Transcriptional Regulation

Regulation of early scalloped expression in the wing disc has not been characterized. Late expression of scalloped appears to be reciprocally activated by vestigial in the wing disc. Late expression of sd is absent in vestigial mutants (Williams, 1993).

Both vestigial and scalloped are overexpressed in shaggy/zeste white mutant clones. shaggy may be acting downstream of localized wingless expression to specify or maintain marginal identity in the wing (Blair, 1994).

The wingless product is required to restrict the expression of the apterous gene to dorsal cells and to promote the expression of the vestigial and scalloped genes that demarcate the wing primordia and act in concert to promote morphogenesis. Two genes expressed along the normal wing margin, vestigial and scalloped, are overexpressed at margin-like levels in shaggy-zeste white 3 clones. This phenotype does not depend upon the formation of ectopic bristle precursors and occurs in clones lacking both shaggy/zeste white 3 and the entire achaete-scute complex. As vestigial and scalloped are both involved in early patterning events prior to the stages of bristle specification, these results strongly suggest that shaggy/zeste white 3 is required for the normal specification or maintenance of regional identity in the developing wing blade. However, the margin-like transformation is only partial, since the expression of apterous (in pupal wings) and wingless and cut (at late third instar) is not reliably altered in shaggy/zeste white 3 clones. shaggy/zeste white 3 may act downstream of localized apterous and wingless expression to specify or maintain margin identity in the wing (Williams, 1993 and Blair, 1994). Expression of scalloped appears to be reciprocally activated by vestigial in the wing disc (Williams, 1993).

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).

Targets of Activity

scalloped appears to be a specific activator of vestigial in the wing disc (Williams 1993).

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

A small number of major regulatory (selector) genes have been identified in animals that control the development of particular organs or complex structures. In Drosophila, the vestigial gene is required for wing formation and is able to induce wing-like outgrowths on other structures. Because ectopic expression of Vg in many imaginal discs induces the outgrowth of wing tissue, the expression of various wing patterning genes was examined to see if they are induced in ectopic growths. Vg is expressed in the entire developing wing pouch whereas Sal and SRF have specific expression patterns within this domain but are not expressed in wild-type leg discs. Targeted expression of Vg with the Gal4-UAS system induces ectopic expression of Sal and SRF in developing leg imaginal discs. Similarly, the nubbin (nub) gene (which is also expressed and required during wing development ) is ectopically induced in leg discs by Vg expression. In each case, only a subset of the cells expressing Vg activate the target gene, which suggests that additional factors control the expression pattern of each gene. In a first step toward elucidating the molecular mechanism by which Vg regulates gene expression, the response of wing-specific enhancers to ectopic Vg expression was examined. Attention was focused on both the boundary and quadrant enhancers of the vg gene and the enhancer from the SRF gene that drives expression specifically in the intervein region between veins three and four. All three enhancers are induced by ectopic Vg expression in leg and other imaginal discs. Importantly, ectopic expression of Vg in clones of cells induces the enhancers only within the clones. However, gene expression is not induced in all cells within clones nor in all clones. In addition, each individual enhancer is expressed in different regions of these discs that appear to correlate with the spatial distribution of the different signaling inputs known to be required for activation of these enhancers (Halder, 1998).

Scalloped is required for Vg function. In the notum primordia of the wing disc, the vg enhancers, as well as the sal, SRF, and nub genes are not induced by ectopic Vg even though the known required extracellular signals are present. Target gene activation could depend then on the function of another gene(s). One candidate for such a factor is the product of the sd gene, which is expressed in a pattern similar to Vg in the wing disc and is required for wing formation and the proper expression of Vg and other genes. In other discs, such as the leg and eye discs, sd is endogenously expressed and is upregulated wherever ectopic Vg is able to induce wing-specific gene expression and trigger wing development. It is noted, however, that a sd enhancer trap line and the SRF-intervein C enhancer transgene are also ectopically induced by Vg in the presumptive notum, although at levels lower than those observed in the wing pouch. This is consistent with the inability of Vg to trigger wing development and induce other wing patterning genes in the developing notum. Indeed, mis-expression studies show that Sd function is required in parallel with Vg in order for Vg to exert its wing inducing activity. The three wing-specific enhancers from the SRF and vg genes are activated synergistically when Sd and Vg are coexpressed in Drosophila S2 cells. Although each individual protein has some effect on reporter gene expression, this is significantly less than that observed in the presence of both Vg and Sd. Titration of the amounts of transfected Vg and Sd plasmids with all enhancers shows that the relative concentration of the two factors is critical and, at any given Vg concentration, high levels of Sd reduce activation (Halder, 1998).

To define the sequences of the enhancers that respond to Vg/Sd, the activation of smaller fragments from the 704-bp SRF intervein C enhancer, the 806-bp vg quadrant enhancer, and the 754-bp vg boundary enhancer in tissue culture were analyzed. A 125-bp fragment (SRF-A) derived from the 5' end of the SRF enhancer is activated, whereas an adjacent 131-bp fragment (SRF-B) is not activated. A 65-bp fragment from the vg quadrant enhancer (MD2) has been identified that, when multimerized, produces an expression pattern very similar to the full-length enhancer in wing discs. When assayed in tissue culture, MD2 is activated by Vg and Sd. Within the vg boundary enhancer, a 120-bp fragment sufficient to drive reporter gene expression in the wing pouch (vg-A) as well as a nonoverlapping 90-bp fragment (vg-B) are also activated synergistically by cotransfection of Vg and Sd. Sd was shown, using mobility shift and DNase I footprinting assays, to bind specifically to essential sites for target gene activation (Halder, 1998).

One possible reason for the importance of the concentration of Sd on Vg function concerns the localization of the Vg protein. It was observed that in S2 cells transfected with the vg expression plasmid alone, the Vg protein appears to be localized to both the cytoplasm and the nucleus. In contrast, in cells cotransfected with the Vg and Sd expression plasmids, Vg is clearly localized to nuclei. Vg localization is more diffuse in sd mutant clones than in sd+ cells; this is also true of ectopic Vg localization in regions of imaginal discs that lack endogenous Sd expression. Furthermore, deletion of the Sd interaction domain of Vg results in cytoplasmic accumulation of Vg in vivo. Thus, Sd may facilitate the transport or retention of Vg protein in the nucleus and, coupled with the concentration-dependent, synergistic effects of Vg and Sd on target gene expression, these results suggest that the proteins form a complex in vivo (Halder, 1998).

These results demonstrate that the activation of several genes in the wing field requires Vg/Sd function. It is also known that for each of the cis-regulatory elements analyzed here, direct input(s) of particular signaling pathways are also required. Specifically, the activation of the SRF intervein C element requires both Vg/Sd and Hh signaling; the activation of the vg boundary enhancer requires Vg/Sd and N signaling, and the activation of the vg quadrant enhancer requires Vg/Sd and Dpp signaling. Because these regulatory elements are not expressed in all tissues in which the signals are active, nor in all wing cells in which Vg/Sd are active, it is deduced that neither the input of various signals nor of Vg/Sd alone are sufficient for gene activation in vivo. Rather, the results suggest that the various wing-specific cis-regulatory elements require a combination of direct inputs, comprising the Vg/Sd selector function, which restricts expression to the wing field, and at least one signal transducer that mediates signaling inputs and hence, the pattern of gene expression within the wing field. One prediction of this model is that gene expression patterns within the wing field may be changed by altering the signal-transducer binding sites within a cis-response element. To test this, the Suppressor of Hairless [Su(H)] binding site that mediates the N input in the vg boundary enhancer was changed to sites for the Cubitus interruptus (Ci) protein that transduces Hh signaling. This switches the pattern of gene expression from a N-induced dorsoventral stripe to a Hh-induced anteroposterior stripe while retaining the restriction of gene activation to the wing disc (Halder, 1998 and references).

These results demonstrate that the role of the Vg/Sd selector function is to directly regulate wing-specific cis-regulatory elements that also require particular signaling inputs. The patterns of gene expression induced in the wing disc are limited to cells in which both the selector genes and specific signaling pathways are active. The response of the SRF-A, vg boundary, and vg quadrant enhancers to Hh, N, and Dpp signaling are limited to the wing pouch by Vg/Sd and occur in different patterns because of their direct regulation by the Ci, Su(H), and Mad proteins, respectively. Furthermore, the finding that the changing of the Su(H) binding site into a Ci binding site in the vg boundary enhancer switches the pattern from a wing-specific dorsoventral N-regulated stripe to a wing-specific anteroposterior Hh-regulated stripe suggests that spatial expression patterns are determined by the sites for individual DNA-binding signal transducers. One corollary of this model is that for any given signaling protein, different selector proteins may be involved in directing tissue-specific responses in different organs and tissues. For example, other studies have shown that tissue-specific enhancers in the embryo that are regulated by Dpp also require the action of the Labial/Extradenticle or Tinman selector proteins to limit expression to the endoderm or mesoderm, respectively. It is suggested that, in general, combinatorial control by selector proteins and common signal transducers at a cis-regulatory level is required for the tissue- and organ-specific responses of target genes to widely deployed signaling systems (Halder, 1998 and references).

The development of the Drosophila wing involves progressive patterning events. In the second larval instar, cells of the wing disc are allotted wing or notum fates by a wingless-mediated process and dorsal or ventral fates by the action of apterous and wingless. Notch-mediated signaling is required for the expression of the genes vestigial and scalloped in the presumptive wing blade. Later, wingless, Notch and cut are involved in cell fate specification along the wing margin. The function of scalloped in this process is not well understood and is the focus of this study. Patterning downstream of Notch and wingless pathways is altered in scalloped mutants. Reduction in scalloped expression results in a loss of expression of wing blade- and margin-specific markers. An enhancer element in the second intron of the vg gene is found influence the level of sd expression. Misexpression of scalloped in the presumptive wing causes misexpression of scalloped, vestigial and wingless reporter genes. However, high levels of scalloped expression have a negative influence on wingless, vestigial and its own expression. These results demonstrate that scalloped functions in a level-dependent manner in the presumptive wing blade in a loop that involves vestigial and itself. It is suggested that wing development requires the regulated expression of scalloped, together with vestigial: the 'wing formation' effects of Vestigial in other imaginal discs are probably due to its interaction with the scalloped gene product normally expressed in these discs. sc and vg respond to wg and N signaling in a manner very similar to that characterized for vg in the developing wing. This activation is maintained by auto-regulatory events. It is also suggested that sd and vg serve to regulate and modulate wg expression and to change the pattern observed in the second larval instar to the very different pattern seen in the third instar wing disc (Varadarajan, 1999).

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).

blistered is expressed in the precursors of the terminal tracheal cells and in the future intervein territories of the third instar wing imaginal disc. Dissection of the blistered regulatory region reveals that a single enhancer element, which is under the control of the fibroblast growth factor (FGF)-receptor signaling pathway, is sufficient to induce blistered expression in the terminal tracheal cells. In contrast, two separate enhancers direct expression in distinct intervein sectors of the wing imaginal disc. One element is active in the central intervein sector and is induced by the Hedgehog signaling pathway. The other element is under the control of Decapentaplegic and is active in two separate territories, which roughly correspond to the intervein sectors flanking the central sector. Hence, each of the three characterized enhancers constitutes a molecular link between a specific territory induced by a morphogen signal and the localized expression of a gene required for the final differentiation of this territory (Nussbaumer, 2000).

An blistered enhancer (the boundary enhancer) has been identified that is activated by Hh signaling in the cells anterior to the A/P compartment boundary. In agreement with previous reports demonstrating a direct morphogenic role of Hh in the central region of the wing, this might indicate that the Hh signaling is required to trigger intervein differentiation through blistered expression in the intervein C domain. However, in contradiction to the activity of the boundary enhancer, blistered is expressed in smo mutant clones analyzed during pupal development. Noteworthy, the clones that were generated were analyzed during third instar, whereas blistered expression is detected later, 24-36 h after puparium formation. At this time, gene interactions between vein- and intervein-specific genes might be sufficient to maintain their respective, mutually exclusive expression domains. Thus, Hh would be required only for the early setting of blistered expression as a result of patterning the intervein sector C. Indeed, beta-galactosidase expression directed by the boundary enhancer is not detected in the wing of newly emerged flies. This indicates that in the presumptive intervein sector C, the early setting of blistered is controlled through the boundary enhancer, whereas the later expression might recruit another cis-regulatory element. The fact that the expression of blistered is observed in the posterior compartment of pupal wings, whereas the boundary enhancer is restricted to the anterior compartment in third instar discs, further supports this idea (Nussbaumer, 2000).

