(H) complex, and instructive genes, for which Notch plays an essential role in activation by recruiting specific coactivators. These differences presumably depend on the combinatorial code of transcription factors that regulate each promoter. This study shows that vgBE, an enhancer previously placed in the permissive category, as well as wg, require Med12 and Med13 for their repression but not their activation. During eye development, the proneural gene atonal is likewise regulated permissively by Notch, and ectopically expressed in skd or kto mutant clones. Unexpectedly, this study found that Gro, previously thought to be a cofactor through which Hairless mediates repression, is not required for the repression of vgBE or wg. Hairless may repress target genes at the wing margin through CtBP, its other binding partner. Alternatively, Gro may affect the expression of other upstream regulators of wing margin fate, masking its repressive effect on the genes that were examined (Janody, 2011).

It was also show in this study that instructive Notch target genes can be further subdivided into two classes based on their requirement for kinase module subunits; E(spl)m8 requires all four subunits, while cut requires Med12 and Med13, but not Cdk8 and CycC. Cdk8 and CycC may simply increase the ability of the mediator complex to recruit Pol II or promote transcriptional initiation; this model would suggest that E(spl)m8 has a higher activation threshold than cut. Alternatively, Cdk8 and CycC might enhance the function of a transcription factor that is specifically required for the expression of E(spl)m8 but not cut. Good candidates for such factors would be the proneural proteins Achaete or Scute or their partner Daughterless (Janody, 2011).

The mechanism by which the kinase module is recruited to promote the activation of instructive target genes is not yet clear. Although Mam proteins are well-characterized coactivators for Nintra, this study found that Mam is necessary for the activation of both instructive and permissive genes. It may thus have a general function in transcriptional activation, such as recruiting histone acetyltransferases or stabilizing the Notch-Su(H) complex. A coactivator that recruits Med12 and Med13 specifically to instructive target genes to promote activation may remain to be identified. The current results, like recent reports demonstrating that the arrangement of Su(H) binding sites can affect the interactions between Notch and its coactivators, highlight the complexity in the mechanisms through which promoter elements respond to Notch signaling (Janody, 2011).

Regeneration, transdetermination and apoptosis: The role of wingless and its regulation

Imaginal discs of Drosophila have the remarkable ability to regenerate. After fragmentation wound healing occurs, ectopic wg is induced and a blastema is formed. In some, but not all fragments, the blastema will replace missing structures and a few cells can become more plastic and transdetermine to structures of other discs. A series of systematic cuts through the first leg disc revealed that a cut must transect the dorsal-proximal disc area and that the fragment must also include wg-competent cells. Fragments that fail to both transdetermine and regenerate missing structures will do both when provided with exogenous Wg, demonstrating the necessity of Wg in regenerative processes. In intact leg discs ubiquitously expressed low levels of Wg also leads to blastema formation, regeneration and transdetermination. Two days after exogenous wg induction the endogenous gene is activated, leading to elevated levels of Wg in the dorsal aspect of the leg disc. A wg enhancer was identified that regulates ectopic wg expression. Deletion of this enhancer increases transdetermination, but lowers the amount of ectopic Wg. It is speculated that this lessens repression of dpp dorsally, and thus creates a permissive condition under which the balance of ectopic Wg and Dpp is favorable for transdetermination (Schubiger, 2010).

The systematic analysis of a series of different cuts presented in this study has led to the conclusion that for TD to occur a cut must go through the dorsal region of the disc and the fragment must also contain wg-responsive cells. After fragmentation the peripodial epithelium transiently fuses with the columnar epithelium. In the first leg disc the dorsal peripodial cells express hh, and will signal to the anterior columnar cells during initial wound healing to activate wg-responsive cells. In the posterior compartment in contrast, wg is not activated and thus this fragment will not transdetermine (Schubiger, 2010).

