REGULATION (part 3/3)

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

Third instar wing discs stained with an antibody directed against the N-terminal portion of the Spitz protein show a strong expression of Spi along the DV boundary and weaker expression throughout the disc. This elevated protein level at the DV boundary is likely to reflect post-transcriptional control, since work from several laboratories has shown that SPI mRNA is expressed ubiquitously at low levels throughout the wing disc. To determine if the growth promoting activity of the Notch pathway is mediated by Egfr, the dpp-Gal4 driver system was used to ectopically express an activated, secreted form of Spi (sSpi) that would activate Egfr along this boundary. This results in an extensive outgrowth of the wing pouch. When these discs are stained with an a-Vg antibody, the overproliferating cells are found to express Vg. These results can either imply that Vg expression is activated by the Egfr pathway leading to cell proliferation or that Egfr activation results in random proliferation of cells within the pouch, which then secondarily express the Vg protein. To distinguish between these possibilities, the vg 1 mutant allele in which Vg expression is reduced but not eliminated, was used. In dpp-Gal4/UAS-sspi;vg 1/vg 1 flies there is no expansion of the wing pouch. It is concluded that activation of EGFR leads to expression of Vg, which functions downstream of or parallel to the Egfr pathway for the proper proliferation of cells in the pouch (Nagaraj, 1999).

To address the early role of Egfr in wing patterning, four alternative strategies involving loss-of-function mutations in components of the Egfr pathway were used. First, a temperature-sensitive allele of the Egfr gene (EGFR ts) was used to inactivate the pathway. The heteroallelic combination EGFR ts /EGFR top1 gives rise to a null phenotype at the non-permissive temperature and shows decreased Vg expression in the wing pouch. The expression of Vg in the folds outside the pouch region is unaffected and is therefore not responsive to Egfr signaling. In these discs, the notum is significantly reduced in size, consistent with the fact that the alternative Egfr ligand, Vein, is expressed in the third instar notum. As a second approach, a dominant negative version of Egfr (EGFR DN) was expressed using the UAS/Gal4 system. Beginning in early third instar stages, the A9-Gal4 element causes expression of a reporter gene mostly in the dorsal compartment of the wing pouch and at lower levels in the ventral compartment. A9-Gal4; UAS-EGFR DN wing discs show a dramatic reduction in the dorsal compartment of the wing pouch. This is not a secondary consequence of a perturbation in DV boundary specification since the expression pattern of Wg along the DV boundary is maintained. Rather this effect is mediated through the control of vg, since the expression of boundary enhancers and quadrant enhancers are dramatically reduced. As a third approach to attenuate Egfr signaling during development, a hypomorphic allele of pointed (pnt) was used. This allele encodes an ETS domain transcription factor that functions as a downstream member of the Egfr pathway. pnt mutant wing discs show reduced pouch size when compared with wild type, again supporting the conclusion from previous experiments that the Egfr pathway is necessary for the growth of the wing pouch. Finally, a loss of function in genes belonging to the Egfr pathway interact genetically with vg. Egfr signaling is shown to activate the boundary enhancer and represses the quadrant enhancer of vg (Nagaraj, 1999).

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

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

A common consequence of Notch signaling in Drosophila is the transcriptional activation of seven Enhancer of split [E(spl)] genes, which encode a family of closely related basic-helix-loop-helix transcriptional repressors. Different E(spl) proteins can functionally substitute for each other, hampering loss-of-function genetic analysis and raising the question of whether any specialization exists within the family. Each individual E(spl) gene was expressed using the GAL4-UAS system in order to analyse each gene's effect in a number of cell fate decisions taking place in the wing imaginal disk. A focus was placed on sensory organ precursor determination, wing vein determination and wing pattern formation. All of the E(spl) proteins affect the first two processes in the same way: they antagonize neural precursor and vein fates. Yet the efficacy of this antagonism is quite distinct: E(spl)mbeta, which is normally expressed in intervein regions, has the strongest vein suppression effect, whereas E(spl)m8 and E(spl)m7 are the most active bristle suppressors. While E(spl)m8 is more effective in abolishing the notum microchaeta fate, E(spl)m7 is most active against wing margin bristles (Ligoxygakis, 1999).

During wing patterning, Notch activity orchestrates a complex sequence of events that define the dorsoventral boundary of the wing. Two phases within this process have been discerned, based on the sensitivity of N loss-of-function phenotypes to concomitant expression of E(spl) genes. E(spl) proteins are initially involved in repression of the vg quadrant enhancer, whereas later they appear to relay the Notch signal that triggers activation of cut expression. Of the seven proteins, E(spl)mgamma is most active in both of these processes (Ligoxygakis, 1999).

How do E(spl) proteins, implicated in gene processing, come to activate cut expression? The present work suggests that cut expression requires Gro and partly also depends on E(spl)bHLH factors. One possibility is that E(spl) [at least E(spl)mgamma and E(spl)mdelta], like a number of other transcription factors, might have a dual function as either a transcriptional repressor or an activator, depending on context. Such an activation role has never been suggested before for either E(spl) or Gro. Alternatively, two models can be envisaged that reconcile a repressor activity of E(spl) proteins with their role in cut activation. In one, E(spl) can act by repressing a negative regulator of cut transcription. In the other, E(spl) can repress a negative regulator of Notch signaling. In the latter case, E(spl) expression would promote a positive feedback loop to enhance Notch signaling, thus increasing the signaling output from the severely compromised Nts1 receptor at the restrictive temperature. The fact that no restoration is observed in the expression of two other Notch targets, namely wg and E(spl)m8-lacZ , argues against this hypothesis. A direct role of E(spl)mgamma and E(spl)mdelta in cut expression is favored, either as activators or as repressors of a repressor, but not as general positive regulators of Notch signaling (Ligoxygakis, 1999).

Ectopic expression of E(spl)mgamma/E(spl)mdelta is not sufficient for cut expression in a wild-type background. Rather, it appears that the ability of ectopic E(spl)mgamma/E(spl)mdelta to induce cut is spatially restricted to the normal domain of cut expression. Since activated Notch is sufficient to ectopically turn on cut, it follows that some other Notch-responsive event, other than E(spl) expression, must also contribute to cut expression. This is consistent with findings that early reduction of Notch activity abolishes cut expression despite concomitant ectopic expression of E(spl)mdelta. Molecular analysis has shown that cut expression requires the transcription factor Scalloped (Sd). sd is a candidate target gene of Vg, which in turn is initially activated by Notch independent of E(spl). It is possible that expression of vg and sd at the wing margin during early L3 could make these cells competent for cut expression. This would only be initiated later, when a second pulse of Notch signaling during mid-L3 activates (or relieves the repression of) cut via E(spl)mgamma or another Gro-interacting protein. In conclusion, E(spl) proteins have partially redundant functions, yet they have evolved distinct preferences in implementing different cell fate decisions, which closely match their individual normal expression patterns (Ligoxygakis, 1999).

In Drosophila, the imaginal discs are the primordia for adult appendages. Their proper formation is dependent on the activation of the decapentaplegic gene in a stripe of cells just anterior to the compartment boundary. In imaginal discs, the dpp gene has been shown to be activated by Hedgehog signal transduction. However, an initial analysis of its enhancer region suggests that its regulation is complex and depends on additional factors. In order to understand how multiple factors regulate dpp expression, focus was placed on a single dpp enhancer element, the dpp heldout enhancer, from the 3' cis regulatory disc region of the dpp locus. A molecular analysis of this 358 bp wing- and haltere-specific dpp enhancer is presented that demonstrates a direct transcriptional requirement for the Cubitus interruptus (Ci) protein. The results suggest that, in addition to regulation by Ci, expression of the dpp heldout enhancer is spatially determined by Drosophila TCF (dTCF) and the Vestigial/Scalloped selector system and that temporal control is provided by dpp autoregulation. Consistent with the unexpectedly complex regulation of the dpp heldout enhancer, analysis of a Ci consensus site reporter construct suggests that Ci, a mediator of Hedgehog transcriptional activation, can only transactivate in concert with other factors (Hepker, 1999).