The boundary enhancer is directly regulated by Vestigial (Vg) and Scalloped (Sd) which form a complex on a 120-bp DNA sub-element. The wing-specific Vg-Sd complex restricts the activation of the boundary enhancer to the future wing, consistent with the finding that Ci can only activate it in the pouch region. Hence, the boundary enhancer integrates positional cues from the Vg-Sd transcriptional complex and the Hh signal. The gene knot/collier (kn), which encodes a putative DNA-binding protein acting downstream of the Hh signaling pathway, has been found to be required for the expression of blistered in the intervein sector C. Therefore, the Hh responsiveness of the boundary enhancer may be indirect and mediated by Kn. Alternatively, activation of the enhancer may require a molecular interaction between Ci and Kn. Therefore, it will be of prior interest to determine whether Kn and Ci directly regulate the boundary enhancer and cooperate for its activation. Further analysis of how the boundary enhancer integrates input from the Vg-Sd complex and Hh signaling will contribute to a molecular understanding of the synergistic activation of enhancers by signaling input and selector genes, a strategy that may be widely used to regulate gene expression during development (Nussbaumer, 2000).

In the Drosophila wing the cut gene is activated by Notch signaling along the dorso-ventral boundary but not in other cell types. Additional regulatory components, scalloped and strawberry notch, that are targets of the Notch pathway, are expressed specifically within the wing anlagen. As suggested by physical interactions, these proteins could be co-factors of the cut trans-regulator Vestigial. Additional regulatory input comes from the Wingless pathway. These data support a model whereby context specific involvement of distinct co-regulators modulates Notch target gene activation (Nagel, 2001).

These data show that the complex regulation of ct along the D/V boundary is based on a bifurcation of the Notch signaling pathway. Most signals from the Notch pathway are mediated by Su(H), which seems to act as a repressor on its own that is converted to an activator by Nact. Since Su(H) has the capacity to bind directly to the ct wing margin enhancer, the repression of ct by Su(H) and the activation by Nact/Su(H) might be direct. However, although sufficient for the activation of ct along the D/V boundary, a number of additional factors downstream of Nact are required. These include the products of wing fate selector genes vg and sd, that seem to be, together with Sno, part of a multi-factor trans-activation complex that binds to the ct wing margin enhancer. Thereby, Sd binds directly to the ct promoter, presumably recruiting the other factors by protein-protein interactions. In agreement with this hypothesis, respective physical interactions are observed between Vg and Sd or Sno. However, all three genes are targets of the Notch signaling pathway and are activated upon the overexpression of Su(H) specifically within presumptive wing tissue. Activation of Vg is also observed also within the wing pouch, although Su(H) acts as a repressor on the vg quadrant enhancer, indicating that the isolated enhancer elements reveal only a subset of the normal pattern and might contribute differently in a wild type context (Nagel, 2001).

A combination of Sd and Su(H) binding sites is sufficient to drive expression along the D/V boundary within the wing anlagen. This synthetic enhancer is too simplified to faithfully model ct regulation. Since the overexpression of Su(H) affects the accumulation of all the important trans-activator components, Vg, Sd, Sno and Su(H) itself, ct expression would be expected. Instead, repression of ct is observed: this might be due to a lack of Nact as co-activator of Su(H). However, repression can be overcome by concurrent expression of Wg resulting in strong ct activation. It is concluded that factors downstream of the Wg signaling cascade are able to convert Su(H) from a repressor to an activator, maybe by supplying a respective co-activator or by a cooperative combinatorial activity, e.g. together with the Wg signaling mediator dTCF, in accordance with a presumptive dTCF binding site within the ct wing margin enhancer (Nagel, 2001).

These signaling events appear to be unique to the activation of ct along the D/V boundary of the wing disc. Another important role of ct is the specification of external sensory organ cells during embryogenesis and imaginal development alike. Although Notch signaling is essential for setting up the correct number of neuronal cells in the peripheral nervous system by lateral specification, it appears not to be involved in the transcriptional activation of ct within these cells. The complex mechanism of ct trans-activation from the wing margin enhancer is, therefore, not a general paradigm for ct gene regulation. Moreover, neither wg, sd nor sno are under the direct regulatory influence of the Notch pathway in various embryonic tissues suggesting that this remarkably complex control is strictly tissue specific (Nagel, 2001).

These data confirm and extend the model of context dependent activity of Notch signaling towards the regulation of ct expression along the presumptive wing margin. The regulation of ct requires the combined input of components downstream of Su(H) and Wg, including Vg, Sd and Sno. The latter three components have the potential to form a multi-protein complex which seems to be a pre-requisite for the trans-activation of the ct wing margin enhancer. Whether Su(H) is part of this specific complex or other, similar complexes has to be elucidated in the future. Although there are no indications for direct interactions between Su(H) and Sd, Vg or Sno, Su(H) has the capacity to bind to the ct wing margin enhancer and act in a combinatorial manner together with the Sd/Vg/Sno transactivation complex and components of the Wg pathway. Presumably, in many instances of Notch signaling, where Su(H) acts as a DNA-binding molecule and signal transducer, a number of additional positive or negative co-regulators confers tissue and cell specificity. Therefore, the identification of corresponding factors should help to further the understanding of the context dependent outcome of Notch signaling events (Nagel, 2001).

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).

There are several potential mechanisms whereby selector proteins and signaling effectors might operate in a combinatorial manner to regulate transcription. One mechanism is through cooperative interactions that increase the occupancy of transcription factor-binding sites on the DNA. Such a scenario appears unlikely in this case, because it would require that each selector protein be able to interact directly with many different signaling pathway transcriptional effectors. Furthermore, cooperative filling of binding sites alone is insufficient to explain selector-signal synergy, because Sd alone binds cooperatively to DNA, and yet the presence of multiple Sd-binding sites alone is insufficient to generate transcriptional activation (Guss, 2001).

A second, more likely mechanism underlying selector-signal synergy is the formation of complexes between the two classes of transcription factors and required transcriptional coactivators. Coactivators facilitate transcription by relieving repression by chromatin and/or by mediating interactions with the basal transcriptional machinery. It is suggested that gene activation by selector proteins and signaling pathways may require both of these activities, and these proteins may form complexes with coactivators on the cis-regulatory DNA. These complexes could include coactivators such as the multifunctional protein CBP, which has been shown to interact directly with three signaling pathway transcriptional effectors, Mad, Ci, and Pangolin, and also appears to interact with Sd. Alternatively, synergy between Sd and signaling pathway transcriptional effectors could be mediated by different coactivators, with independent functions. The obligate requirement for combined inputs from selector genes and signaling pathways, seen here in the wing, may be a general mechanism whereby a universally deployed set of signals can elicit field, tissue, and cell type-specific genetic responses (Guss, 2001).

A regulatory code for neuron-specific odor receptor expression

Olfactory receptor neurons (ORNs) must select (from a large repertoire) which odor receptors to express. In Drosophila, most ORNs express one of 60 Or genes, and most Or genes are expressed in a single ORN class in a process that produces a stereotyped receptor-to-neuron map. The construction of this map poses a problem of receptor gene regulation that is remarkable in its dimension and about which little is known. By using a phylogenetic approach and the genome sequences of 12 Drosophila species, regulatory elements were systematically identfied that are evolutionarily conserved and specific for individual Or genes of the maxillary palp. Genetic analysis of these elements supports a model in which each receptor gene contains a zip code, consisting of elements that act positively to promote expression in a subset of ORN classes, and elements that restrict expression to a single ORN class. A transcription factor, Scalloped, was identifed that mediates repression. Some elements are used in other chemosensory organs, and some are conserved upstream of axon-guidance genes. Surprisingly, the odor response spectra and organization of maxillary palp ORNs have been extremely well-conserved for tens of millions of years, even though the amino acid sequences of the receptors are not highly conserved. These results, taken together, define the logic by which individual ORNs in the maxillary palp select which odor receptors to express (Ray, 2008).

The spatial organization was examined of ORN classes in the maxillary palp. First, an anti-Elav antibody was used to illustrate the distribution of the entire population of ORN nuclei of the maxillary palp. Second, a multiple-label experiment was carried out to differentially mark ORNs of the three types of sensilla: ORNs of the pb1A class were labeled in green, pb2B in yellow, and pb3A in red. The three classes of ORNs show extensive spatial overlap. These results are consistent with the intermingling of sensillum types that are observed when recordings are taken from sensillar shafts. The spatial overlap of ORN nuclei indicates that the identity of an ORN and, by extension, its choice of a receptor gene, are not dictated solely by its spatial position in a field (Ray, 2008).

The upstream regions of the two Or genes coexpressed in pb2A have been compared to identify regulatory sequences shared by these two genes, but not by any other maxillary palp Or gene. To identify upstream regulatory elements for the other five maxillary palp Or genes, a different strategy was used based on phylogenetic analysis (Ray, 2008).

D. melanogaster and D. pseudoobscura diverged tens of millions of years ago and contain orthologous receptor genes. The upstream regions of orthologous Or genes were examined for conserved elements shared by the members of each orthologous pair, but not by any of the other maxillary palp Or genes. Accordingly, all conserved upstream sequences were identified greater than 6 base pairs (bp) in length for each pair of orthologs using DOT-PLOT analysis, and from these conserved elements, those were selected that were specific to each gene. The analysis was focused on the 500 bp that are upstream of the translational start site, because in a previous study, this extent of DNA was sufficient to confer faithful expression to a GAL4 reporter gene in the case of each of two maxillary palp Or genes analyzed in detail. One pair of orthologs, Or85d and its D. pseudoobscura counterpart, was exceptionally well-conserved in the 500-bp upstream region, showing 80% identity. To identify discrete conserved elements within the region upstream of Or85d, the analysis was expanded to include a more divergent species, D. virilis (Ray, 2008).

Conserved, gene-specific elements were identified for each of the five Or genes analyzed. The number of such elements varies: Or59c contains one, whereas Or42a contains six. In the special case of Or85d, two elements are shared by D. virilis and D. melanogaster upstream of Or85d, but are not found upstream of any other maxillary palp Or gene (Ray, 2008).

To identify the best candidate for a regulatory element for each of these receptor genes, a powerful bioinformatic approach was used that takes advantage of the recent sequencing of the genomes of ten other Drosophila species: D. simulans, D. sechellia, D. yakuba, D. erecta, D. ananassae, D. persimilis, D. willistoni, D. virilis, D. mojavensis, and D. grimshawi. The upstream regulatory regions of the orthologous receptor genes from all 12 species were aligned using the genome browser at the University of California Santa Cruz, and each of the elements was mapped onto the alignment. Using this approach, it was possible to identify the gene-specific element with the highest sequence conservation for each of the receptor genes; in the case of Or42a, two elements were nearly identical in their extent of conservation, and both were analyzed (Ray, 2008).

To determine whether the evolutionarily conserved, gene-specific elements have a regulatory function, they were tested in vivo using two complementary approaches, one based on a loss of function and one on a gain of function. For each gene, the element was analyzed with the highest sequence conservation. Or85d elements were not analyzed because no faithful Or85d-GAL4 driver was available (Ray, 2008).

Or46a is expressed in the pb2B neuron, and its upstream region contains two conserved, gene-specific elements. One of these elements, 46a1, is more highly conserved. It is 10 bp long, its sequence shows 93% identity across the 12 species, and its position is conserved. A 1.9-kb region of DNA upstream of Or46a drives faithful expression of a GAL4 reporter in pb2B. However, when the 46a1 element is mutated, the 1.9-kb region no longer drives expression. In most cases, no cells are labeled; in rare cases, a single ORN is labeled. The simplest interpretation of these results is that the 46a1 element is necessary for Or46a expression in pb2B (Ray, 2008).

It was then asked whether the 46a1 element can drive expression in the context of a minimal promoter. Four copies of 46a1 upstream were placed of a TATA box, and it was found that this small construct can in fact drive expression in maxillary palp cells. Many, if not all, of the cells could be identified as ORNs, because they contain dendrites and axons; their identity is considered further below. Expression from this artificial promoter could also be detected in a small subset of neurons in the main gustatory organ, the labellum (Ray, 2008).

Or71a is expressed in pb1B. Its upstream region contains multiple gene-specific elements, of which the longest and best conserved is 71a3, consisting of 16 bp and showing 97% sequence identity. This element was tested in the context of the Or71a 5' + 3' construct, which contains sequences both upstream and downstream of Or71a. This construct drives faithful expression of GAL4 when the 71a3 element is intact, but not when it is mutated. When multiple copies of 71a3 were placed upstream of a TATA box, the construct drove GAL4 expression in maxillary palp cells that can be identified as ORNs by virtue of their dendrites and axons. Low levels of expression could also be detected in a small subset of cells in the labellum (Ray, 2008).

Or59c is expressed in pb3A, and its upstream region contains a single gene-specific conserved element, 59c1, which is 11 bp long and shows 97% sequence identity across nine species; the region containing the 59c1 sequences could not be identified in three of the most distantly related species, D. virilis, D. mojavensis and D. grimshawi. Its function was tested by placing multiple copies upstream of a TATA box and it was found that this minimal promoter drives robust expression of GAL4 in the maxillary palp. Expression was not detected in the labellum (Ray, 2008).