Direct contacts between leg and wing have been mapped to the dorsal anterior aspect of the leg disc. This is the same region that transdetermines after ubiquitous wg expression. Recently, it has been shown that only cells in the dorsal region of the leg disc could be induced to form eyes after ectopically expressing retina-determining genes. Thus the cells in this region appear to have greater developmental plasticity, and the region has been called the 'weak point.' This region of higher plasticity is overlaid by the hh expressing peripodial cells. A cut through hh positive peripodial cells is required for TD and this study has experimentally defined the region capable of inducing TD. Yet after fragmentation transdetermined structures arise within the newly regenerated epithelium and not from dorsal cells in the original piece. This has led to some confusion about the term 'weak point.' Why do the cells in the original fragment fail to transdetermine but can be induced to do so in the intact disc when wg or, for example, some selector genes are ectopically expressed? One reason may be that the cells at the dorsal cut rarely contribute to the regenerate, indicating that they are not participating in the proliferating blastema. The induction of proliferation however is a prerequisite for regeneration and TD. In addition WG and Dpp must interact for TD to occur, but at the dorsal cut wg is not expressed. It was shown that Wg is expressed in the blastema this study has shown that ectopic Wg is expressed, in this study has proposed that as new dorsal leg disc cells (i.e., dpp expressing cells) are regenerated, Wg and Dpp can interact in the blastema to allow TD (Schubiger, 2010).

Lost or damaged tissues are repaired by different mechanisms depending on the organism. But in many cases Wg or Wnt has an essential function. During morphallactic regeneration in Hydra, Wnt expression correlates with cell movements and head regeneration (Philipp, 2009). In planaria where regeneration depends on stem cells, blocking Wnt-signalling has no effect on blastema formation, but drastically changes the type of structures formed. In this case Wnt functions to pattern the regeneration blastema (Schubiger, 2010).

Canonical Wg signaling is one of the early signals necessary and sufficient in epimorphic limb or fin regeneration. This study observed that one of the first changes in disc fragments is the ectopic expression of Wg near the wound area preceding blastema formation. The critical role of Wg signaling was shown in amputated limbs of chick embryos where wound healing but not regeneration occurs. However when activated β-catenin is induced in the wound cap, an apical ectodermal ridge is formed and regeneration now can proceed (Schubiger, 2010).

This study demonstrated that the absence of Wg in the 1/2 L fragment is a major reason for its failure to regenerate, and as in chick limb regeneration, induced Wg-signaling allows regeneration. Moreover, Wg- signaling led to TD, indicating that cells became more plastic (Schubiger, 2010).

Duplicated leg structures after inducing ectopic wg expression has been reported. Twelve hours after they induced expression, Wg was observed at low levels throughout the disc, but surprisingly all defects in the adult leg were dorsal. This study showed that after 2–3 days Wg becomes highly expressed in the dorsal leg disc where outgrowth occurs. The initial low levels of ubiquitous Wg activated the endogenous wg-promoter in this part of the leg disc. Thus uniform wg expression in the disc can activate the endogenous gene in specific regions. It is not known if Wg signaling directly activates wg, as has been reported in the embryo, or indirectly, but it was possible to show that the wg-enhancer BRV118 reacts to the induced low levels of Wg. The BRV118 enhancer is also activated in the blastema after fragmentation, demonstrating that it responds to regeneration signals in general, and suggests that BRV118 regulates wg expression during regeneration (Schubiger, 2010).

The BRV118 enhancer is largely deleted in the wg1 allele. The wg1 mutation is not 100% penetrant, and although the majority of flies are missing one or both wings, some animals do have two perfect wings. Thus other enhancer regions must be able to promote wing blade formation at a low frequency. When TD frequency was tested in wg1/wg1 leg discs after ubiquitous wg expressionm a significantly higher rate of TD was observed, compared to ubiquitous wg expression in a wild-type background. At first glance this might seem unexpected since wg1/wg1 animals are characterized by the loss of wings. Previous work showed that varying amounts of ectopic Wg controlled the rate of leg to wing TD. Moderate ectopic Wg led to high TD rates, whereas high Wg signaling down-regulated dpp expression and led to ventralization, loss of dorsal structures and low TD rate. The current results show that wg1 reduces ectopic Wg levels in the leg disc, most dramatically evident by the absence of discs with a mirror image Wg pattern. It is proposed that in the absence of the BRV118 regulatory sequence ectopic Wg reaches a threshold permissive for TD but too low for extensive loss of dorsal structures, structures from which TD will occur (Schubiger, 2010).