The dppho enhancer (so named because mutations in the region result in a 'held out' wing phenotype) was chosen for detailed analysis because this small region contains a cluster of putative transcription factor binding sites that is conserved in Drosophila virilis. The dppho enhancer is located from map position 111.9 to 112.3, approximately 18 kb from the 3' terminus of the dpp structural gene. The enhancer shares 52% sequence identity with the homologous region from D. virilis. Within the conserved sequences are found reasonable matches for the binding sites of several known transcription factors, including Engrailed, Ci, dTCF, Mothers against Decapentaplegic (Mad) and Scalloped. Of particular interest is the presence of potential Ci consensus binding sites. Gel mobility shift assays were performed with the DNA-binding domain of Ci and they demonstrated sequence-specific binding to the dppho fragment (Hepker, 1999).

The expression pattern of dppho-lacZ is consistent with this reporter being restricted by the extent of overlap between Hh and Wg signals in the wing pouch. For example, the dppho enhancer directs expression of lacZ reporter in a stripe coincident with high-level full-length Ci and endogenous dpp expression in the wing primordium of the wing imaginal disc. Furthermore, its expression is most robust in early larval stages and fades in a manner complementary to the dynamic pattern of wg expression in the wing disc. This enhancer also directs expression of a reporter ventrally in an analogous stripe in the haltere disc. Indeed ectopic expression data, together with clonal analysis, demonstrates that Ci and dTCF regulate dppho-lacZ expression, and this regulation is shown to be direct (Hepker, 1999).

Regulation of the dppho enhancer cannot be solely dependent on Wg and Hh signals since this element directs expression specifically in presumptive wing tissue. A candidate for a wing-specific factor involved in dppho regulation is the Vestigial/Scalloped transcriptional complex. The dppho sequence contains a weak match to the Sd/TEA DNA-binding site consensus, therefore a test was performed to see whether the Vg/Sd selector system is involved in restricting dppho-lacZ expression to the wing. A 30AGAL4 or an apterousGAL4 driver was used to direct expression of UAS-vg. In both cases, ectopic expression of vg induces expression of dppho-lacZ, but only near the A/P boundary. Similar experiments performed with UAS-sd result in loss of dppho-lacZ expression (Hepker, 1999).

The Drosophila wing imaginal disc gives rise to three body parts along the proximo-distal axis: the wing blade, the wing hinge and the mesonotum. wingless is required for hinge development, but wg does not activate vg in the hinge as it does in the blade, but instead activates homothorax, which is required for blade development. hth also limits where along the D/V compartment boundary wing blade development can initiate, thus helping to define the size and position of the wing blade within the disc epithelium. The gene teashirt, which is coexpressed with hth throughout most of wing disc development, collaborates with hth to repress vg and block wing blade development. These results suggest that tsh and hth block wing blade development by repressing some of the activities of the Notch pathway at the D/V compartment boundary (Casares, 2000).

Notch signaling and the determination of appendage identity

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

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

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

Brinker requires two corepressors for maximal and versatile repression in Dpp signalling

Responses to graded Dpp activity requires an input from a complementary and opposing gradient of Brinker (Brk), a transcriptional repressor protein encoded by a Dpp target gene. Brk harbours a functional and transferable repression domain, through which it recruits the corepressors Groucho and CtBP. By analysing transcriptional outcomes arising from the genetic removal of these corepressors, and by ectopically expressing Brk variants in the embryo, it has been demonstrated that these corepressors are alternatively used by Brk for repressing some Dpp-responsive genes, whereas for repressing other distinct target genes they are not required. These results show that Brk utilizes multiple means to repress its endogenous target genes, allowing repression of a multitude of complex Dpp target promoters (Hasson, 2001).

In the wing imaginal disc, cells in the posterior compartment are programmed by the engrailed selector gene product to secrete Hedgehog (Hh), which induces dpp in a stripe of anterior cells along the A/P boundary. Dpp then acts as a long-range morphogen that governs patterning across the entire imaginal disc field. To determine whether Gro participates in the implementation of Hh signaling, clones overexpressing gro, or clones that are homozygous for the strong groE48 allele, were stained for dpp-lacZ expression. In all clones, even those overlapping with the Hh activity domain, there are no noticeable alterations in the dpp expression pattern, indicating that Gro is not required downstream of Hh for dpp transcriptional regulation. In striking contrast, however, three distinct targets of the Dpp pathway, expressed either in the wing pouch [optomotor-blind (omb) and vestigial (vg) or in the periphery of the wing disc (brk)], are repressed in clones overexpressing gro. Expression of omb-lacZ, as well as that of a lacZ reporter driven by vg's Dpp-responsive enhancer (vgQ-lacZ), is completely abrogated in these clones, whereas expression of brk-lacZ is only reduced. All three Dpp targets are repressed in a cell autonomous manner, i.e. only in the clones but never in adjacent cells. These results, together with an extensive gro loss-of-function clonal analysis detailed below, implicate Gro specifically as a downstream effector of Dpp signaling (Hasson, 2001).

Recent genetic and molecular studies have shown that brk encodes a repressor acting downstream of the Dpp pathway, which helps define the low end of the Dpp gradient. In particular, the Dpp targets omb and vgQ are both derepressed in brk- mutant clones and in brk- wing imaginal discs, suggesting that they are normally subjected to Brk repression. More directly, Brk binds to specific sequences within defined omb and vgQ enhancer elements, bringing about their silencing by outcompeting the Mad-Medea complex, or some other activator, from binding to overlapping DNA sites (Hasson, 2001).

To establish whether Brk represses vgQ via Gro, CtBP or both, vgQ-lacZ expression was monitored in gro- and CtBP- single, and CtBP-; gro- double mutant clones. In this instance, a mandatory requirement for Gro, but not for CtBP is found; in gro- clones, vgQ is upregulated. Importantly, as is the case for brk- clones, the cell-autonomous upregulation of vgQ is seen only in gro- clones close to the periphery of the disc, suggesting that the observed effects are Brk dependent. In contrast, in CtBP- mutant clones vgQ expression is downregulated, in the Brk territory but also outside it, at the centre of the disc, indicating that these effects are Brk independent and that CtBP is positively required for vg expression. CtBP-;gro- double mutant clones show a composite effect: ectopic expression and upregulation of vgQ in clones in the brk expression domain, and a phenotype resembling that of CtBP- clones at the middle of the disc, where brk is not expressed. Thus, Brk repression of vgQ is Gro- but not CtBP-dependent (Hasson, 2001).

omb and vgQ expression is completely shut off in clones of cells overexpressing gro, whereas that of brk is only reduced, suggesting that Brk might be repressing its own transcription via a negative autoregulatory loop. To establish whether, in negating its own expression, Brk is assisted by Gro and/or CtBP, gro- and CtBP- single, or CtBP-, gro- double mutant clones were stained for brk-lacZ expression. brk is never ectopically expressed in any of the single mutant clones, whereas ectopic brk expression is clearly observable in double mutant clones. Thus, in the absence of one corepressor, repression is adequately mediated by the other, suggesting that negative autoregulation by Brk is robust, relying on either Gro or CtBP (Hasson, 2001).