Earlier studies have shown that the expression of a subset of the maxillary palp Or genes requires the POU domain transcription factor Acj6, which is expressed in all ORNs of the maxillary palp. Acj6 also controls axon targeting specificity of a subset of maxillary palp ORNs . The 46a1, 71a3, and 59c1 elements do not contain predicted Acj6 binding sites, and the transcription factors that act on these sequences are unknown. To test whether the factors that act on these neuron-specific elements are dependent on acj6, the expression of the minimal promoter constructs was examined in an acj66 background (Ray, 2008).

In the acj66 mutant, although the expression of the Or46a-GAL4 driver is lost, which is consistent with the loss of Or46a mRNA observed previously, the expression of the 46a1 minimal promoter construct is still strong. These results suggest that the factors that direct expression from the 46a1 motif are independent of acj6 for their expression and function. An alternative possibility is that another transcription factor can compensate for the loss of acj6 (Ray, 2008).

Expression of the Or71a-GAL4 driver can be detected in acj6, and the expression of the 71a3 minimal promoter construct can also be detected. These results suggest that the factors binding to 71a3 do not require acj6 for their expression or function (Ray, 2008).

In the case of Or59c, it was found that acj6 is required both for expression of the gene and for the minimal promoter. These results suggest that acj6 is required directly or indirectly for the expression of the 59c1 binding factor or for its function at the 59c1 site (Ray, 2008).

Or42a is expressed in pb1A, and 4.1 kb of upstream DNA drives faithful expression of GAL4 in maxillary palp ORNs. Two elements are nearly identical in their high conservation: 42a4 (98%) and 42a6 (98%), and the function of both elements was tested in vivo. 42a6 maps only three bp from 42a5. A small deletion was constructed that eliminates both 42a6 and 42a5 elements, and no effect was found on Or42a-GAL4 expression (Ray, 2008).

The longer of the two most highly conserved elements at Or42a, 42a4, contains an inverted repeat: AGTGTAAAAGTTTACACTT. Surprisingly mutation of this element led to a 2-fold increase in the number of labeled maxillary palp cells, from 18.2 ± 1.8 to 33.2 ± 3.7. The simplest interpretation of this result is that 42a4 is a negative regulatory element that represses Or42a in a subset of ORNs. To test this interpretation, a double-label experiment was carried out using probes for the endogenous Or42a mRNA and for the green fluorescent protein (GFP) that is driven by the mutant promoter via GAL4. It was found that all Or42a+ cells express GFP, but that GFP is also expressed in an additional subset of cells (Ray, 2008).

To identify the cells that ectopically express GFP, a series of additional double-label experiments was undertaken. It was found that the GFP+ cells do not express Or59c mRNA, indicating that they are not pb3A neurons, nor are they paired with cells that express Or59c mRNA, indicating that they are not pb3B neurons. In another experiment, GFP+ cells did not label with an Or33c probe, indicating that they are not pb2A neurons; however, GFP+ cells were often found paired with Or33c+ cells, indicating that many GFP+ cells are pb2B neurons. The identity of these GFP+ cells as pb2B neurons was confirmed directly in another double-label experiment using a probe for Or46a mRNA (Ray, 2008).

The simplest interpretation of these results is that positive regulatory elements in the Or42a upstream region are capable of driving expression not only in the pb1A neuron but also in the pb2B neuron. The 42a4 element represses expression in pb2B neurons, thereby restricting expression to a single ORN class, pb1A (Ray, 2008).

The ectopic expression of an Or42a promoter in Or46a+ neurons suggested a relationship between these two genes. Further evidence for a relationship came from analysis of the minimal promoter containing multiple copies of 46a1. This promoter drove GFP expression in more ORNs than could be accounted for by Or46a+ neurons alone. A double-label experiment showed that while most of the GFP+ cells are in fact Or46a+, some are Or42a+ (Ray, 2008).

The reciprocal relationship between Or42a and Or46a misexpression suggests that Or42a may contain an unidentified positive regulatory element, 42ax, that is similar in sequence to 46a1, with both sites able to bind a transcription factor present in both pb1A and pb2B. To test this interpretation, the 500 bp upstream region of Or42a was examined for an element similar, but not identical, to 46a1 (GACATTTTAA). A sequence, TATATTTTAA, was identified identical to 46a1 at the 8 underlined positions, at -455 bp. Moreover, these two sequences share an ATTTTA core, which has been shown to function as a binding site for basic helix-loop-helix transcription factors at other loci. TATATTTTAA is not found upstream of any other maxillary palp Or genes. This 42ax sequence is conserved in sequence (80% identity) and location in seven of the 12 Drosophila species. It will be interesting to identify the transcription factor that binds 46a1 and then test directly its binding to 42ax (Ray, 2008).

When DNA upstream of Or59c was fused to GAL4, expression of the reporter GFP was not faithful; the same result was obtained when upstream regions of varying lengths were used (either 2.1 kb, which extends to the next upstream gene, or 5.2 kb, which includes upstream coding sequences). Double-label experiments using an Or59c probe revealed misexpression in many Or59c cells; moreover, many Or59c+ cells did not express GFP. Some of the misexpressing cells are the neighboring pb3B neurons, which can be seen to be paired with Or59c+ pb3A cells. To identify the other ORNs that ectopically express the Or59c-GAL4 construct, double-label experiments were carried out with other Or genes. Misexpression was also observed in pb1A cells, which express Or42a, but not in the pb1B cells, nor in the pb2A or B cells. In summary, misexpression is specific to pb1A and pb3B (Ray, 2008).

Because neither of the varying lengths of upstream DNA sequences were sufficient to restrict GAL4 expression to the Or59c+ cells, 3' sequences were added to the construct. Initially, 500 bp of DNA taken directly from the region immediately downstream from the Or59c stop codon was added downstream of the GAL4 coding region. Between the downstream sequences of Or59c and the GAL4 coding region was the Hsp70 3' untranslated region (UTR), which is present in the GAL4 vector and which is often present in promoter-GAL4 analysis (Ray, 2008).

This Or59c 5' + 3' construct showed much less misexpression in Or59c cells. The total number of GFP+ cells declined from 49.7 ± 1.3 to 27.3 ± 2.1. However, some misexpression remained, and only 62% of the Or59c+ neurons were GFP+. Then the Hsp70 3' UTR sequences were removed, such that the Or59c downstream sequences were in close proximity to the 3' end of the GAL4 coding region and the Or59c 3' UTR is used. This construct drove faithful expression. Thus, there is a negative regulatory element downstream of Or59c that restricts expression of this gene to pb3A neurons, and either there is a requirement that the native 3' UTR be used, or else there is a regulatory factor that acts on this element in a context-dependent fashion in order to achieve this negative regulation. It is noted with interest that the inclusion of the downstream sequences, without the Hsp70 sequences, also drove expression in Or59c+ neurons that had previously failed to express the reporter, suggesting that the downstream sequences are required for positive as well as negative regulation of Or59c (Ray, 2008).

Inspection of the sequences downstream of Or59c that repressed misexpression revealed a binding site for the transcription factor Scalloped (Sd), AAATATTT. This site is well-conserved among a number of other species. Sd has been shown to be expressed in olfactory organs. To confirm and extend the description of sd expression an enhancer trap line, sdETX4 was used, and it was confirmed that sd is expressed in a subset of cells in the maxillary palp (Ray, 2008).

To test whether sd represses Or59c, in situ hybridizations were carried to the maxillary palp of a hypomorphic sd mutant, sd1. A 40% increase was found in the number of Or59c+ neurons. By contrast, there was no increase in the number of Or42a+ neurons. There was, however, an increase in the number of Or85d+ cells, and it is noted with interest that there is another type of Sd binding site, TAAAATTA, 737 bp downstream from the stop codon of Or85d (Ray, 2008).

The Or59c-GAL4 construct that contains only upstream sequences, Or59c 5', misexpresses in two ORN classes, the neighboring pb3B cell (Or85d+) and pb1A (Or42a+). It was asked whether sd is expressed in these two ORN classes. Using an Or59c probe, which labels the pb3A cell, it was found that sd is in fact expressed in neighboring cells, but not in pb1A cells, which express Or42a. These results suggest that Sd may repress the Or59c gene in pb3B. If so, it would be expected that in an sd mutant, cells would be observed that coexpress Or59c and Or85d. This possibility was tested by carrying out double-label in situ hybridizations in two different hypomorphic alleles of sd, sd1, and sdSG29.1. In both alleles, Or59c+ Or85d+ cells were found, but not Or59c+ Or42a+. Thus repression of Or59c in the neighboring pb3B cell requires both a Sd binding site and Sd (Ray, 2008).

Since Sd represses Or59c in pb3B, why doesn't Sd also repress Or85d in pb3B, given that both Or genes have Sd binding sites? The simplest explanation is that the two Sd binding sites are distinct. There are several potential interacting partners with which Sd may interact to form a functional transcription factor, and the pb3B cell may contain a partner necessary for repression at the Or59c binding site but not a partner necessary for repression at the Or85d binding site. If a faithful Or85d-GAL4 construct becomes available, it will be interesting to replace the Or85d-type Sd binding site with the Or59c-type Sd binding site, to determine whether the Or59c-type site confers repression in the pb3B cell (Ray, 2008).

It is noted that Or85d-GAL4 constructs containing only the 5' regions of Or85d, which lack the Sd binding site, drive misexpression in a number of non-neuronal cells of the maxillary palp. Most of the labeled cells lack dendrites and axons, and when labeled with a membrane-bound GFP, as opposed to with RNA probes that label the cell bodies, these cells appear larger than ORNs. These results suggest that Sd may interact with a binding partner in non-neuronal cells to repress Or85d expression in these cells (Ray, 2008).

Or42a is expressed in the larval olfactory system as well as in the maxillary palp. The Or42a-GAL4 construct shows expression in one ORN in each of the bilaterally symmetric larval olfactory organs, the dorsal organs. Expression was also observed in two neurons of the labellum, the taste organ on the adult head. To determine whether the conserved elements identified in analysis of maxillary palp receptor choice can act in these other chemosensory organs, Or42a-GAL4 constructs were examined in which these elements were mutated. A mutation that affects both 42a6 and 42a5, which did not affect expression in the maxillary palp, had no effect on expression in these other organs. However, mutation of 42a4, which relieved repression of Or42a in other maxillary palp ORNs, also relieved repression of Or42a-GAL4 in the larval olfactory organs and the labellum: in both cases supernumerary neurons were labeled. In the labellum, ~8-10 pairs of neurons were labeled. These results suggest that the molecular mechanisms underlying receptor gene choice in the maxillary palp overlap with those specifying receptor expression in other chemosensory organs (Ray, 2008).

This study has identified and functionally characterized a number of regulatory elements that operate in directing the formation of the receptor-to-neuron map of D. melanogaster. Because the newly defined elements analyzed in this study are conserved in sequence and position among Drosophila species, it is predicted that the programmed regulation leading to the formation of receptor-to-neuron maps would be conserved as well. To test this prediction, a physiological analysis of the D. pseudoobscura maxillary palp was carrie out . Although each of the seven Or genes expressed in the maxillary palp has an ortholog expressed in the D. pseudoobscura maxillary palp, it is expected that their odor response profiles would have diverged a great deal over the course of tens of millions of years. It was not known a priori whether it would be possible to correlate D. pseudoobscura ORNs with D. melanogaster counterparts (Ray, 2008).

It was surprising to find that the profiles of the maxillary palp ORNs are remarkably well conserved between these two species. Despite the tens of millions of years of separation, each ORN class in D. melanogaster has a counterpart in D. pseudoobscura, and their responses to a panel of ten diverse odorants are strikingly similar. Not only are the magnitudes of the responses well conserved, but the modes of the responses, i.e., excitation versus inhibition, are conserved. For example, both the pb2B ORN of D. melanogaster and its D. pseudoobscura counterpart are excited by 4-methyl phenol and inhibited by 3-octanol. The orthologous receptors show amino acid identity as low as 59% in the case of Or71a, and in no case exceeded 84%, the identity determined for Or42a. Thus pb1B in D. melanogaster, which expresses Or71a, shows the same specificity for 4-methyl phenol and 4-propyl phenol as the corresponding ORN in D. pseudoobscura, although Or71a is only 59% identical between the two species (Ray, 2008).

The conservation of odor response spectra allows determination of whether the stereotyped pairing of ORNs is also conserved in the two species. The results suggest that not only are the response spectra of the odor receptors conserved with respect to a diverse panel of odorants, but that the program of receptor gene expression is also conserved between these distantly related species (Ray, 2008).