Cell clones induced in regenerating anterior leg disc fragments are composed of both En and non-En expressing cells, thus clearly breaking their compartment identity. Once some posterior cells are formed the compartmental boundary is reestablished even before all missing pattern elements of the posterior compartment are reformed. This study has shown that during the early stages of regeneration of the 1/4UM fragment, cells co-expressed Ci and En. This indicates that cells in the blastema do not revert to an embryonic stage before compartment identity is established. Since anterior cells must be reprogrammed to make posterior cells it is speculated that a novel pathway is activated as a consequence of wound healing and activation of the JNK-pathway that reduces polycomb function. Polycomb group (PcG) genes are required to maintain the anterior/posterior compartment boundary. In the absence of the PcG gene ph, mutant clones in the anterior compartment will express en. It is proposed that during regeneration decreased PcG function destabilizes anteriorness and allows the expression of en. Once en is activated it will inhibit Ci expression leading to en only expression. Since the anterior/posterior compartment is reestablished during regeneration, PcG function is normalized again, and the new anterior/posterior compartment is maintained. It has been reported that during normal dorsal closure an anterior cell at the leading edge of the ectoderm is reprogrammed and moves to the posterior compartment. The JNK pathway is required for the change. During regeneration of the posterior compartment in disc fragments the JNK-pathway is also involved, opening the possibility that in both processes the switch from anterior to posterior may be similarly regulated (Schubiger, 2010).

Earlier work also supports the idea that new developmental programs are initiated during regeneration. For TD from leg to wing to occur, this study has shown that the vg boundary enhancer is essential, and not the quadrant enhancer which is required earlier in wing development, namely during the second instar. The clearest case for following a novel program during regeneration comes from transdetermining leg disc cells that transiently adopt a novel cell-cycle profile, different from the profile of younger disc cells (Schubiger, 2010).

In summary these results indicate that successful regeneration does not require cells to re-capitulate embryonic programs, but is more likely to involve programs not normally used during disc development. In amphibians it was recently shown that in the regenerating axolotl limbs progenitor cells in the blastema do not dedifferentiate and re-enter an embryonic state. Elegant labeling studies revealed that dedifferention leads to progenitor cells with restricted potential rather than to pluripotency. It will be interesting to see if such cells also embark on a novel program during regeneration (Schubiger, 2010).

Wingless in apoptosis and apoptosis-induced proliferation

Irradiated or injured cells enter apoptosis, and in turn, promote proliferation of surrounding unaffected cells. In Drosophila, apoptotic cells have an active role in proliferation, where the caspase Dronc and p53 induce mitogen expression and growth in the surrounding tissues. The Drosophila p53 gene structure is conserved and encodes at least two protein isoforms: a full-length isoform (Dp53) and an N-terminally truncated isoform (DΔNp53). Historically, DΔNp53 was the first p53 isoform identified and was thought to be responsible for all p53 biological activities. It was shown that DΔNp53 induces apoptosis by inducing the expression of IAP antagonists, such as Reaper. This study investigated the roles of Dp53 and DΔNp53 in apoptosis and apoptosis-induced proliferation. It was found that both isoforms were capable of activating apoptosis, but that they each induced distinct IAP antagonists. Expression of DΔNp53 induced Wingless (Wg) expression and enhanced proliferation in both 'undead cells' and in 'genuine' apoptotic cells. In contrast to DΔNp53, Dp53 did not induce Wg expression in the absence of the endogenous p53 gene. Thus, it is proposed that DΔNp53 is the main isoform that regulates apoptosis-induced proliferation. Understanding the roles of Drosophila p53 isoforms in apoptosis and in apoptosis-induced proliferation may shed new light on the roles of p53 isoforms in humans, with important implications in cancer biology (Dichtel-Danjoy, 2012)