Brk utilizes a self-reliant mechanism, which need not depend on tethered corepressors, by competing with activators over coinciding DNA-binding sites. In the absence of both Gro and CtBP, Brk represses not only omb and zen, but also sal, suggesting that the Brk-binding site(s) in the sal promoter overlap with those employed by activators. Transcription of both sal and vgQ requires activation by Mad, yet, although both promoters are exposed to identical levels of pMad, the sal expression domain is spatially more restricted than that of vgQ, presumably because activation of sal requires higher levels of pMad than that of vgQ. Hence, 'passive' competition-based repression should efficiently block activation of sal but may not be sufficient for promoters like vgQ, which are activated even by low amounts of Mad. For silencing such promoters, alternative mechanisms such as recruitment of corepressors have evolved and are employed (Hasson, 2001).

Brk represses its distinct endogenous target genes by recruiting Gro and/or CtBP differentially. For the silencing of many target promoters, Gro alone is sufficient (vg, tld and pnr) but, for fully repressing others, Brk depends on both corepressors. Thus, in the case of dpp and Sxl, when CtBP is lacking, a decrease in Brk's overall repressor capacity is apparent and, in the absence of Gro, repression is almost completely impaired. Importantly, for negating its own transcription, Brk can utilize either corepressor (Hasson, 2001).

In summary, these data suggest that Brk uses multiple means to negate target gene expression, such as competition and the varied recruitment of long- and short-range corepressors. It is proposed that this versatility is, biologically, most significant given Brk's role in Dpp signaling, since it facilitates the negative regulation of diverse, complex Dpp target promoters (Hasson, 2001).

Control of a genetic regulatory network by a selector gene

The formation of many complex structures is controlled by a special class of transcription factors encoded by selector genes. It has been shown that Scalloped, the DNA binding component of the selector protein complex for the Drosophila wing field, binds to and directly regulates the cis-regulatory elements of many individual target genes within the genetic regulatory network controlling wing development. Furthermore, combinations of binding sites for Scalloped and transcriptional effectors of signaling pathways are necessary and sufficient to specify wing-specific responses to different signaling pathways. The obligate integration of selector and signaling protein inputs on cis-regulatory DNA may be a general mechanism by which selector proteins control extensive genetic regulatory networks during development (Guss, 2001).

The discovery of genes whose products control the formation and identity of various fields, dubbed 'selector genes', has enabled the recognition and redefinition of fields as discrete territories of selector gene activity. Although the term has been used somewhat liberally, two kinds of selector genes have been of central interest to understanding the development of embryonic fields. These include the Hox genes, whose products differentiate the identity of homologous fields, and field-specific selector genes such as eyeless, Distal-less, and vestigial-Scalloped (vg-sd) whose products have the unique property of directing the formation of entire complex structures. The mechanisms by which field-specific selector proteins direct the development of these structures are not well understood. In principle, selector proteins could directly regulate the expression of only a few genes, thus exerting much of their effect indirectly, or they may regulate the transcription of many genes distributed throughout genetic regulatory networks (Guss, 2001).

In the Drosophila wing imaginal disc, the Vg-Sd selector protein complex regulates wing formation and identity. Sd is a TEA-domain protein that binds to DNA in a sequence-specific manner, whereas Vg, a novel nuclear protein, functions as a trans-activator. To determine whether direct regulation by Sd is widely required for gene expression in the wing field, the regulation of several genes that represent different nodes in the wing genetic regulatory network and that control the development of different wing pattern elements were analyzed. Focus was placed in particular on genes for which cis-regulatory elements that control expression in the wing imaginal disc have been isolated, including cut, spalt (sal), and vg (Guss, 2001).

First it was tested whether sd gene function is required for the expression of various genes in the wing field. Mitotic clones of cells homozygous for a strong hypomorphic allele of sd were generated and the expression of gene products or reporter genes was assessed within these clones. Reduction of sd function reduces or eliminates the expression of the Cut and Wingless (Wg) proteins and of reporter genes under the control of the sal 10.2-kb and the vg quadrant enhancers, demonstrating a cell-autonomous requirement for selector gene function for the expression of these genes in the wing field (Guss, 2001).

These results, however, do not distinguish between the direct and indirect regulation of target gene expression by Vg-Sd. To differentiate between these possibilities, whether the DNA binding domain of Sd could bind to specific sequences in cut, sal, and vg wing-specific cis-regulatory elements were tested. Using DNase I footprinting, Sd-binding sites were identified in all of the elements assayed. Thus, Sd may control the expression of these genes by binding to their cis-regulatory elements (Guss, 2001).

To determine whether Sd binding to these sites is necessary for the function of these cis-regulatory elements in vivo, specific Sd-binding sites within each of the elements were mutated such that they reduced or abolished Sd binding in gel mobility-shift assays. The mutation of tandem Sd-binding sites in the cut and sal elements results in complete loss of reporter gene expression in vivo. Similarly, mutation of the four single Sd-binding sites identified in the vg quadrant enhancer eliminated or dramatically reduced reporter gene expression. These results show that Sd binds to and directly regulates the expression of four genes (cut, sal, vg, and DSRF) in the wing genetic regulatory network. This molecular analysis and the genetic requirement for Sd function for the expression of other genes suggest a widespread requirement for direct Vg-Sd regulation of genes expressed in the wing field (Guss, 2001).

Each of the Sd targets analyzed is activated in only a portion of the wing field, in patterns controlled by specific signaling pathways. For instance, cut is a target of Notch signaling along the dorsoventral boundary, and the sal and vg quadrant enhancers are targets of Dpp signaling along the anteroposterior axis. Binding sites for the transcriptional effectors of the Notch- and Dpp-signaling pathways, Suppressor of Hairless [Su(H)], and Mothers Against Dpp (Mad), and Medea (Med), respectively, have been shown to be necessary for the activity of a number of wing-specific cis-regulatory elements, and occur in these elements. This observation, coupled with the data demonstrating a direct requirement for Sd binding, suggests that gene expression in the wing field requires two discrete inputs on the cis-regulatory DNA: one from the selector proteins that define the field, and one from the signaling pathway that patterns the field (Guss, 2001).

These findings also raised the possibility that the combination of selector and signal inputs may be sufficient to drive field-specific, patterned gene expression. To test this, there were built a number of synthetic regulatory elements comprised of combinations of Sd binding sites with binding sites for Su(H) or Mad/Med. The activity of these elements was compared with those composed of tandem arrays of just selector- or signal effector-binding sites, or combinations of different signal effector sites. Each of the binding sites used in these constructs was selected from sequences found in native Drosophila cis-regulatory elements that have been demonstrated to function in vivo (Guss, 2001).

Elements containing only single classes of binding sites for the selector or signal effectors were unable to drive reporter gene expression in the wing. In contrast, the synthetic elements in which binding sites for both selector and signal effector were combined drove field-specific expression restricted to the wing and haltere discs in patterns predicted by the specific signaling inputs to each element. That is, the [Sd]2 [Su(H)]2 element drove wing-specific expression along the dorsoventral margin, consistent with Notch activation along this boundary, and the [Sd]2 [Mad/Med] element drove expression in a broad domain oriented with respect to the anteroposterior axis of the disc, consistent with Dpp-signaling activity along this boundary. These patterns of expression are similar to those of the native cut and vg quadrant cis-regulatory elements that also respond to Notch- and Dpp-signaling inputs, respectively. However, regulatory elements containing a combination of Su(H) and Mad/Med sites were not active in vivo, demonstrating that combinatorial input in the absence of selector input is not sufficient to drive gene expression. These results suggest that the Vg-Sd complex provides a qualitatively distinct function required to generate a wing-specific response to signaling pathways (Guss, 2001).