Given the success in identifying gene-specific elements required for the expression of individual Or genes in individual classes of ORNs, it was asked whether the same approach could be used to identify sensillum-specific elements required uniquely by the Or genes that are expressed in the neighboring ORNs of a common sensillum. Sensillum-specific elements were sought conserved in the upstream regions of D. melanogaster and D. pseudoobscura Or genes. Only one element, AAATCAATTA, was found upstream of all orthologs expressed in a particular sensillum type. Mutational analysis of this element in the Or42a promoter did not, however, appear to affect expression. Furthermore, expression was not affected by mutation of the more proximal of the two copies of this element in the Or71a upstream region. These results suggest that this element is not required for expression in the pb1 sensillum (Ray, 2008).

This study has concentrated on receptor gene choice in the maxillary palp, on account of its numerical simplicity. Does a system of molecular zip codes also underlie the process of receptor gene choice across the entire odor receptor repertoire? In addition to the seven maxillary palp receptors, the Or gene family contains 53 other members expressed in the antenna or the larval olfactory system. Using a comparative bioinformatic approach, a large-scale analysis was performed of sequence conservation in the 500 bp upstream of each of 42 Or genes across all 12 Drosophila species. Great diversity was found in the number, lengths, and distribution of highly conserved upstream regions. Within the most highly conserved of these regions a variety of elements were identified that are shared among subsets of Or genes. This analysis, then, reveals a combinatorial structure to the organization of shared elements upstream of these receptor genes. This pattern supports a model in which a combinatorial code of positive and negative regulatory elements dictates the proper expression of each Or gene (Ray, 2008).

What kind of proteins accomplish this regulation? In C. elegans, several kinds of transcription factors have been elegantly shown to play roles in specifying ORN identity and receptor expression. In the mouse, a LIM-homeodomain protein, Lhx2, is required for normal ORN differentiation and expression of OR genes. In Drosophila the POU domain protein Acj6 is required for the expression of a subset of Or genes. This study has shown that Sd, a TEA domain-containing transcription factor, is critical in restricting the expression of some Or genes to their proper ORNs. Sd has been shown to act as a repressor in other systems and in fact is required for normal taste behavior in both larvae and adults. Another aspect of receptor gene choice depends on proteins of the Notch pathway: receptor choice in neighboring ORNs of a sensillum appears to be coordinated via asymmetric segregation of regulatory factors from a common progenitor (Ray, 2008).

Some elements that are essential to odor receptor gene choice are also located upstream of genes required for axon guidance and sorting. The presence and positions of these elements have been conserved for tens of millions of years of evolution. The presence of Or regulatory elements upstream of ORN axon-guidance genes could reflect a relationship between receptor gene choice and axon targeting. In addition to selecting particular Or genes for expression, ORNs send axons to particular glomeruli in the antennal lobe of the brain. ORNs that express the same Or gene send axons to the same glomerulus. Thus the olfactory system contains both a stereotyped receptor-to-neuron map and a stereotyped connectivity map in the antennal lobes. The tight coordination between receptor gene choice and axonal projection could in principle arise in part from overlap in the mechanisms underlying these processes. In mammals, odor receptors play a role in ORN targeting. In Drosophila, ORN targeting does not require the receptors, but could require the regulatory apparatus used to express the receptors. Acj6 provides an example of a link between the two processes: it acts both in receptor expression and ORN axon targeting. Moreover, it was found that Acj6 is required for the activity of one of the regulatory elements identified in this study (Ray, 2008).

This study found a remarkable similarity of function between the maxillary palp ORNs of two species that diverged more than tens of millions of years ago. It had been expected that over this time interval, the odor specificities of the ORNs would have diverged markedly to serve differing needs of the two evolving species. Instead, every ORN class showed strikingly similar responses, with few exceptions. The results show that two odor receptors can differ a great deal in amino acid sequence and still exhibit a very similar odor specificity (Ray, 2008).

The organization of the organ in the two species is also identical, in that corresponding ORNs are combined according to the same pairing rules. This high degree of conservation suggests a critical role for the maxillary palp in odor coding and in the generation of olfactory-driven behavior. The conservation of regulatory elements and organization also suggests that the two species use common mechanisms to specify the receptor-to-neuron map (Ray, 2008).

The regulatory challenge confronted by the Drosophila olfactory system represents an extreme among problems of gene regulation. It requires the storage and deployment of a great deal of information. These data support a model in which Or gene expression is controlled by a system of molecular zip codes. Each Or gene contains elements that dictate expression in the proper olfactory organ, positive regulatory elements that specify expression in a subset of ORN classes, and negative regulatory elements that restrict expression to a single ORN class. This logic and the components that execute it have solved such a challenging problem with such efficiency that they have apparently been well conserved for tens of millions of years (Ray, 2008).

Cooperation between dE2F1 and Yki/Sd defines a distinct transcriptional program necessary to bypass cell cycle exit

The Hippo signaling pathway regulates organ size homeostasis, while its inactivation leads to severe hyperplasia in flies and mammals. The transcriptional coactivator Yorkie (Yki) mediates transcriptional output of the Hippo signaling. Yki lacks a DNA-binding domain and is recruited to its target promoters as a complex with DNA-binding proteins such as Scalloped (Sd). In spite of recent progress, an open question in the field is the mechanism through which the Yki/Sd transcriptional signature is defined. This study reports that Yki/Sd synergizes with and requires the transcription factor dE2F1 to induce a specific transcriptional program necessary to bypass the cell cycle exit. Yki/Sd and dE2F1 bind directly to the promoters of the Yki/Sd-dE2F1 shared target genes and activate their expression in a strong cooperative manner. Consistently, RBF, a negative regulator of dE2F1, negates this synergy and limits the overall level of expression of the Yki/Sd-dE2F1 target genes. Significantly, dE2F1 is needed for Yki/Sd-dependent full activation of these target genes, and a e2f1 mutation strongly blocks yki-induced proliferation in vivo. Thus, the Yki transcriptional program is determined through functional interactions with other transcription factors directly at target promoters. It is suggested that such functional interactions would influence Yki activity and help diversify the transcriptional output of the Hippo pathway (Nicolay, 2011).

While recent work has provided insight into how the regulation of Yki occurs via the location within the cell through protein-protein interactions, less is known about how Yki-mediated transcription is regulated. The results presented in this study suggest that Yki may rely on a combinatorial network of transcription factors to modulate transcriptional output in response to Hippo pathway signaling. One such transcription factor is dE2F1, which is required for the full activation of specific target genes by Yki/Sd (Nicolay, 2011).

These studies were prompted by the strong enhancement of the wts mutant phenotype by an rbf mutation. Both the pRB and Hippo pathways are negative regulators of cell proliferation. In flies, RBF functions to limit the activity of the transcriptional activator dE2F1, while the Wts kinase inhibits the transcriptional coactivator Yki. Therefore, one possibility is that, in rbf wts double mutants, dE2F1 and Yki are left unchecked to independently induce genes that promote cell proliferation. However, the data do not support such a trivial explanation. Microarray profiling followed by gene ontology analysis demonstrated that the rbf wts double mutant gene expression signature was distinct from that of either rbf or wts single mutants. Importantly, the rbf wts double mutant signature contained a significant number of up-regulated genes involved in cell cycle progression and cell proliferation that were not present in the rbf or wts single mutant signatures. Thus, an alternative explanation, one that is favored, is that, in rbf wts double mutants, hyperactivated dE2F1 and Yki synergistically up-regulate a novel set of genes and establish the distinct gene expression signature needed to overcome terminal cell cycle exit upon differentiation. Importantly, the synergy results from a direct binding and cooperation between the two factors on the target promoters, since both can be detected by ChIP on dE2F1-Yki/Sd coregulated genes. Consistently, inhibition of dE2F1 by RBF, which is also present on the same set of promoters, is sufficient to limit this synergistic activation by dE2F1 and Yki/Sd (Nicolay, 2011).

Previous studies demonstrated that, in the absence of de2f1, Yki fails to drive inappropriate proliferation, indicating that Yki alone is not sufficient to induce the transcriptional program to prevent cell cycle exit. Importantly, Yki is still active and capable of inducing other Yki-dependent target genes, such as dIAP1. Thus, it appears that the interplay between Yki/Sd and dE2F1 is highly specific to the activation of a distinct set of target genes and is not simply a reflection of a Yki transcription program gone awry. It is suggested that Yki requires an assist from dE2F1 to up-regulate some, if not all, of the dE2F1-Yki/Sd target genes. This assist is critical, since, in the absence of dE2F1, Yki is unable to fully activate these genes to a level sufficient to bypass the cell cycle exit and undergo inappropriate proliferation. Such an interpretation is supported by the transcriptional reporter assays demonstrating that the activation potential of Yki/Sd is reduced in dE2F1-depleted cells. It is noteed that the dE2F1-Yki/Sd target genes are regulated primarily through activation. It remains unclear why RBF/dE2F2 complexes are bound at promoters that are regulated by dE2F1, yet these genes remain insensitive to RBF/dE2F2-mediated repression. Interestingly, two of the dE2F1-Yki/Sd target genes, dDP and cdc2c, were isolated in a genome-wide RNAi screen for factors that are required for Yki to activate a synthetic reporter (Ribeiro, 2010). Given that de2f1 is a transcriptional target of Yki activity as well, it is tempting to speculate that a positively reinforcing signaling loop occurs between Yki/Sd and dE2F1 (Nicolay, 2011).

Yki is a potent oncogene and can elicit a dramatic effect on cell proliferation and apoptosis. Therefore Yki is tightly regulated at multiple levels, including its transcriptional activity, nuclear localization, and degradation. Additionally, it appears that Yki target gene specificity is determined by the transcription factors that interact with Yki and tether it to DNA. For example, Yki partners with Sd and Hth transcription factors. Notably, Hth/Yki transcriptional complexes appear to be important for promoting cell proliferation and survival within the anterior compartment of the eye disc, while in the posterior of the eye disc, Yki switches to partner with Sd to regulate a different set of target genes. The ability of Yki to partner with different DNA-binding proteins in different contexts is thought to provide a basis for altering the transcriptional output of the Hippo pathway. The current results exemplify how, under oncogenic conditions, another transcription factor, such as dE2F1, helps to set up a specific Yki/Sd gene expression signature that is needed to overcome the cell cycle exit. Thus, one conclusion drawn from these results is that the Yki transcriptional program is determined not only by DNA binding proteins that recruit Yki to its target genes, but additionally through interactions with other transcription factors directly at specific target genes. Such functional interactions would influence Yki activity and essentially help to further shape the transcriptional output of the Hippo pathway (Nicolay, 2011).

Another implication of the results is that not only does dE2F1 help to engage a Yki/Sd transcriptional program, but, conversely, a hyperactive Yki/Sd complex contributes to the deregulation of E2F transcription in rbf wts double mutant cells. Given that E2F-dependent transcription is often deregulated in tumor cells, this is an important point. Thus, depending on the identity of other cooperating mutations in pRB-deficient tumor cells, E2F can potentially synergize with a distinct repertoire of transcription factors to engage in transcriptional programs unique to tumor cells of different origins (Nicolay, 2011).

Although initially Yki-induced ectopic proliferation was characterized by an up-regulation in the expression of cyclin E, cyclin A, and cyclin B in flies, this mechanism does not appear to be conserved. In mammals, the up-regulation of cyclin D1 by YAP (the Yki mammalian homolog) is thought to be more critical in promoting inappropriate cell divisions. Thus, it is possible that, in mammals, YAP relies on a different network of transcription factors to promote cell cycle progression than Yki does in flies. Indeed, although YAP has been shown to partner with the Sd homologs TEAD1-4 in mammals, it is also known to interact with other transcription partners (SMAD1 and p73) under specific contexts. Thus, it appears that, similar to Yki, YAP may rely on a distinct repertoire of transcription factors to relay the response to various cellular stimuli (Nicolay, 2011).

Intriguingly, it has been demonstrated that the pRB and Hippo pathways are functionally integrated in human cells. However, the precise mechanism of interaction has seemingly evolved, as it has been shown that inactivation of the Wts homolog LATS2 interferes with the formation of the p130/DREAM repressor complex at E2F target promoters. The inability to repress E2F targets in the absence of LATS2 prevents pRB-induced senescence in human cells . In contrast, the Drosophila dREAM complex appears to be functional in wts mutants (data not shown), and instead the cross-talk between the two pathways occurs at the level of cooperation between Yki and dE2F1. Nonetheless, although the mechanistic paths taken may have diverged between flies and humans, the end point is the same: limit E2F transcriptional activity to prevent inappropriate proliferation (Nicolay, 2011).