The discovery of multiple p53 isoforms raises the question of their functional specificity in the spectrum of p53-mediated biological responses. In Drosophila, as the first and only p53 isoform identified in almost a decade, the truncated DΔNp53 isoform was initialy presumed responsible for all p53 activities. The identification of the full-length Dp53 isoform that contains a full N-terminal transactivation domain challenged this presumption. Using gain-of-function studies, this study examined the role of these two isoforms in apoptosis and apoptosis-induced proliferation. Both Dp53 isoforms were found to activate apoptosis but preferentially activate different DIAP antagonists (Rpr or Hid) for caspase activation DΔNp53 promotes wg expression and cell proliferation, independently of endogenous p53, whereas Dp53 is unable to do so. Dp53 was also found to be primarily responsible for damage-induced transcriptional activation of rpr, whereas DΔNp53 is the p53 isoform dedicated to promoting apoptosis-induced proliferation (Dichtel-Danjoy, 2012)

DΔNp53 binds a DNA damage response element in the rpr regulatory region, which is responsible for the induction of apoptosis in response to irradiation. This study showed that in wing imaginal discs, Dp53 is a stronger inducer of rpr expression than DΔNp53. Moreover, it was shown that DΔNp53 strongly induced hid expression, whereas Dp53 was only a weak inducer. Together, these observations suggest that the transcriptional competence of DΔNp53 differs from that of Dp53, and is consistent with a previous study showing that hid is transcriptionally induced by DΔNp53 in eye and wing imaginal discs. These results also suggest that some intrinsic ability to distinguish its activity for rpr and hid expressions is embedded in the N-terminus of the full length Dp53. Therefore, it is proposed that Dp53 is responsible for the damage-mediated activation of rpr for apoptosis, whereas DΔNp53 promotes apoptosis by inducing expression of hid. The physiological consequences of this functional segregation in apoptosis regulation by p53 isoforms remain to be determined (Dichtel-Danjoy, 2012)

Previous works have shown that apoptotic cells secrete morphogens that induce proliferation of surrounding cells. Although more clearly detected in 'undead cells', mitogen gene expression and extra proliferation have also been detected in genuine apoptotic cells. It was proposed that the initiator caspase Dronc leads to Dp53 expression, which in turn activates mitogen gene expression, but the specific roles of Dp53 and DΔNp53 remain to be established. This study showed that DΔNp53 is a potent inducer of wg expression both in the 'undead cell' and genuine apoptotic cell model. Specifically, this study showed that DΔNp53 induced wg expression independently of dronc. This indicates that DΔNp53 acts downstream of the apoptotic pathway to induce proliferation via the expression of wg. Thus, like JNK, DΔNp53 promotes proliferation independently of the apoptotic cascade. Further analysis will be required to determine the relationship between JNK and p53 isoforms in the induction of proliferation (Dichtel-Danjoy, 2012)

It has been proposed that in the apoptosis-induced proliferation process, there is a feedback loop that activates wg expression in 'undead cells' via Dronc and Dp53. The current results are consistent with such a feedback mechanism in which Dp53 and DΔNp53 induce apoptosis via rpr and hid, which in turn amplifies DΔNp53 via Dronc to promote wg expression. The results also suggest that the feedback loop not only functions in 'undead cells' but also in genuine apoptotic cells. Together, it is proposed that p53 isoforms act both upstream and downstream of the apoptotic pathway to promote wg expression and proliferation (Dichtel-Danjoy, 2012)

The results show that DΔNp53 is a potent inducer of wg expression in both wild-type and p53-null wing discs. In contrast, Dp53 only weakly increased wg expression in wild-type but not in p53-null flies. Therefore, the weak induction of wg expression by Dp53 in wild-type disc is likely dependent on the endogenous p53 gene. Further investigations will be required to determine if DΔNp53 is the only p53 isoform regulating wg expression or if another isoform such as Dp53ΔC or the one encoded by the recently annotated p53-RD transcript contribute as well to the regulation of wg expression (Dichtel-Danjoy, 2012)