Regulation of Wingless and Vestigial expression in wing and haltere discs of Drosophila

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).

Additional evidence is provided that repression of Vg in non-DV cells by Ubx is crucial for haltere development. Overexpression of Vg in haltere discs causes haltere-to-wing transformations. This is particularly significant considering the fact that haltere-to-wing homeotic transformations are always associated with loss of Ubx, by direct removal of Ubx, by activation of its repressors (e.g., polycomb proteins) or by suppression of its activators (e.g. trithorax proteins). Mitotic clones of Ubx alleles in the haltere capitellum normally 'sort out' and often remain as an undifferentiated mass of cells. This is attributed to differential cell-adhesion properties of transformed (Ubx) and non-transformed (Ubx+) cells. No such sorting out of wing-like trichomes was observed in halteres overexpressing Vg. This implies that cells surrounding the wing-like trichomes are also transformed, at least at the level of cell-adhesion properties. This is consistent with observations that removal of Ubx from the DV boundary or over-growth caused by mutations in the tumor-suppressor gene fat confers wing-like cell-adhesion properties to capitellum cells. Since DV signaling is closely associated with the activation of Vg in non-DV cells and Vg is primarily a growth-promoting gene, it is likely that the cell-sorting behavior of Ubx clones is linked to their changed growth properties (Prasad, 2003).

The role of the T-box gene optomotor-blind in patterning the Drosophila wing: omb regulates vg

The development of the Drosophila wing is governed by the action of morphogens encoded by decapentaplegic and wingless that promote cell proliferation and pattern the wing. Along the anterior/posterior (A/P) axis, the precise expression of dpp and its receptors is required for the transcriptional regulation of specific target genes. The function of the T-box gene optomotor-blind (omb), a dpp target gene, was analyzed. The wings of omb mutants have two apparently opposite phenotypes: the central wing is severely reduced and shows massive cell death, mainly in the distal-most wing, and the lateral wing shows extra cell proliferation. Genetic evidence is presented that omb is required to establish the graded expression of the Dpp type I receptor encoded by the gene thick veins (tkv) to repress the expression of the gene master of thick veins and also to activate the expression of spalt (sal) and vestigial (vg), two Dpp target genes. optomotor-blind plays a role in wing development downstream of dpp by controlling the expression of its receptor thick veins and by mediating the activation of target genes required for the correct development of the wing. The lack of omb produces massive cell death in its expression domain, which leads to the mis-activation of the Notch pathway and the overproliferation of lateral wing cells (del Alamo Rodriguez, 2004).

Suppression of Polycomb group proteins by JNK signalling induces wg and vg expression and transdetermination in Drosophila imaginal discs

The PcG proteins function through cis-regulatory elements called PcG response elements (PREs), which enable them to bind and to maintain the state of transcriptional silencing over many cell divisions. PcG proteins operate in two key evolutionarily conserved chromatin complexes, and reduced expression of these complexes, as found in PcG mutants, results in the derepression of PRE-controlled genes. To determine whether PcG silencing is modulated in regenerating tissue, the FLW-1 line, which contains a lacZ reporter gene under the control of the Fab7 PRE, was used. Prothoracic leg discs silent for lacZ expression were fragmented and transplanted into the abdomen of host flies. Flies were fed with 5-bromodeoxyuridine (BrdU) to mark the regenerated tissue (the blastema). In uncut discs, there was little proliferation and expression of lacZ was undetectable. On fragmentation, however, lacZ was expressed in the blastema. To confirm that this derepression was due to a reduction in PcG silencing and not simply to massive proliferation at the wound site, the line LW-1 was used; this line lacks the Fab7 PRE and is normally silent, but it can be activated by induction of GAL4. Neither uncut nor cut leg discs of the LW-1 line showed expression of lacZ after transplantation (Lee, 2005).

To show that transdetermination takes place only in cells with downregulated PcG function, fragmented leg discs of the FLW-1 line were stained for lacZ expression and for Vg in order to visualize the transdetermination to wing fate. It was consistently observed that the Vg staining lay within the lacZ expression domain, suggesting that PcG genes are downregulated in the blastema, enabling PRE-silenced genes to be reactivated according to new morphogenetic cues (Lee, 2005).

To investigate direct targets of PcG regulation that, when reactivated, might contribute to transdetermination, the PREs predicted at the wg and vg genes were tested and both were found to be controlled by PcG proteins. The fact that both the transgenic vg-lacZ reporter construct (which lacks the PRE) and the endogenous vg gene were upregulated in the blastema suggests that PcG proteins may affect vg expression both indirectly (for example, through wg) and directly by means of the vg PRE (Lee, 2005).

JNK signalling in Drosophila is crucial for wound healing and is implicated in many different developmental processes, such as dorsal and thorax closure. hemipterous encodes the JNK kinase (JNKK) that activates the Drosophila JNK Basket. Products of DJun and kayak (the Drosophila homologue of Fos) form the AP-1 transcription factor. A downstream target of JNK signalling is puckered (puc), which encodes a phosphatase that selectively inactivates Basket and thus functions in a negative feedback loop. The expression of puc thus mirrors JNK activity. Because wound healing takes place after fragmentation, it was reasoned that activation of the JNK pathway might be causing the downregulation of PcG proteins in the blastema. The pucE69 line, which carries a P(lacZ) insertion at the puc locus, was used to monitor JNK activity. During the third-instar larval stage puc is not expressed and thus JNK signalling was not activated in leg discs. As expected, however, puc was expressed on fragmentation in all cells at the annealing cut edge (Lee, 2005).

To check whether cells that have activated the JNK pathway also show transdetermination, fragmented leg discs of flies carrying the puc-lacZ reporter and vgBE-Gal4; UAS-GFP constructs were transplanted. In these flies, cells that adopted a wing fate were identified by their expression of green fluorescent protein (GFP). Two days after fragmentation, weak residual puc-lacZ staining was still visible in the central region of the disc. puc-lacZ staining is known to decline rapidly after wound healing is completed. It was found that stronger staining was visible along the cut site, probably owing to ongoing wound healing. On comparison of puc-lacZ staining and GFP fluorescence, JNK-active cells showed a substantial overlap with transdetermined cells; thus, it is concluded that JNK signalling is activated in cells that undergo transdetermination (Lee, 2005).

JNK signalling affects the transcription of numerous genes, including those encoding chromatin regulating factors. Therefore whether JNK signalling can downregulate the PcG proteins required for transdetermination was examined. A constitutively active form of hep was overexpressed in UAS-hepact; hsGal4 flies by a heat-shock pulse. Activating the JNK pathway caused a downregulation of some PcG genes, such as Pc, ph-p and E(Pc). No downregulation of these genes was observed in wild-type larvae before and after heat shock, indicating that this was not an unspecific heat-shock response. Expression was examined of two genes of the Trithorax group (ash1 and brm) that function antagonistically to PcG proteins, but found no upregulation on JNK induction (Lee, 2005).