To date, the most well-defined oncogenic role for YAP, in the context of Hippo pathway signaling, is in the formation of hepatocellular carcinoma (HCC). However, YAP is also capable of transforming immortalized human mammary epithelial cells, which appears to be through an interaction with the EGFR signaling pathway. In the future, it will be interesting to determine how many other signaling networks oncogenic YAP activity is dependent on, and with what degree these interactions are tissue- or cell type-specific. Finally, these findings support a conserved function of the pRB and Hippo pathways and suggest that a complex coordination of gene expression by these two pathways may underlie a key mechanism during oncogenic proliferation (Nicolay, 2011).

Divergent transcriptional regulatory logic at the intersection of tissue growth and developmental patterning

The Yorkie/Yap transcriptional coactivator is a well-known regulator of cellular proliferation in both invertebrates and mammals. As a coactivator, Yorkie (Yki) lacks a DNA binding domain and must partner with sequence-specific DNA binding proteins in the nucleus to regulate gene expression; in Drosophila, the developmental regulators Scalloped (Sd) and Homothorax (Hth) are two such partners. To determine the range of target genes regulated by these three transcription factors, genome-wide chromatin immunoprecipitation experiments for each factor was performed in both the wing and eye-antenna imaginal discs. Strong, tissue-specific binding patterns are observed for Sd and Hth, while Yki binding is remarkably similar across both tissues. Binding events common to the eye and wing are also present for Sd and Hth; these are associated with genes regulating cell proliferation and 'housekeeping' functions. In contrast, tissue-specific binding events for Sd and Hth significantly overlap enhancers that are active in the given tissue, are enriched in Sd and Hth DNA binding sites, respectively, and are associated with genes that are consistent with each factor's tissue-specific functions. Overall these results suggest that both Sd and Hth use distinct strategies to regulate distinct gene sets during development: one strategy is shared between tissues and associated with Yki, while the other is tissue-specific, generally Yki-independent and associated with developmental patterning (Slattery, 2013).

The control of gene expression in multicellular eukaryotes depends on a limited set of transcription factors that are reused in different contexts and combinations to execute a diverse array of cellular functions. To gain insight into this process this study used tissue-specific, genome-wide ChIP to explore the global DNA targeting properties of three transcriptional regulators – Yki, Sd, and Hth. Yki is a transcriptional coactivator that regulates tissue growth in all tissues, and it does so in part through interactions with the DNA binding TFs Sd and Hth. However, in addition to their Yki-dependent roles in promoting tissue growth, Sd and Hth also have highly tissue-specific developmental roles. Thus, this group of regulators provides an ideal starting point for addressing the logic by which TFs execute both tissue-specific and -nonspecific gene regulatory functions in vivo. The implications of the differences this study uncovered between these modes of binding for Hth and Sd are discussed, as well as the unexpectedly large number of shared binding sites for Yki (Slattery, 2013)

Drosophila Yki was initially identified as an essential transcriptional coactivator in the Hippo tumor suppressor pathway. Loss of function clones of yki grow very poorly, while gain of function Yki clones result in tissue overgrowths that are similar to those generated when the upstream kinases (Hippo and Warts) are compromised. These observations suggested that Yki, with the help of DNA binding proteins, would target genes required for cell proliferation and survival, including the known Hippo pathway targets cycE and diap1. Consistent with this expectation, this study observed Yki binding to these and other genes that are regulated by the Hippo pathway. Unexpectedly, however, in addition to known Hippo pathway genes Yki binding to several thousands of genes was observed in both the eye-antenna and wing imaginal discs, implying that Yki targets many more genes than those regulated by the Hippo pathway, or that the Hippo pathway targets many more genes than previously thought. Consistent with the latter possibility, over 1000 of the genes identified as tissue-shared Yki targets in this study are upregulated >2-fold in wts- wing discs relative to wild-type based on recently published RNA-seq data. In addition, Yki was recently shown to bind and activate several genes required for mitochondrial fusion. Moreover, the mammalian homologs of Yki, Yes-associated protein (YAP) and TAZ (transcriptional coactivator with PDZ-binding motif) are thought to regulate many genes in a wide variety of contexts, including human embryonic stem cells and several adult human tissues. Taken together, these results suggest that Yki may be a widely used transcriptional coactivator in Drosophila and vertebrates. The severe cell proliferation defects associated with yki mutant clones may have obscured its other functions in other pathways. These results are consistent with the idea that Yki and its vertebrate orthologs interact with a wide variety of transcription factor. Together, the data imply that DNA binding proteins in addition to Sd and Hth may recruit Yki to a large number of broadly active CRMs (Slattery, 2013)

The view that Yki is recruited to DNA by factors other than Sd was recently questioned by experiments suggesting that, in the eye imaginal disc, sd yki double mutant clones proliferate better than yki single mutant clones. These observations were interpreted to suggest that Sd is a default repressor of proliferation and survival-promoting genes. However, this conclusion is complicated by the observation that both Sd and Yki are also important for specifying non-retinal (peripodial epithelium) fates in the eye imaginal disc: thus, the partially rescued growth of sd yki clones could in part be due to a fate transformation. Further, this study found that the activity of the ban-eye enhancer is not affected in sd clones, but is lost in hth clones, arguing that at least for this direct Hippo pathway target Hth, not Sd, is the primary activator. It is noteworthy that although their activities can be separated, the ban wing and eye enhancers identified in this study are adjacent to each other in the native ban locus. It is plausible that Sd+Yki input provides a basal level of activity in both tissues and that Hth and Sd boost this level in the eye and wing, respectively. Regardless, the improved growth of sd yki clones does not argue against the idea that Yki is recruited to survival genes by Hth in wild type eye discs. Taken together with genome-wide binding and ban enhancer studies, it is suggested that the absence of Sd results in both a fate change and some derepression of survival genes, but that wild type proliferation and gene regulation in the eye disc requires the recruitment of Yki to the DNA by Hth (Slattery, 2013)

In contrast to the widespread and largely tissue-nonspecific binding observe for Yki, Sd and Hth exhibit both tissue-specific and tissue-shared binding events. Multiple characteristics distinguish these types of binding. First, tissue-shared binding by both Sd and Hth is frequently associated with Yki binding and often close to cell cycle and housekeeping genes, while tissue-specific binding is not. These observations are consistent with previous studies showing that Yki controls cell survival and proliferation in all imaginal discs, an activity that is regulated by the Hippo pathway. Second, compared to tissue-shared binding, DNA sequences bound by Sd and Hth in a tissue-specific manner are more conserved, more likely to contain the TF's consensus binding site, less likely to be promoter proximal, and more likely to be associated with key developmental regulatory loci. Third, tissue-specific Sd and Hth binding events are more likely to overlap with enhancers active in the corresponding tissue. To illustrate this point, the newly identified tissue-specific TF-CRM interactions at wg match the known roles for Sd and Hth. Taken together, these results suggest that regulation at the level of TF-DNA binding is a significant mechanism by which Sd and Hth regulate tissue-specific gene expression. Tissue-specific binding could be regulated through direct or indirect interactions with additional transcription factors, through tissue-specific differences in DNA accessibility, or through a combination of these factors (Slattery, 2013)

This study also found that distinct chromatin types are differentially correlated with tissue-specific and -nonspecific binding, even though these chromatin categories were defined in Kc cells. All tissue-shared binding events have a strong tendency to occur in actively transcribed chromatin states. Tissue-specific (W>EA) Sd and Hth binding is also enriched in RED chromatin (see Filion, 2010) but is uniquely enriched in BLUE chromatin. BLUE chromatin is associated with Polycomb-mediated repression. The W>EA Sd and Hth binding in Polycomb-associated chromatin indicate that these factors target tissue-specific enhancers that are also regulated by PcG proteins during development (Slattery, 2013)

Despite the importance of tissue-specific binding as a regulatory mechanism for Sd and Hth activity, both factors also displayed a significant amount of tissue-shared binding. These tissue-shared binding events can be broken down into distinct groups based on the local chromatin environment. The majority of tissue-shared binding occurs in YELLOW chromatin and is associated with ubiquitously expressed housekeeping genes. However, binding that occurs in BLUE chromatin, and to a lesser extent in RED chromatin, is more conserved and more likely to be associated with a TF's motif, both characteristics of tissue-specific binding. In the case of the bantam eye and wing enhancers, Sd and Hth binding in BLUE chromatin is direct and apparently able to drive tissue-specific, rather than ubiquitous, expression patterns. Other examples of enhancers in RED or BLUE chromatin that drive patterned expression and have tissue shared binding are shown in this study. These observations suggest that gene regulation by Sd and Hth may also be controlled at a step beyond DNA binding, perhaps via interactions with additional transcription factors at a given enhancer. Alternatively, some of the binding events called as tissue-shared may turn out to be specific binding events in distinct cell types within each imaginal disc (e.g. hinge, notum, and pouch in the wing disc and antenna, eye progenitor domain, and photoreceptors in the eye-antenna disc). Regardless, the hundreds of Sd- and Hth-CRM interactions identified in this study provide a tremendous resource for further dissecting the mechanisms by which Sd and Hth regulate patterned gene expression (Slattery, 2013)

Notably, few of the above conclusions would have been clear had genome-wide binding been measured in only one of the two tissues. Tissue-specific binding is not the most highly enriched (that is, the signal is generally weaker compared to tissue-shared events) and might have been overlooked had just one tissue been characterized, where the strongest peaks are generally the focal point. The tissue-specific binding events detected in this study may also occur in subsets of cells in the wing or eye-antennal discs, which are also heterogeneous in cell type. This would explain why tissue-specific binding signals may be weaker, because the ChIP data represent an average of all cell types in a single imaginal disc type. If correct, it would be an error to focus on only the strongest peaks when analyzing in vivo TF binding, particularly in heterogeneous tissues. It is possible that ChIP signal is more biologically meaningful in highly homogenous tissues like the blastoderm Drosophila embryo, or in cell culture. Still, distinct TF-DNA binding mechanisms (long residence time versus rapid binding turnover) with different functional outcomes can lead to indistinguishable, strong ChIP peaks, making it difficult to interpret ChIP data on strength of signal alone. Despite their lower intensity, many biologically relevant binding events, such as those identified in this study, may only stand out when looking at the influence of tissue context on binding (Slattery, 2013).

A common set of DNA regulatory elements shapes Drosophila appendages

Animals have body parts made of similar cell types located at different axial positions, such as limbs. The identity and distinct morphology of each structure is often specified by the activity of different 'master regulator' transcription factors. Although similarities in gene expression have been observed between body parts made of similar cell types, how regulatory information in the genome is differentially utilized to create morphologically diverse structures in development is not known. This study used genome-wide open chromatin profiling to show that among the Drosophila appendages, the same DNA regulatory modules are accessible throughout the genome at a given stage of development, except at the loci encoding the master regulators themselves. In addition, open chromatin profiles change over developmental time, and these changes are coordinated between different appendages. It is proposed that master regulators create morphologically distinct structures by differentially influencing the function of the same set of DNA regulatory modules (McKay, 2013).

This paper addresses a long-standing question in developmental biology: how does a single genome give rise to a diversity of structures? The results indicate that the combination of transcription factors expressed in each thoracic appendage acts upon a shared set of enhancers to create different morphological outputs, rather than operating on a set of enhancers that is specific to each tissue. This conclusion is based upon the surprising observation that the open chromatin profiles of the developing appendages are nearly identical at a given developmental stage. Therefore, rather than each master regulator operating on a set of enhancers that is specific to each tissue, the master regulators instead have access to the same set of enhancers in different tissues, which they differentially regulate. It was also found that tissues composed of similar combinations of cell types have very similar open chromatin profiles, suggesting that a limited number of distinct open chromatin profiles may exist at a given stage of development, dependent on cell-type identity (McKay, 2013).

Different tissues were dissected from developing flies to compare their open chromatin profiles. These tissues are composed of different cell types, each with its own gene expression profile. Formaldehyde-assisted isolation of regulatory elements (FAIRE) data thus represent the average signal across all cells present in a sample. However, data from embryos and imaginal discs indicate that FAIRE is a very sensitive detector of functional DNA regulatory elements. For example, the Dll01 enhancer is active in 2–4 neurons of the leg imaginal disc; yet, the FAIRE signal at Dll01 is as strong as the Dll04 enhancer, which is active in hundreds of cells of the wing pouch. Thus, FAIRE may detect nearly all of the DNA regulatory elements that are in use among the cells of an imaginal disc. This study does not rule out the existence of DNA regulatory elements that are not marked by open chromatin or are otherwise not detected by FAIRE (McKay, 2013).

Despite this sensitivity, the approach of this study does not identify which cells within the tissue have a particular open chromatin profile. For a given locus, it is possible that all cells in the tissue share a single open chromatin profile or that the FAIRE signal originates from only a subset of cells in which a given enhancer is active. Comparisons between eye-antennal discs, larval CNS, and thoracic discs suggest that the latter scenario is most likely, with open chromatin profiles among cells within a tissue shared by cells with similar identities at a given developmental stage (McKay, 2013).