One of the most intensely debated questions regarding Drosophila ΔNp53 isoforms is whether they have their own biological activity or exert a dominant negative activity on p53. The fact that DΔNp53 induced Wg expression independently of endogenous p53 gene indicates that DΔNp53 does not require p53 for this function. In vertebrate studies, zebrafish Δ113p53 and human Δ133p53 do not act exclusively in a dominant-negative manner toward p53 but differentially regulate p53 target gene expression to modulate p53 function. Similarly, the current results show that Drosophila p53 isoforms have the capacity to use distinct targets to orchestrate their biological functions; Dp53 promotes rpr expression, whereas DΔNp53 activates Hid and Wg expression in wing epithelium. Overall, it is proposed that balancing apoptosis and apoptosis-induced proliferation may represent one primordial function of the TP53 gene family, and that this function requires the expression of Dp53 and DΔNp53 isoforms in a tightly controlled manner. In vertebrate, this primordial functional capacity may be differently exploited by TP53, TP63 and TP73 to regulate specific aspects of death/proliferation in the equilibrium, depending upon tissues and physiological contexts (Dichtel-Danjoy, 2012)

Steep differences in wingless signaling trigger Myc-independent competitive cell interactions

Wnt signaling is a key regulator of development that is often associated with cancer. Wingless, a Drosophila Wnt homolog, has been reported to be a survival factor in wing imaginal discs. However, it was found that prospective wing cells survive in the absence of Wingless as long as they are not surrounded by Wingless-responding cells. Moreover, local autonomous overactivation of Wg signaling (as a result of a mutation in APC or axin) leads to the elimination of surrounding normal cells. Therefore, relative differences in Wingless signaling lead to competitive cell interactions. This process does not involve Myc, a well-established cell competition factor. It does, however, require Notum, a conserved secreted feedback inhibitor of Wnt signaling. It is suggested that Notum could amplify local differences in Wingless signaling, thus serving as an early trigger of Wg signaling-dependent competition (Vincent, 2011).

One conclusion from this work is that Wg signaling is not intrinsically required for wing cell survival and that, instead, competitive cell interactions triggered by local differences in Wingless signal transduction influence survival decisions. Such local differences can arise between clones that either cannot transduce the signal (e.g., fz fz2 or arrow mutant) or overactivate signaling (e.g., axin or APC mutant). In both cases, the low signaling cells are eliminated. It has been suggested that other forms of cell competition could be relevant to cancer. Moreover, mutations in axin and APC are found in a variety of cancers. Therefore, it is conceivable that humans precancerous APC or axin mutant cells could acquire a competitive advantage that enables them to clear surrounding normal tissue, thus contributing to tissue colonization. As this study has shown, this is not mediated by local differences in the activity of Myc, a key regulator of ribosomal activity and a well-established factor of cell competition. In fact, the competitive nature of axin mutant cells was boosted by experimentally increasing their relative content of functional ribosomes. By analogy, in humans, loss of axin (or APC) and increased translational potential are two features that could have additive effects in boosting early tumor progression and enabling tumors to overcome preexisting barriers to tissue growth (Vincent, 2011).

Although the cell biological basis of Wg signaling-induced competition remains to be elucidated, this study has identified one important mediator, the secreted phospholipase encoded by notum. notum knockdown prevents axin mutant cells from taking over the wing pouch even though these cells are themselves insensitive to Notum activity. Therefore, the overgrowth of axin mutant cells is not solely an autonomous consequence of overactive Wg signaling. As a result of high signaling activity, axin mutant cells secrete Notum, which inhibits signaling in neighboring wildtype cells. Thus, an initial signaling difference is amplified and then transduced into downstream events that lead to the elimination of normal cells, which is required for axin mutant cells to overgrow and take over the tissue (Vincent, 2011).

Transcriptional Regulation Table of contents

wingless continued: Biological Overview | Evolutionary Homologs | Targets of Activity | Protein Interactions | mRNA Transport | Developmental Biology | Effects of Mutation | References

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