To show further that JNK has a specific effect on PcG proteins, the analogous experiment was carried out in mammalian cells. The JNK pathway can be activated in mouse embryonic fibroblasts by exposing the cells to ultraviolet light. The expression of MPh2 (mouse polyhomeotic2) was examined because this mammalian PcG gene is expressed in these cells. The expression of MPh2 was decreased on JNK induction, but after treatment with a specific JNK inhibitor it was partially restored. In addition, to show that the downregulation of PcG genes is directly controlled by AP-1, chromatin immunoprecipitation was carried out using antibodies against Fos on chromatin from UAS-hepact; hsG4 and kay1 mutant flies. Enrichment of Fos on the promoter region of ph-p was observed, but no enrichment in chromatin from flies lacking Fos. This finding suggests that AP-1 binds directly to this region to regulate negatively the transcription of ph-p (Lee, 2005).

If activation of JNK signalling in the blastema indeed leads to a downregulation of PcG genes, then impairment of the JNK pathway should result in reduced efficiency of transdetermination. The transdetermination behaviour of wild-type discs was compared with that of discs bearing mutations in the JNKK hep. The transdetermination events were classified into three categories: large regions, small regions, and no regions of transdetermination. In wild-type discs only large regions were detected. In males hemizygous for hep1 (a weak hypomorphic allele), most transplanted leg discs had large transdetermined regions; however, a substantial proportion showed only small regions of transdetermination and a few showed no transdetermination event. In flies heterozygous for hepr75 (a null allele which is hemizygous lethal), most discs showed no or only small regions of transdetermination, and large regions were rarely seen. The morphology of the regenerated discs seemed unaffected in these mutants, indicating that the decline of transdetermination efficiency was not due to inefficient wound healing (Lee, 2005).

This study has shown that PcG genes are downregulated by JNK signalling. Because many developmental regulators need to be switched, the role of PcG downregulation may be to render the cells susceptible to a change in cell identity by shifting the chromatin to a reprogrammable state. Transdetermination has been ascribed to the action of ectopic morphogens, which induce cells to activate incorrect gene cascades. Without doubt, wg and decapentaplegic signalling must be crucially involved in this process, because transdetermination does not result from any random cut but occurs preferentially when cuts are made through particular regions of the disc called 'weak points', which are regions of high morphogen. Inappropriate or overextreme downregulation of the PcG system by JNK in sensitive cells of the weak points thus may create such aberrant local patterns. Indeed, the data indicate that at least the two patterning genes, wg and vg, may be direct targets of the PcG. Notably, hyperactive Wnt signalling can also induce a switch in lineage commitment in mammals, implying that signalling pathways are a potent inducer of cell fate changes in many organisms (Lee, 2005).

Another study has shown that regenerating and transdetermining cells in the blastema have a distinct cell-cycle profile in contrast to the surrounding normal disc cells. It has been proposed that this change in cell-cycle regulation is a prerequisite for the change in cell fate. Indeed, PcG targets include genes involved in cell-cycle regulation, suggesting that this initial step is part of the complete reprogramming cascade required for the regenerating cells to achieve multipotency. Downregulation of PcG silencing by JNK seems to be a fundamental, evolutionarily conserved mechanism of cell fate change and thus may also have implications for studies of stem cell plasticity and tissue remodelling (Lee, 2005).

Regulation of vg expression: Fat and Wingless signaling oppositely regulate epithelial cell-cell adhesion and distal wing development

Development of organ-specific size and shape demands tight coordination between tissue growth and cell-cell adhesion. Dynamic regulation of cell adhesion proteins thus plays an important role during organogenesis. In Drosophila, the homophilic cell adhesion protein DE-Cadherin regulates epithelial cell-cell adhesion at adherens junctions (AJs). This study shows that along the proximodistal (PD) axis of the developing wing epithelium, apical cell shapes and expression of DE-Cad are graded in response to Wingless, a morphogen secreted from the dorsoventral (DV) organizer in distal wing, suggesting a PD gradient of cell-cell adhesion. The Fat (Ft) tumor suppressor, by contrast, represses DE-Cad expression. In genetic tests, ft behaves as a suppressor of Wg signaling. Cytoplasmic pool of ß-catenin/Arm, the intracellular transducer of Wg signaling, is negatively correlated with the activity of Ft. Moreover, unlike that of Wg, signaling by Ft negatively regulates the expression of Distalless (Dll) and Vestigial (Vg). Finally, Ft is shown to intersect Wnt/Wg signaling, downstream of the Wg ligand. Fat and Wg signaling thus exert opposing regulation to coordinate cell-cell adhesion and patterning along the PD axis of Drosophila wing (Jaiswal, 2006).

In both loss- and gain-of-function assays, this study shows that Ft downregulates Dll and Vg/Q-vg-lacZ in the distal wing. Although Vg/Q-vg-lacZ and Dll have not been ascertained to be the direct targets of Wg, all available evidence so far suggests that these targets positively respond to Wg signaling. These results also show that Ft and Wg signaling intersect and control distal wing growth and pattern, presumably through their opposing regulation of a common set of targets, namely, DE-Cad, Vg and Dll. Apart from Wg signaling, Dpp signaling also regulates Q-vg-lacZ; however, its long-range target, Omb is not upregulated in ft mutant clones, suggesting that regulation of distal wing targets by Ft is mediated by its intersection with Wg signaling (Jaiswal, 2006).

The results show that Ft negatively regulates Wg signaling. Loss or gain of Ft induces a telltale sign of perturbations in Wg signaling, namely, changes in the cellular pool of ß-catenin/Arm, consistent with its role as a suppressor of Wg signaling in genetic tests. The results further reveal intersection of Ft with Wg signaling downstream of the Wg ligand, while with respect to its receptor, Ft is likely to act either upstream of or parallel to Fz/Fz2. It is interesting to note here that the role of Ft in PCP regulation has also been suggested to be either parallel to or upstream of the Fz receptor. It is also noted that Ft co-localizes with neither Fz nor Fz2 and does not mediate their subcellular localization, thereby suggesting that Ft interacts with Fz indirectly. Unraveling the genetic and molecular basis of this interaction may explain how Ft straddles both the canonical (growth and cell-cell adhesion) and non-canonical (PCP) Wnt signaling pathways (Jaiswal, 2006).

One of the remarkable aspects of development of an organ primordium is that a stereotypic PCP is achieved even while it passes through dynamic changes in its size and shape. The fact that changing organ sizes/shapes does not alter PCP suggests an in-built mechanism to regulate constancy of PCP during animal development. A link between PCP and growth through the activity of Ft has been speculated, since it regulates both. Intersection of Ft and the canonical Wg signaling seen here might provide a mechanism to coordinate PCP and organ growth (Jaiswal, 2006).

Drosophila wing growth is under dynamic spatial and temporal regulation by Wg signaling. Furthermore, different thresholds of Wg signaling impact cell proliferation in their characteristic ways and activate distinct sets of PD markers. Although at a very high threshold, Wg signaling inhibits cell proliferation, at a modest threshold it has been shown to stimulate growth. It is noted that loss of Ft fails to activate Wg targets that demand a high threshold of Wg signaling, e.g., Ac, which is required for wing margin specific bristle development. Conversely, overexpression of Ft also does not lead to loss of margin bristles, suggesting that it is not a strong repressor of Wg signaling either. The short-range Wg target, fz3-lacZ, which responds to a high threshold of Wg signaling, is also not upregulated by loss of Ft. Dll responds to a higher threshold of Wg signaling than that required for Vg/Q-vg. Dll and Vg display modest and strong upregulation respectively, following loss of Ft. These results suggest that loss of Ft upregulates Wg signaling to only modest thresholds, consistent with the growth-promoting effect of the latter (Jaiswal, 2006).