The observation that halteres and wings share open chromatin profiles demonstrates that Hox proteins like Ubx can differentially interpret the DNA sequence within the same subset of enhancers to modify one structure into another. This is consistent with the idea that morphological differences are largely dependent on the precise location, duration, and magnitude of expression of similar genes, and it is further supported by the similarity in gene expression profiles observed between Drosophila appendages and observed between vertebrate limbs. However, that such dramatic differences in morphology could be achieved by using the same subset of DNA regulatory modules in different tissues genome-wide was not known. The current findings provide a molecular framework to support the hypothesis that Hox factors function as 'versatile generalists,' rather than stable binary switches. The similarity in open chromatin profiles between wings and legs suggests that this framework also extends to other classes of master regulators beyond the Hox genes. It is also noted that, like the Drosophila appendages, vertebrate limbs are composed of similar combinations of cell types that differ in their pattern of organization. Moreover, the Drosophila appendage master regulators share a common evolutionary origin with the master regulators of vertebrate limb development, suggesting that the concept of shared open chromatin profiles may also apply to human development (McKay, 2013).

The data suggest that open chromatin profiles vary both over time for a given lineage and between cell types at a given stage of development. Given the dramatic differences in the FAIRE landscape observed during embryogenesis and between the CNS and the appendage imaginal discs during larval stages, it appears as though the alteration of the chromatin landscape is especially important for specifying different cell types from a single genome. After cell-type specification, open chromatin profiles in the appendages continued to change as they proceeded toward terminal differentiation, suggesting that stage-specific functions require significant opening of new sites or the closing of existing sites. These findings contrast with those investigating hormone-induced changes in chromatin accessibility, in which the majority of open chromatin sites did not change after hormone treatment, including sites of de novo hormone-receptor binding. Thus, it may be that genome-wide remodeling of chromatin accessibility is reserved for the longer timescales and eventual permanence of developmental processes rather than the shorter timescales and transience of environmental responses (McKay, 2013).

Different combinations of 'master regulator' transcription factors, often termed selector genes, are expressed in the developing appendages. Selectors are thought to specify the identity of distinct regions of developing animals by regulating the expression of transcription factors, signaling pathway components, and other genes that act as effectors of identity. One property attributed to selectors to explain their unique power to specify identity during development is the ability to act as pioneer transcription factors. In such models, selectors are the first factors to bind target genes; once bound, selectors then create a permissive chromatin environment for other transcription factors to bind. The finding that the same set of enhancers are accessible for use in all three appendages, with the exception of the enhancers that control expression of the selector genes themselves and other primary determinants of appendage identity, suggests that the selectors expressed in each appendage do not absolutely control the chromatin accessibility profile; otherwise, the haltere chromatin profile (for example) would differ from that of the wing because of the expression of Ubx (McKay, 2013).

What then determines the appendage open chromatin profiles? Because open chromatin is likely a consequence of transcription factor binding, two nonexclusive models are possible. First, different combinations of transcription factors could specify the same open chromatin profiles. In this scenario, each appendage's selectors would bind to the same enhancers across the genome. For example, the wing selector Vg, with its DNA binding partner Sd, would bind the same enhancers in the wing as Dll and Sp1 bind in the leg. In the second model, transcription factors other than the selectors could specify the appendage open chromatin profiles. Selector genes are a small fraction of the total number of transcription factors expressed in the appendages. Many of the non-selector transcription factors are expressed at similar levels in each appendage, and thermodynamic models would predict them to bind the same enhancers. This model could also help to explain how the appendage open chromatin profiles coordinately change over developmental time despite the steady expression of the appendage selector genes during this same period. It is possible that stage-specific transcription factors determine which enhancers are accessible at a given stage of development. This would help to explain the temporal specificity of target genes observed for selectors such as Ubx. Recent work supports the role of hormone-dependent transcription factors in specifying the temporal identity of target genes in the developing appendages (Mou, 2012). Further experiments, including ChIP of the selectors from each of the appendages, will be required to determine the extent to which either of these models is correct (McKay, 2013).

Binding of Ubx results in differential activity of enhancers in the haltere imaginal disc relative to the wing, despite equivalent accessibility of the enhancers in both discs, indicating that master regulators control morphogenesis by differentially regulating the activity of the same set of enhancers. It is likely that functional specificity of enhancers is achieved through multiple mechanisms. These include differential recruitment of coactivators and corepressors, modulation of binding specificity through interactions with cofactors, differential utilization of binding sites within a single enhancer, or regulation of binding dynamics through an altered chromatin context. This last mechanism would allow for epigenetic modifications early in development to affect subsequent gene regulatory events. For example, the activity of Ubx enhancers in the early embryo may control recruitment of Trithorax or Polycomb complexes to the PREs within the Ubx locus, which then maintain Ubx in the ON or OFF state at subsequent stages of development. Consistent with this model, Ubx enhancers active in the early embryo are only accessible in the 2-4 hr time point, whereas the accessibility of Ubx PREs varies little across developmental time or between tissues at a given developmental stage (McKay, 2013).

The current results also have implications for the evolution of morphological diversity. Halteres and wings are considered to have a common evolutionary origin, but the relationship between insect wings and legs is unresolved. The observation that wings and legs share open chromatin profiles supports the hypothesis that wings and legs also share a common evolutionary origin in flies. Because legs appear in the fossil record before wings, the similarity in their open chromatin profiles suggests that the existing leg cis-regulatory network was co-opted for use in creation of dorsal appendages during insect evolution (McKay, 2013).

The Hippo pathway regulates hematopoiesis in Drosophila melanogaster

The Salvador-Warts-Hippo (Hippo) pathway is an evolutionarily conserved regulator of organ growth and cell fate. It performs these functions in epithelial and neural tissues of both insects and mammals, as well as in mammalian organs such as the liver and heart. Despite rapid advances in Hippo pathway research, a definitive role for this pathway in hematopoiesis has remained enigmatic. The hematopoietic compartments of Drosophila melanogaster and mammals possess several conserved features. D. melanogaster possess three types of hematopoietic cells that most closely resemble mammalian myeloid cells: plasmatocytes (macrophage-like cells), crystal cells (involved in wound healing), and lamellocytes (which encapsulate parasites). The proteins that control differentiation of these cells also control important blood lineage decisions in mammals. This study defines the Hippo pathway as a key mediator of hematopoiesis by showing that it controls differentiation and proliferation of the two major types of D. melanogaster blood cells, plasmatocytes and crystal cells. In animals lacking the downstream Hippo pathway kinase Warts, lymph gland cells overproliferated, differentiated prematurely, and often adopted a mixed lineage fate. The Hippo pathway regulated crystal cell numbers by both cell-autonomous and non-cell-autonomous mechanisms. Yorkie and its partner transcription factor Scalloped were found to regulate transcription of the Runx family transcription factor Lozenge, which is a key regulator of crystal cell fate. Further, Yorkie or Scalloped hyperactivation induced ectopic crystal cells in a non-cell-autonomous and Notch-pathway-dependent fashion (Milton, 2014).

Impaired Hippo signaling promotes Rho1-JNK-dependent growth

The Hippo and c-Jun N-terminal kinase (JNK) pathway both regulate growth and contribute to tumorigenesis when dysregulated. Whereas the Hippo pathway acts via the transcription coactivator Yki/YAP to regulate target gene expression, JNK signaling, triggered by various modulators including Rho GTPases, activates the transcription factors Jun and Fos. This study shows that impaired Hippo signaling induces JNK activation through Rho1. Blocking Rho1-JNK signaling suppressed Yki-induced overgrowth in the wing disk, whereas ectopic Rho1 expression promoted tissue growth when apoptosis was prohibited. Furthermore, Yki directly regulates Rho1 transcription via the transcription factor Sd. These results identify a novel molecular link between the Hippo and JNK pathways and implicate the essential role of the JNK pathway in Hippo signaling-related tumorigenesis (Ma, 2005).

Recent studies have revealed a complex interaction network between Hippo and other key signaling pathways, including TGF- β /SMAD and Wnt/β-catenin pathways, whereas its communication with JNK signaling remains elusive. This study provides genetic evidences that impaired Hippo signaling promotes overgrowth through Rho1-JNK signaling in Drosophila. First, loss of Hippo signaling induces JNK activation and its target gene expression. Second, Yki-induced overgrowth is suppressed by blocking Rho1-JNK signaling. Third, ectopic Rho1 expression phenocopies Yki-triggered overgrowth and proliferation when cell death is compromised (Ma, 2005).

Yki/YAP's ability in promoting tissue growth depends on transcription factors, including Sd/TEADs and SMADs. Consistent with this notion, this study found Sd, but not Mad, is essential for Yki-induced JNK activation, whereas ectopic Sd expression is sufficient to activate JNK signaling by itself. The Rho1 GTPase was further implicated as the critical factor that bridges the interaction between Hippo and JNK signaling. Rho1 not only mediates Yki-induced JNK activation and overgrowth, but also serves as a direct transcriptional target of Yki/Sd complex. Intriguingly, Rho1 activation was also found to promote nuclear translocation of Yki in wing discs, and reducing Yki activity significantly impeded Rho1 induced growth, implying the existence of a potential positive feedback loop to amplify Yki-induced overgrowth and to help maintain signaling in a steady state. Consistent with thi observation, recent studies reported that GPCRs could activate YAP/TAZ through RhoA in mammals, whereas elevated JNK signaling in Drosophila could stimulate Yki nuclear translocation during regeneration and tissue growth. Thus, these results provide the other side of the story about a novel cross-talk between Hippo and JNK signaling (Ma, 2005).

Intriguingly, it was found that ectopic Yki expression driven by ptc-Gal4 induced MMP1 activation, puc-LacZ expression, rho1 transcription, and Yki target gene transcription predominantly in the proximal region of wing disk, but not that of the dorsal/ventral boundary. This is consistent with a recently published paper showing that tension in the center region of Drosophila wing disk is lower than that in the periphery, which correlates with lower Yki activity. It is also worth noting that despite the requirement of JNK signaling in Yki-induced wing overgrowth, JNK was not activated strictly in an autonomous manner upon Yki overexpression. This could be caused by supercompetitive activity of Yki expression clones, or, alternatively, through a propagation of JNK signal into neighboring cells, which would be very interesting to study further (Ma, 2005).

Apart from its role in growth control, the Hippo pathway also regulates tumor invasion and metastasis. Similarly, JNK signaling plays a major role in modulating metastasis in both flies and mammals. Rho1 was also reported to cooperate with oncogenic Ras to induce large invasive tumors. Hence, it is likely that Rho1 also acts as the molecular link between Yki and JNK signaling in modulating metastasis as well (Ma, 2005).

Protein Interactions

The two genes vestigial and scalloped are required for wing development in Drosophila. They present similar patterns of expression in second and third instar wing discs and similar wing mutant phenotypes. vg encodes a nuclear protein without any recognized nucleic acid-binding motif. Sd is a transcription factor homologous to the human TEF-1 factor, whose promoter activity depends on cell-specific cofactors. It is postulated that Vg could be a cofactor of Sd in the wing morphogenetic process and that, together, they could constitute a functional transcription complex. Genetic interactions between the two genes have been investigated. vg and sd are shown to co-operate in vivo in a manner dependent on the structure of the Vg protein. The vg79d5 mutation is recessive. Surprisingly, in an sdETX4 background, the flies heterozygous for the vg79d5 allele exhibit a mutant phenotype that is almost as strong as that of the sdETX4 flies homozygous for this allele. The same phenotype is observed in a sd1 background. Morover, the sdETX4 flies heterozygous for the vgnull homozygous mutants have a very extreme phenotype. Therefore, in a hypomorphic sd background, the vg79d5 mutation has a dominant effect that leads to an enhancement of the sd mutant phenotype. The vg79d5 allele is known to encode a protein with an internal deletion corresponding to the 5' end of exon 3, which includes a poly-alinine-rich region, the correct reading frame being preserved. When the expression of sd is reduced, the presence of the vg79d5 allele encoding such a deleted protein is more drastic than the complete loss of one vg allele. Therefore, the genetic interactions observed between vg and sd are dependent on the structure of the Vg protein (Paumard-Rigal, 1998).

vg was ectopically expressed in patched (ptc) domains. Wing-like outgrowths induced by ectopic expression of vg are severely reduced in vg or sd mutant backgrounds. Accordingly, it was demonstrated that ptc-GAL4-driven expression of vg induces both expressions of the endogenous vg and sd genes and that the two Vg and Sd proteins have to be produced together to promote wing proliferation. It is unknown whether vg alone is able to initiate sd expression in the ptc domain or if vg requires Sd protein. Indeed, both proteins are ubiquitously expressed at a low level throughout the wing disc in early second instar larvae. It is concluded that vg activates its own transcription Futhermore, an interaction between the two proteins is demonstrated by double hybrid experiments in yeast. The C-terminal region of the Vg protein contains the domain involved in the formation of the Vg-Sd complex. Therefore, these results support the hypothesis that Sd and Vg directly interact in vivo to form a complex regulating the proliferation of wing tissue (Paumard-Rigal, 1998).

The formation and identity of organs and appendages are regulated by specific selector genes that encode transcription factors that regulate potentially large sets of target genes. The DNA-binding domains of selector proteins often exhibit relatively low DNA-binding specificity in vitro. It is not understood how the target selectivity of most selector proteins is determined in vivo. The Scalloped selector protein controls wing development in Drosophila by regulating the expression of numerous target genes and forming a complex with the Vestigial protein. The binding of Vestigial to Scalloped switches the DNA-binding selectivity of Scalloped. Two conserved domains of the Vestigial protein that are not required for Scalloped binding in solution are required for the formation of the heterotetrameric Vestigial-Scalloped complex on DNA. It is suggested that Vestigial affects the conformation of Scalloped to create a wing cell-specific DNA-binding selectivity. The modification of selector protein DNA-binding specificity by co-factors appears to be a general mechanism for regulating their target selectivity in vivo (Halder, 2001).

Essential native Sd-binding sites have been identified in several cis-regulatory elements that control the wing field-specific expression of Sd-regulated target genes. These sites were identified by DNaseI footprinting using the TEA domain of Sd. In these analyses, the finding that essential sites occurred most often as tandem double sites, for example, in the cut, spalt and DSRF (bs) genes, was particularly striking. Despite substantial differences in sequence, the TEA domain of Sd binds cooperatively to all of these doublet sites with high affinity, and with similar affinity to single, nonessential sites and to native single vertebrate TEF-1-binding sites in muscle-specific cis-regulatory elements and the SV40 enhancer. From these studies, a consensus binding site sequence of T/A A/G A/G T/A AT G/T T for the TEA domain of Sd, which is very similar to that of the TEA domain of TEF-1, has been inferred (Halder, 2001).

In contrast to the isolated TEA domain, however, the full-length Sd protein (produced by in vitro translation) does not bind equivalently to all of these sites but rather shows a restricted DNA-binding specificity. Full-length Sd binds specifically to the doublet site in the DSRF enhancer and to most of the single binding sites, but binding to the cut, sal, kni and other native templates with doublet sites is weak or nearly undetectable. The difference in DNA-binding activity between the TEA domain and Sd protein indicates that there are motifs within the native Sd protein that affect the activity of the TEA domain and restrict its binding to certain sites. Sites that are bound by Sd are referred to here as A-sites (Halder, 2001).

The finding that most of the doublet-binding sites are not bound by the full-length Sd protein is surprising, considering that these templates are bound with high affinity by the TEA domain and that these sites are essential for enhancer activity in vivo. The observations that the activity of these cis-regulatory elements in vivo and in cell culture depends on co-expression of Vg with Sd, and the finding that Vg and Sd interact physically, raises the possibility that interaction of Vg with Sd changes Sd's DNA-binding properties and enables binding to these sites. However, previous Vg-Sd protein interaction studies have been performed in the absence of DNA and the possible effect of the interaction between Vg and Sd on DNA-binding has thus not been addressed. Whether Sd and Vg form a complex on DNA in vitro and whether this complex has different DNA-binding properties from the Sd protein alone has now been tested (Halder, 2001).

Co-translation of Sd with Vg produces a Vg-Sd complex that binds to these other sites (referred to as B-sites). In contrast to Sd alone, complexes containing Sd and Vg bind strongly to the cut, sal and kni elements. Quantification of the bound complexes shows that Vg increases Sd binding to these doublet sites by about 10-fold. In addition to enabling binding to B-sites, interaction with Vg reduces Sd binding to the single site templates by at least fivefold. Importantly, binding of Vg alone to any of the binding sites described in this report or to any other DNA templates tested has not been detected. Therefore, Vg binding to Sd switches the DNA target preference of Sd from the single A-sites to the doublet B-sites (Halder, 2001).

Four key findings are reported in this study: (1) it was found that the Sd protein has a more restricted DNA-binding specificity than its isolated TEA domain; (2) the Vg-Sd complex binds well to sites in native cis-regulatory elements to which Sd alone does not bind well; (3) two domains of the Vg protein are required for Vg-Sd complex formation on DNA that are not required for Vg binding to Sd in solution, and (4) that this complex is a heterotetramer on DNA while apparently a heterodimer in solution. A mechanistic model is presented for the control of Vg-Sd DNA target selectivity that considers these findings (Halder, 2001).

This model proposes that Vg binding to Sd switches the DNA target selectivity of Sd. The Sd protein alone binds to sites with a particular composition, termed A-sites, which exist singly or as doublets. In the latter case, Sd may bind cooperatively if the two sites are arranged in tandem. When Vg is also present, Vg and Sd interact and form a dimer in solution. This complex has two distinct properties. First, the Vg-Sd dimer has a greatly reduced affinity for A-sites. Vg may either induce a conformational change in Sd that inhibits the TEA domain from interacting with DNA, or Vg could directly mask the TEA domain. Second, the dimer forms a higher order complex on a different set of binding sites, termed B-sites. These two activities of Vg are distinguished by their structural requirements. While the Sd-interaction (SID) domain of Vg is sufficient to inhibit Sd DNA-binding to A-sites, additional domains N- and C-terminal to the SID are required for complex formation on B-sites. Importantly, B-sites are poorly bound by Sd in the absence of Vg. Thus, Vg binding to Sd inhibits binding to A-sites while enabling binding to B-sites, that is, Vg switches the DNA-binding preference from A-sites to B-sites (Halder, 2001).

How does Vg binding affect the target selection of Sd? Two, not necessarily mutually exclusive models, may be postulated. First, Vg may influence Sd through global effects on Sd DNA binding. That is, Vg may act to reduce the DNA binding affinity of Sd to any target DNA, while also enhancing cooperativity of neighboring Vg-Sd complexes on DNA. It was found that Vg and Sd form dimers in solution and that these dimers do not bind single A-sites. In spite of the negative effect of Vg on DNA binding, two Vg-Sd dimers bind strongly to doublet B-sites. Apparently, strong cooperative interactions between two Vg-Sd dimers allow binding to B-sites. The N- and C-terminal protein domains of Vg that are required in addition to the SID for complex formation on DNA may be required for these interactions, which could involve Vg-Sd and/or Vg-Vg interactions between the two dimers on DNA (Halder, 2001).

Alternatively, Vg interaction may specifically enhance binding to doublet B-sites. This model is favored because Vg-Sd has a similar affinity for several B-sites such as those in cut and 2xGT, even though 2xGT is a much better Sd binding site. The affinities of Sd for these sites therefore do not translate directly into the relative affinities observed for Sd-Vg binding, as would be expected if Vg only enhances cooperativity. In addition, it was found that the TEA domain binds several A- and B-sites with high affinity, but that full-length Sd has a strong preference for A-sites over B-sites. Thus, in the absence of any co-factor, Sd is in a conformation in which a domain of Sd separate from the TEA domain inhibits the TEA domain from binding to B-sites specifically. In vitro, Vg interaction appears to be able to alleviate this inhibition because Vg-Sd complexes bind strongly to B-sites. This alleviation only occurs when complexes form on doublet sites, since Vg-Sd complexes do not bind to DNA as a dimers. It is suggested that some sort of conformational change is associated with binding to doublet B-sites. The model is supported by the finding that the region of Sd that binds to the SID of Vg is homologous to a region of the vertebrate TEF-1 that negatively affects DNA binding. This model is analogous in part to the role of Exd overcoming the inhibitory effect of the YKWM motif in the Labial Hox protein (Halder, 2001).

It has been argued that Sd and the Vg-Sd complex differentiate between A- and B-sites. What then are the distinguishing features of these sites? The sequences of the A- and B-sites are quite diverse and their alignment does not reveal different consensus sequence motifs. However, Sd clearly prefers binding to A-sites, and the inability of Sd to bind strongly to B-sites, such as that in the cut element, must therefore be due to the sequence of the template site. Vg-Sd complexes bind with high affinity to only two sites when arranged in tandem, and do not form on single A- or B-sites. Thus, Sd discriminates between A- and B-sites based on sequence, while the binding of Vg-Sd complex depends both on sequence and the arrangement of the sites. Two sites, DSRF and 2xGT, have been identified that have A- as well as B-site properties, so these properties are not mutually exclusive. However, many sites exist that are bound well by Sd or Vg-Sd, but not by both. Most of the essential sites for Vg-Sd regulation in vivo have mainly B-site character and are bound poorly by Sd. The identification of the exact sequence requirements that distinguish native essential Sd sites from the known Vg-Sd target sites will require some knowledge of Sd-regulated target genes in other tissues (Halder, 2001).

Vg binding and its effect on the DNA target selectivity of Sd plays a major role in distinguishing the biological specificity of Sd action in the developing wing from Sd function in other tissues. Sd is required for the development of tissues other than the wing -- for example, the eye and the PNS -- where it is not co-expressed with Vg. Based on the results of this study, it is postulated that Sd selects a different set of target genes in organs other than the wing, at least in part because its DNA-binding specificity is different in the absence of Vg (Halder, 2001).

No direct target genes for Sd in these other tissues have been identified. However, many target genes for the vertebrate Sd homolog TEF-1 are known. Sd and TEF-1 may function very similarly, since their TEA domains are 99% identical and have indistinguishable DNA-binding properties in vitro, and TEF-1 can substitute for Sd in Drosophila. In mammals, TEF-1 directly regulates many genes expressed during muscle differentiation by binding to A-sites containing the so-called 'm-CAT' motif (CATTCCT). Importantly, this motif is bound by a single TEF-1 molecule. Two of these m-CAT sites were tested for Sd binding and it was found that, as for other single A-sites, Sd alone binds well, but the presence of Vg inhibits Sd binding and does not result in complex formation on DNA. Because these sites are in vivo targets of TEF-1, this suggests that TEF-1 and Sd may directly regulate gene expression by binding to single A-sites alone or in complexes with other factors, but not in complexes containing the Vg/Fdu proteins. Interestingly, it has been found that vertebrate TEF-1 forms a complex with the bHLH protein Max in vivo, and that Max, or another bHLH protein, may be an obligatory co-factor for TEF-1 function during muscle differentiation. Because Max contacts DNA sequence specifically, it increases the target selectivity of TEF-1 in muscle cells. The association of TEF-1 with Max may present another example of a tissue-specific co-factor that differentiates the DNA-target selectivity of a TEF transcription factor family member between different tissues (Halder, 2001).

One of the major aims of genome sequence analysis is to decipher genetic regulatory sequences involved in development and differentiation. One critical challenge in achieving this goal is the ability to correctly predict the in vivo target genes of transcription factors. Several types of data may be considered for such predictions, including the presence or absence of transcription factor binding sites in potential regulatory regions, gene expression profiles and detailed protein function studies. Searching genomic sequences for binding sites is obviously important; however, binding site consensus sequences are often short and degenerate, so that potential binding sites are predicted to occur in regulatory regions of virtually any gene. This also holds true for Sd. The consensus binding site of the TEA domain (T/A A/G A/G T/A AT G/T T) is found once about every 2 kb, on average. However, many, if not all, Vg-Sd-regulated target genes possess a doublet of Sd-binding sites. Requiring a second binding site in tandem decreases the frequency of potential biologically relevant Vg-Sd binding sites by a factor of ~2000. The fact that most of the Vg-Sd sites would not have been found using full-length Sd protein in footprint assays and that the Sd DNA-binding domain alone binds promiscuously therefore sounds a note of caution. Understanding the role of tissue-specific co-factors may be imperative to deciphering transcription factor-regulated networks on a genome-wide scale. Efforts are under way, using these new insights into the selectivity of the Vg-Sd complex, towards defining the network of Vg-Sd-regulated genes in the developing wing (Halder, 2001).

The development of the Drosophila wing requires both scalloped and vestigial functions. Using a fusion between full-length Vestigial and the Scalloped TEA domain, the fusion protein can rescue scalloped wing mutations because within wing development, Scalloped and Vestigial cooperatively act as a transcription complex. Scalloped provides the necessary DNA binding function via the TEA domain and Vestigial promotes the activation of target genes. The putative nuclear localization signal contained in the TEA domain of Scalloped is likely responsible for the nuclear localization of Vestigial. The fusion protein is also capable of activating a known target gene of the native complex and thus represents a tool that will be helpful in rapidly identifying target genes of the Sd/Vg complex that are involved in wing differentiation. The functionality of the fusion suggests that only the TEA domain of Scalloped is critical for wing development and the rest of the protein (about 70%) is dispensable. This result is novel and should stimulate further studies of sd in other tissues in view of the fact that scalloped is a vital gene in Drosophila (Srivastava, 2002).