Cell cycle genes regulate vestigial and scalloped to ensure normal proliferation in the wing disc of Drosophila

In Drosophila, the Vestigial-Scalloped (VG-SD) dimeric transcription factor is required for wing cell identity and proliferation. Previous results have shown that VG-SD controls expression of the cell cycle positive regulator dE2F1 during wing development. Since wing disc growth is a homeostatic process, the possibility was investigated that genes involved in cell cycle progression regulate vg and sd expression in feedback loops. The experiments focused on two major regulators of cell cycle progression: dE2F1 and the antagonist Dacapo (Dap). The results reinforce the idea that VG/SD stoichiometry is critical for correct development and that an excess in SD over VG disrupts wing growth. Transcriptional activity of VG-SD and the VG/SD ratio are both modulated by down-expression of cell cycle genes. A dap-induced sd up-regulation was detected that disrupts wing growth. Moreover, a rescue was observed of a vg hypomorphic mutant phenotype by dE2F1 that is concomitant with vg and sd induction. This regulation of the VG-SD activity by dE2F1 is dependent on the vg genetic background. The results support the hypothesis that cell cycle genes fine-tune wing growth and cell proliferation, in part, through control of the VG/SD stoichiometry and activity. This points to a homeostatic feedback regulation between proliferation regulators and the VG-SD wing selector (Legent, 2006).

Cell proliferation relies on the tight control of cell cycle genes, and, in the wing pouch, VG-SD is also critically required. Accordingly, vg up-regulates dE2F1 expression and antagonizes the CKI dap. This study investigated the effects of these two antagonistic proliferation regulators in the wing pouch of the disc, and tested the hypothesis that cell cycle genes fine-tune proliferation, through regulation of the respective expressions of vg and sd and VG-SD dimer activity, thereby providing a feedback control (Legent, 2006).

Combined loss and gain of function experiments has ascertained the requirement of a precise VG/SD ratio for normal wing development and has shown that an excess in SD disrupts VG-SD function in wing growth, and probably acts as a dominant-negative through titration of functional VG-SD dimers. Therefore, sd induction may efficiently restrain VG-SD function in vivo, and a similar effect may also be physiologically achieved down-regulating vg. Moreover, since SD DNA target selectivity is modified upon binding of VG to SD in vitro, the hypothesis cannot be discarded that, in vivo too, VG-SD targets might be different from the targets of SD alone. This could explain to some extent the phenotypes observed when sd is induced (Legent, 2006).

The results show that the CKI member DAP, homogeneously expressed in the wing disc, regulates VG-SD function. dap heterozygotes display a wild type wing phenotype, reduced levels of both vg and sd transcripts, but an almost normal vg/sd ratio, thus VG-SD activity is normal. Consistently, no abnormal wing phenotype could be detected. Therefore, the relative vg/sd stoichiometry, rather than absolute vg and sd expression levels, determines wing growth. Interestingly, it had been observed that dap homozygous mutant adult escapers display duplication of the wing margin, indicating a role of DAP at the D/V boundary. This phenotype could be linked to an enhanced proliferation due to the absence of CKI function. Moreover, D/V-specific over-expression of dap alters wing margin structures. This dap over-expression triggers both ectopic expression of sd and subsequent impairment of VG-SD activity associated with a proliferation decrease.The associated wing phenotype is clearly enhanced in vg heterozygous flies, providing evidence that dap over-expression affects VG/SD stoichiometry and represses VG-SD activity in wing development. This reveals a model in which, in the wing pouch, cell proliferation down-regulation through cyclin/CDK inhibition by DAP, may be enhanced by an additive reduction of VG-SD proliferation function. Such a mechanism probably participates in vivo in the control of balanced wing growth (Legent, 2006).

The results also demonstrate that dE2F1-DP regulates VG-SD: the dE2F1 heterozygote displays a reduced vg/sd ratio due to a decrease in vg and an increase in sd transcripts, associated with reduced dimer activity, comparable to the vgnull/+ context. Thus, dE2F1 is required for vg normal expression. This supports the hypothesis that the slower proliferation observed in these contexts is linked to an imbalance in the dimer ratio (Legent, 2006).

Conversely, over-expressing dE2F1-DP-P35, in a vg83b27 hypomorphic mutant context, rescues expression of both vg and sd and normal VG-SD function, wing appendage specification and growth. This is not observed in vgnull flies implying the necessity for vg sequences, but the second intron, missing in the vg83b27 mutant. In addition, it was ascertained that not all the genes triggering cell cycle progression or cell proliferation can induce vg expression. Neither ectopic expression of CYC E, which promotes dE2F1-induced G1/S cell cycle transition, nor the growth regulator Insulin receptor (InR) is sufficient to elicit VG expression and wing growth in the vg83b27 mutant. These results demonstrate that vg induction is a prerequisite for vg83b27 wing pouch growth in response to dE2F1 activity (Legent, 2006).

In a vg+ genetic background, dE2F1 over-expression induces only sd, disrupting VG/SD stoichiometry. Consistently, at the D/V boundary, wing notching was observed. Therefore, although dE2F1 basically displays a positive role in proliferation, this sd induction in response to dE2F1 over-expression is clearly associated with wing growth impairment. This effect is significantly weaker in a vg heterozygote background, and a rescue of the wing phenotype was observed, supporting the hypothesis that VG/SD stoichiometry is restored. Therefore, sd induction by dE2F1 depends on the vg genetic context. This indicates that the effects of over-expressing dE2F1 differ depending on the growth-state of the wing pouch, which is tightly linked with the vg genotype (Legent, 2006).

Clearly, feedback regulations rule the growth of the wing disc. Regulation has been noted in three different vg genetic contexts that can be analyzed in the light of a homeostasis hypothesis. In the vg83b27under-proliferative wing pouch, ectopic dE2F1 expression coordinately increases vg and sd expression in a positive feedback loop. This triggers VG-SD activity, and induces both cell proliferation and wing specification in the mutant. Conversely, no such crosstalk occurs in a correctly grown vg+ disc, where over-growth should be prevented. In this latter case, sd induction (VG/SD decrease) probably restrains the proliferation function of dE2F1. Consistently, wings were not overgrown, but notches were observed. This phenotype was partially suppressed in a vg heterozygote background. As a whole, these results support the hypothesis that VG-SD/dE2F1 coordination tends to ensure normal wing growth and that the dimer does not trigger unrestricted cell proliferation in a vg+ context, since an excess in dE2F1 attenuates VG-SD function in a negative feedback loop. Thus, molecular interactions between dE2F1, vg and sd, display a clear plasticity depending on the vg genetic context (Legent, 2006).

Establishing and maintaining homeostasis is critical during development. This is achieved in part through a balance between cell proliferation and death. In mammals E2F1 and p21, the dacapo homolog, play a key role in this process. In the wing disc compensatory proliferation induced by cell death has been observed. However, the role of cell cycle genes in this process has not yet been established. How patterns of cell proliferation are generated during development is still unclear. It seems nevertheless likely that the gene responsible for regulating differentiation also regulates proliferation and growth. For instance, Hedgehog (HH) induces the expression of Cyclins D and E. This mediates the ability of HH to drive growth and proliferation. In the same way, other data support a direct regulation of dE2F1 by the Caudal homeodomain protein required for anterio-posterior axis formation and gut development. Wingless (WG) also displays both patterning and a cell cycle regulator function during Drosophila development (Legent, 2006).

Growth control in the wing pouch seems to be achieved through both positive and negative feedback regulations linking dE2F1 and VG-SD, but also via additive impairment of VG-SD by DAP. In fact, in a vg+ background, over-expression of both dap and dE2F1 induces sd, impairs VG-SD and alters wing development. Nevertheless, clear opposite behaviors are observed in vgnull/+ flies where dap-induced nicks are enhanced, while those of dE2F1 are partially rescued. This highlights the functional discrepancy between these two types of feedback regulation. It is suggested that dap expression inhibits cell proliferation through a process involving both Cyclin-CDK inhibition and VG-SD impairment in the wing pouch. In contrast, it is proposed that dE2F1 over-expression triggers a homeostatic response. It will either induce vg and sd to ensure proliferation (in a vg83b27 genotype), or decrease the VG/SD ratio in a vg+ context. In this latter genotype, down-regulation probably counteracts fundamental proliferative properties of dE2F1 and governs homeostatic wing disc growth (Legent, 2006).

At late third instar, wing discs display a Zone of Non-proliferating Cells (ZNC) along the wing pouch D/V boundary. It has been shown that, although dE2F1-DP is expressed in this area, its proliferative function is inactivated late, because of RBF1-induced G1 arrest. Accordingly, although expression of vg and sd presents a peak at the D/V boundary, in late third instar, VG-SD activity is decreased in D/V cells, and it was suggested to result from an excess of SD. Therefore, the ZNC setting may also reflect a VG-SD/dE2F1 coordinated dialogue that triggers a decrease in proliferation signals in this area (Legent, 2006).

Previous studies of homeostatic control of cell proliferation in the wing reported that, to some extent, over-expression of positive or negative cell cycle regulators only weakly affects the overall division rate. For instance, although dap over-expression alters dE2F1 function in G1-S cell cycle transition, it also promotes dE2F1 expression and function in G2-M transition, preventing a decrease in the overall rate of cell division. Strikingly, the cells seemed able to monitor each phase length and maintain cell cycle duration and normal proliferation in the wing pouch of the disc. Therefore, dE2F1 is a central component that enables cells to ensure normal proliferation in the wing disc and prevents imbalance in the process. The fact that dE2F1 triggers quite different or opposite responses in vg+ or vg hypomorphic contexts suggests that the VG-SD/dE2F1 crosstalk plays a role in the same sort of homeostatic process that ensures entire wing growth (Legent, 2006).

Such regulations are likely to reveal a precise physiological fine-tuning of vg and sd by cell cycle effectors, promoting an exquisite control of wing growth. Feedback loops between the developmental selector VG-SD and cell cycle effectors may stand for a control mechanism to guarantee that the tissue can sustain balanced growth and a reproducible size. Such a subtle mechanism, on a local scale, would correct the alterations in cell proliferation that may occur during development (Legent, 2006).

A feed-forward circuit linking wingless, fat-dachsous signaling, and the warts-hippo pathway to Drosophila wing growth

During development, the Drosophila wing primordium undergoes a dramatic increase in cell number and mass under the control of the long-range morphogens Wingless (Wg, a Wnt) and Decapentaplegic (Dpp, a BMP). This process depends in part on the capacity of wing cells to recruit neighboring, non-wing cells into the wing primordium. Wing cells are defined by activity of the selector gene vestigial (vg) and recruitment entails the production of a vg-dependent 'feed-forward signal' that acts together with morphogen to induce vg expression in neighboring non-wing cells. This study identifies the protocadherins Fat (Ft) and Dachsous (Ds), the Warts-Hippo tumor suppressor pathway, and the transcriptional co-activator Yorkie (Yki, a YES associated protein, or YAP) as components of the feed-forward signaling mechanism; this mechanism promotes wing growth in response to Wg. vg generates the feed-forward signal by creating a steep differential in Ft-Ds signaling between wing and non-wing cells. This differential down-regulates Warts-Hippo pathway activity in non-wing cells, leading to a burst of Yki activity and the induction of vg in response to Wg. It is posited that Wg propels wing growth at least in part by fueling a wave front of Ft-Ds signaling that propagates vg expression from one cell to the next (Zecca, 2010).

During larval life, the Drosophila wing primordium undergoes a dramatic ~200-fold increase in cell number and mass driven by the morphogens Wg and Dpp. Focusing on Wg, it has been established that this increase depends at least in part on a reiterative process of recruitment in which wing cells send a feed-forward (FF) signal that induces neighboring cells to join the primordium in response to morphogen. The present results identify Ft-Ds signaling, the Wts-Hpo tumor suppressor pathway, and the transcriptional co-activator Yki as essential components of the FF process and define the circuitry by which it propagates from one cell to the next. This discussion considers, in turn, the nature of the circuit, the parallels between FF signaling and PCP, and the implications for the control of organ growth by morphogen (Zecca, 2010).

Several lines of evidence are presented that expression of the wing selector gene vg drives production of the FF signal by promoting a non-autonomous signaling activity of Ft. First, it was shown that vg acts both to up-regulate fj and down-regulate ds, two outputs known to elevate an outgoing, signaling activity of Ft in PCP. Second, it was demonstrated that experimental manipulations that elevate Ft signaling -- specifically, over-expression of Ft or removal of Ds -- generate ectopic FF signal. Third, and most incisively, it was shown that ft is normally essential in wing cells to send FF signal (Zecca, 2010).

Ft and Ds are both required in non-wing cells to receive the FF signal, functioning in this capacity to prevent the activation of vg unless countermanded by FF input. Notably, the removal of either Ft or Ds from non-wing cells constitutively activates the FF signal transduction pathway, mimicking receipt of the FF signal. However, the pathway is only weakly activated in this condition and the cells are refractory to any further elevation in pathway activity (Zecca, 2010).

Previous studies have defined a transduction pathway that links Ft-Ds signaling via the atypical myosin myosin Dachs (D) to suppression of the Wts kinase and enhanced nuclear import of Yki. Likewise, Ft and Ds operate through the same pathway to transduce the FF signal. Specifically, it was shown that manipulations of the pathway that increase nuclear activity of Yki (over-expression of D or Yki, or loss of Wts or Ex) cause non-wing cells to adopt the wing state. Conversely, removal of D, an intervention that precludes down-regulation of Wts by Ft-Ds signaling, prevents non-wing cells from being recruited into the wing primordium (Zecca, 2010).

To induce non-wing cells to become wing cells, transduction of the FF signal has to activate vg transcription. Activation is mediated by the vg QE and depends on binding sites for Scalloped (Sd), a member of the TEAD/TEF family of DNA binding proteins that can combine with either Yki or Vg to form a transcriptional activator Hence, it is posited that Yki transduces the FF signal by entering the nucleus and combining with Sd to activate vg. In addition, it is posited that once sufficient Vg produced under Yki-Sd control accumulates, it can substitute for Yki to generate a stable auto-regulatory loop in which Vg, operating in complex with Sd, sustains its own expression. Accordingly, recruitment is viewed as a ratchet mechanism. Once the auto-regulatory loop is established, neither FF signaling nor the resulting elevation in Yki activity would be required to sustain vg expression and maintain the wing state (Zecca, 2010).

Both the activation of the QE by Yki as well as the maintenance of its activity by Vg depend on Wg and Dpp input and hence define distinct circuits of vg auto-regulation fueled by morphogen. For activation, the circuit is inter-cellular, depending on Ft-Ds signaling for vg activity to propagate from one cell to the next. For maintenance, the circuit is intra-cellular, depending on Vg to sustain its own expression. Accordingly, it is posited that growth of the wing primordium is propelled by the progressive expansion in the range of morphogen, which acts both to recruit and to retain cells in the primordium (Zecca, 2010).

To date, Ft-Ds signaling has been studied in two contexts: the control of Yki target genes in tissue growth and the orientation of cell structures in PCP. Most work on tissue growth has focused on Yki target genes that control basic cell parameters, such as survival, mass increase, and proliferation (e.g., diap, bantam, and cyclinE). In this context, Ds and Ft are thought to function as a ligand-receptor pair, with tissue-wide gradients of Ds signal serving to activate Ft to appropriate levels within each cell. In contrast, Ft and Ds behave as dual ligands and receptors in PCP, each protein having intrinsic and opposite signaling activity and both proteins being required to receive and orient cells in response to each signal (Zecca, 2010).

This study has analyzed a different, Yki-dependent aspect of growth, namely the control of organ size by the regulation of a selector gene, vg. In this case, Ft appears to correspond to a ligand, the FF signal, and Ds to a receptor required to receive the ligand -- the opposite of the Ds-Ft ligand-receptor relationship inferred to regulate other Yki target genes. Moreover, as in PCP, evidence was found that Ft and Ds operate as bidirectional ligands and receptors: like Ds, Ft is also required for receipt of the FF signal, possibly in response to an opposing signal conferred by Ds (Zecca, 2010).

Studies of Ft-Ds interactions, both in vivo and in cell culture, have established that Ft and Ds interact in trans to form hetero-dimeric bridges between neighboring cells, the ratio of Ft to Ds presented on the surface of any given cell influencing the engagement of Ds and Ft on the abutting surfaces of its neighbors. These interactions are thought, in turn, to polarize the sub-cellular accumulation and activity of D. Accordingly, it is posited that vg activity generates the FF signal by driving steep and opposing differentials of Ft and Ds signaling activity between wing (vgON) and non-wing (vgOFF) cells. Further, it is posited that these differentials are transduced in cells undergoing recruitment by the resulting polarization of D activity, acting through the Wts-Hpo pathway and Yki to activate vg (Zecca, 2010).

Thus, it is proposeed that FF propagation and PCP depend on a common mechanism in which opposing Ft and Ds signals polarize D activity, both proteins acting as dual ligands and receptors for each other. However, the two processes differ in the downstream consequences of D polarization. For FF propagation, the degree of polarization governs a transcriptional response, via regulation of the Wts-Hpo pathway and Yki. For PCP, the direction of polarization controls an asymmetry in cell behavior, through a presently unknown molecular pathway (Zecca, 2010).

FF propagation and PCP may also differ in their threshold responses to D polarization. vg expression is graded, albeit weakly, within the wing primordium, due to the response of the QE to graded Wg and Dpp inputs. Hence, a shallow differential of Ft-Ds signaling reflecting that of Vg may be sufficient to orient cells in most of the prospective wing territories, but only cells in the vicinity of the recruitment interface may experience a steep enough differential to induce Yki to enter the nucleus and activate vg (Zecca, 2010).

Finally, FF propagation and PCP differ in at least one other respect, namely, that they exhibit different dependent relationships between Ft and Ds signaling. In PCP, clonal removal of either Ft or Ds generates ectopic polarizing activity, apparently by creating an abrupt disparity in the balance of Ft-to-Ds signaling activity presented by mutant cells relative to that of their wild type neighbors. By contrast, in FF propagation, only the removal of Ds, and not that of Ft, generates ectopic FF signal. This difference is attributed to the underlying dependence of Ft and Ds signaling activity on vg. In dso cells, Ft signaling activity is promoted both by the absence of Ds and by the Vg-dependent up-regulation of fj. However, in fto cells, Ft is absent and Vg down-regulates ds, rendering the cells equivalent to dso fto cells (which are devoid of signaling activity in PCP). Thus, for FF propagation, the underlying circuitry creates a context in which only the loss of Ds, but not that of Ft, generates a strong, ectopic signal. For PCP, no such circuit bias applies (Zecca, 2010).

Morphogens organize gene expression and cell pattern by dictating distinct transcriptional responses at different threshold concentrations, a process that is understood conceptually, if not in molecular detail. At the same time, they also govern the rate at which developing tissues gain mass and proliferate, a process that continues to defy explanation (Zecca, 2010).

One long-standing proposal, the 'steepness hypothesis,' is that the slope of a morphogen gradient can be perceived locally as a difference in morphogen concentration across the diameter of each cell, providing a scalar value that dictates the rate of growth. Indeed, in the context of the Drosophila wing, it has been proposed that the Dpp gradient directs opposing, tissue-wide gradients of fj and ds transcription, with the local differential of Ft-Ds signaling across every cell acting via D, the Wts-Hpo pathway, and Yki to control the rate of cell growth and proliferation. The steepness hypothesis has been challenged, however, by experiments in which uniform distributions of morphogen, or uniform activation of their receptor systems, appear to cause extra, rather than reduced, organ growth (Zecca, 2010).

The current results provide an alternative interpretation (see The vestigial feed-forward circuit, and the control of wing growth by morphogen). It is posited that 'steepness,' as conferred by the local differential of Ft-Ds signaling across each cell, is not a direct reflection of morphogen slope but rather an indirect response governed by vg activity. Moreover, it is proposed that it promotes wing growth not by functioning as a relatively constant parameter to set a given level of Wts-Hpo pathway activity in all cells but rather by acting as a local, inductive cue to suppress Wts-Hpo pathway activity and recruit non-wing cells into the wing primordium (Zecca, 2010).

How important is such local Ft-Ds signaling and FF propagation to the control of wing growth by morphogen? In the absence of D, cells are severely compromised for the capacity to transduce the FF signal, and the wing primordium gives rise to an adult appendage that is around a third the normal size, albeit normally patterned and proportioned. A similar reduction in size is also observed when QE-dependent vg expression is obviated by other means. Both findings indicate that FF signaling makes a significant contribution to the expansion of the wing primordium driven by Wg and Dpp. Nevertheless, wings formed in the absence of D are still larger than wings formed when either Wg or Dpp signaling is compromised. Hence, both morphogens must operate through additional mechanisms to promote wing growth (Zecca, 2010).

At least three other outputs of signaling by Wg (and likely Dpp) have been identified that work in conjunction with FF propagation. First, as discussed above, Wg is required to maintain vg expression in wing cells once they are recruited by FF signaling, and hence to retain them within the wing primordium. Second, it functions to provide a tonic signal necessary for wing cells to survive, gain mass, and proliferate at a characteristic rate. And third, it acts indirectly, via the capacity of wing cells, to stimulate the growth and proliferation of neighboring non-wing cells, the source population from which new wing cells will be recruited. All of these outputs, as well as FF propagation, depend on, and are fueled by, the outward spread of Wg and Dpp from D-V and A-P border cells. Accordingly, it is thought that wing growth is governed by the progressive expansion in the range of Wg and Dpp signaling (Zecca, 2010).

Identification of Ft-Ds signaling, the Wts-Hpo pathway, and Yki as key components of the FF recruitment process provides a striking parallel with the recently discovered involvement of the Wts-Hpo pathway and Yki/YAP in regulating primordial cell populations in vertebrates, notably the segregation of trophectoderm and inner cell mass in early mammalian embryos and that of neural and endodermal progenitor cells into spinal cord neurons and gut. As in the Drosophila wing, Wts-Hpo activity and YAP appear to function in these contexts in a manner that is distinct from their generic roles in the regulation of cell survival, growth, and proliferation, namely as part of an intercellular signaling mechanism that specifies cell type. It is suggested that this novel employment of the pathway constitutes a new, and potentially general, mechanism for regulating tissue and organ size (Zecca, 2010).

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).

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vestigial: Biological Overview | Evolutionary Homologs | Targets of Activity and Protein Interactions | Developmental Biology | Effects of Mutation | References

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