Activation of the knirps locus links patterning to morphogenesis of the second wing vein in Drosophila

The adjacent knirps and knirps-related (knrl) genes encode functionally related zinc finger transcription factors that collaborate to initiate development of the second longitudinal wing vein (L2). kni and knrl are expressed in the third instar larval wing disc in a narrow stripe of cells just anterior to the broad central zone of cells expressing high levels of the related spalt genes. A 1.4 kb cis-acting enhancer element from the kni locus has been identified that faithfully directs gene expression in the L2 primordium. Three independent ri alleles have alterations mapping within the L2-enhancer element; two of these observed lesions eliminate the ability of the enhancer element to direct gene expression in the L2 primordium. The L2 enhancer can be subdivided into distinct activation and repression domains. The activation domain mediates the combined action of the general wing activator Scalloped and a putative locally provided factor, the activity of which is abrogated by a single nucleotide alteration in the ri53j mutant. Misexpression of genes in L2 that are normally expressed in veins other than L2 results in abnormal L2 development. These experiments provide a mechanistic basis for understanding how kni and knrl link AP patterning to morphogenesis of the L2 vein by orchestrating the expression of a selective subset of vein-promoting genes in the L2 primordium (Lunde, 2003).

An enhancer element upstream of the kni coding region selectively directs gene expression in the L2 primordium in third instar larval wing discs. Three separate ri alleles have defects mapping within a minimal 1.4 kb L2 enhancer element. Two of these mutations eliminate activity of the L2 enhancer, kniri[1], which contains a 252 bp deletion, and kniri[53j], which harbors a single base-pair substitution. Truncation of the minimal L2 enhancer to a 0.69 kb fragment leads to ectopic reporter gene expression in the extreme anterior and posterior regions of the wing, indicating that repression contributes to restricting activation of the L2 enhancer. In addition, the general wing promoting transcription factor Scalloped (Sd) binds with high affinity to several sites in the L2 enhancer and sd is required for kni expression in the wing disc. The L2 enhancer element has been employed as a tool to drive expression of various UAS transgenes in the L2 primordium. The loss of the L2 vein in ri mutants can be rescued by L2-specific expression of either the kni or knrl genes, or the downstream target gene rho. In addition, misexpression of genes in the L2 primordium that are normally expressed in veins other than L2 results in abnormal L2 development. These results provide a framework for understanding how positional information is converted into morphogenesis of the L2 wing vein by 'vein organizing genes' such as kni and knrl (Lunde, 2003).

A hypothetical transcription factor that binds the kni promoter and mediates an inductive signal presumably collaborates with the more generally required wing selector Sd, since mutation of four of the Sd binding sites (the doublet and two single sites) in the L2 activation domain completely eliminates enhancer activity in the wing disc. Clonal analysis with a hypomorphic sd allele also indicates that sd is required for high-level expression of the full 4.8 kb L2 enhancer element in the wing disc. It is notable that the reduction in lacZ expression in these clones is not as dramatic as the complete loss of L2 activity observed when Sd binding sites in the activation domain are mutated. There are several possible explanations for this discrepancy. (1) The sd mutation used in these experiments is a hypomorphic allele and therefore has residual activity. Unfortunately, stronger sd alleles produce even smaller viable clones in the wing disc and thus were not used. (2) Since only small clones can be generated, they must typically have been produced with only two or three intervening cycles of cell division. Consequently, the sd- cells may still contain functional levels of wild type Sd (protein perdurance). (3) Another possibility is that other activators can partially substitute for Sd, at least in certain regions of the wing. Based on the absence of L2 activity when Sd binding sites are mutated and the reduction in L2 activity in sd- hypomorphic clones, it is concluded that Sd plays an important role as an activator of the L2 enhancer. These results support the view that Sd functions as a general transcriptional activator of genes expressed in the wing field (Lunde, 2003).

An in vivo analysis of the vestigial gene in Drosophila melanogaster defines the domains required for vg function

Considerable evidence indicates an obligate partnership of Vestigial (Vg) and Scalloped (Sd) proteins within the context of wing development. It is evident that Sd and Vg act together as a transcriptional complex during wing formation, wherein Sd provides the DNA-binding activity and nuclear localization signal, while Vg provides the activation function. A 56-amino-acid motif within Vg is necessary and sufficient for binding of Vg with Sd. While the importance of this Sd-binding domain has been clearly demonstrated both in vitro and in vivo, the remaining portions of Vg have not been examined for their in vivo function(s). Herein, additional regions within Vg were tested for possible in vivo functions. The results identify two additional domains that must be present for optimal Vg function as measured 1) by the loss of ability to rescue vg mutants, 2) by the ability to induce ectopic sd expression, and 3) by the ability to perform other normal Vg functions when these domains are deleted. An in vivo study such as this one is fundamentally important because it identifies domains of Vg that are necessary in the cellular context in which wing development actually occurs. The results also indicate that an additional large portion of Vg, outside of these two domains and the Sd-binding domain, is dispensable in the execution of these normal Vg functions (MacKay, 2003).

From results using ectopic sd-lacZ induction (which measures the ability of ectopic vg to induce ectopic sd expression), the ability to rescue vg mutations, and the ability to carry out other functions associated with normal vg, it can be discerned that certain portions of the vg ORF, in addition to the Sd-binding domain, are necessary to accomplish normal Vg function. These appear to be the critical regions, since other portions can be deleted without effect. More specifically, the N-terminal amino acids (approximately the first 65) and C-terminal residues from 335 to 453 seem to play an important role in the induction of sd-lacZ. When the N-terminal deletion Delta5'-5 (deleting amino acids 2-65) is assayed, the ectopic expression ability is reduced markedly compared to that seen with the full-length vg construct, although it is not eliminated completely. Moreover, the larger N-terminal deletions (amino acids 2-170 and 2-278, respectively) do not further lower the ability to express sd. Thus, it seems that the fundamentally important region is already removed with the Delta5'-5 construct. For C-terminal deletions Delta1-4 and Delta1-2 (amino acids 356-453 and 335-426, respectively), the ability to ectopically express sd is much less than that produced by full-length vg but somewhat stronger than that produced when the N-terminal deletion constructs are assayed. Deletions Delta5'-5, Delta5'-6, and Delta5'-7 retain the encoded amino acids missing from Delta1-4 and Delta1-2 and vice versa. Taken together, these data suggest the presence of two important functional domains for Vg: one within amino acids 1-65 (domain 1) and the other within amino acids 336-453 (domain 2). Although the precise boundaries of these domains have not yet been determined, domain 1 is very likely within the first 65 amino acids (deleted in vgDelta5'-5) since this is the region most highly conserved between D. melanogaster and the mosquito Aedes egyptii. There is 82% identity over the first 66 amino acids, but over the next 20 amino acids the identity drops to 35% and drops even further beyond that. In agreement with this notion, the extent of 'functional' loss in UASDeltavg 5'-6 and 5'-7 is no stronger than that exhibited by UASDeltavg 5'-5, which deletes the first 65 amino acids only. The activity of domain 2 appears to be weaker, since domain 1 deletions produce a slightly more drastic impairment of Vg function than do domain 2 deletions (amino acids 356-453 or 335-426). However, homology between Drosophila and mosquito Vg is also high within the Sd-binding domain of Vg and, in fact, remains strong to the carboxyl terminus of Vg (82% identity from residue 335 to 453. The data define the presence of two necessary functional domains for the Vg protein in vivo. These domains correlate well with data that predict two activation regions using in vitro experiments, including yeast one-hybrid assays. The regions identified in this study also complement more recent in vitro data, implicating these regions of Vg as necessary for binding of the Vg/Sd complex to target genes (MacKay, 2003).

Control of apterous by vestigial drives indirect flight muscle development in Drosophila

Drosophila thoracic muscles are comprised of both direct flight muscles (DFMs) and indirect flight muscles (IFMs). The IFMs can be further subdivided into dorsolongitudinal muscles (DLMs) and dorsoventral muscles (DVMs). The correct patterning of each category of muscles requires the coordination of specific executive regulatory programs. DFM development requires key regulatory genes such as cut (ct) and apterous (ap), whereas IFM development requires vestigial (vg). Using a new vgnull mutant, a total absence of vg is shown to lead to DLM degeneration through an apoptotic process and to a total absence of DVMs in the adult. vg and scalloped (sd), the only known Vg transcriptional coactivator, are coexpressed during IFM development. Moreover, an ectopic expression of ct and ap, two markers of DFM development, is observed in developing IFMs of vgnull pupae. In addition, in vgnull adult flies, degenerating DLMs express twist (twi) ectopically. Evidence is provided that ap ectopic expression can induce per se ectopic twi expression and muscle degeneration. All these data seem to indicate that, in the absence of vg, the IFM developmental program switches into the DFM developmental program. Moreover, the muscle phenotype of vgnull flies can be rescued by using the activity of ap promoter to drive Vg expression. Thus, vg appears to be a key regulatory gene of IFM development (Bernard, 2003).

Vg interacts with Sd to form a transcription factor that binds DNA through the Sd TEA/ATTS domain and activates transcription through the Vg activation domain. Since vgnull mutants show drastic muscle degeneration phenotypes, Vg and sd expressions were examined. Vg is expressed in adepithelial cells. Vg is expressed in myoblasts around the forming DLMs and in some of the DLM nuclei. Moreover, sd expression is expressed in adepithelial cells and developing IFMs. Vg is present in all DLM nuclei and sd is coexpressed with Vg. It is therefore likely that in muscle, as in the wing disc, Sd and Vg are obligate partners. This result is supported by indirect arguments: (1) Vg dimerization with Sd is necessary for Vg activity. Protein interaction has been shown between Vg and Strawberry Notch (SNO), but the function of this new partner remains unknown; (2) Vg is localized to the nucleus in muscles, and nuclear relocalization of Vg in S2 cells requires the presence of Sd. However, no muscle phenotypes were found in sd strong hypomorphic viable mutants (sd58 and sd3L). It is concluded that if Sd is required for muscle development, a very low level of sd product is sufficient to fulfill its function. There is some precedent for this type of situation: for example, whereas Ct is necessary for DFMs development, viable ct mutant alleles do not exhibit any muscle phenotypes (Bernard, 2003).

The TEAD/TEF family protein Scalloped mediates transcriptional output of the Hippo growth-regulatory pathway

The Hippo (Hpo) kinase cascade restricts tissue growth by inactivating the transcriptional coactivator Yorkie (Yki), which regulates the expression of target genes such as the cell death inhibitor diap1 by unknown mechanisms. The TEAD/TEF family protein Scalloped (Sd) is a DNA-binding transcription factor that partners with Yki to mediate the transcriptional output of the Hpo growth-regulatory pathway. The diap1 (th) locus harbors a minimal Sd-binding Hpo Responsive Element (HRE) that mediates transcriptional regulation by the Hpo pathway. Sd binds directly to Yki, and a Yki missense mutation that abrogates Sd-Yki binding also inactivates Yki function in vivo. sd is required for yki-induced tissue overgrowth and target gene expression, and that sd activity is conserved in its mammalian homolog. These results uncover a heretofore missing link in the Hpo signaling pathway and provide a glimpse of the molecular events on a Hpo-responsive enhancer element (Wu, 2008).

The Hpo signaling pathway has emerged as a central and highly conserved mechanism that regulates organ size in animals. At the core of this pathway is a kinase cascade that impinges on the transcriptional coactivator Yki to regulate the transcription of target genes involved in cell growth, proliferation, and survival. Given Yki's pivotal position in the Hpo pathway, understanding the mechanisms by which Yki regulates target gene expression should provide important mechanistic insights that can facilitate therapeutic manipulation of this crucial size-control pathway (Wu, 2008).

A major gap in understanding of the Hpo signaling pathway concerns how Yki regulates target gene transcription. This study shows that Sd represents a crucial missing link between Yki and the regulatory DNA of Hpo pathway target genes. First, an unbiased dissection of diap1 regulatory region revealed a minimal Sd-binding enhancer element (HRE) that confers Hpo-responsive regulation. The HRE not only responded to Yki activity in vivo, but also conferred Sd-dependent and Yki-dependent transcriptional activity in cultured Drosophila cells. In a parallel line of experiments, Sd was identified in an unbiased screen for proteins that bind to a critical N-terminal Yki domain defined by a missense allele, ykiP88L. The fact that this missense mutation disrupted binding of Yki to Sd supports the physiological relevance of a Sd-Yki transcription complex in vivo. The identification of Sd as a cognate Yki partner using two unbiased approaches, combined with the genetic interactions between sd and yki, provide strong evidence that Sd is a critical DNA-binding factor that mediates the transcriptional output of the Hpo signaling pathway. It is worth noting that the requirement for sd in yki-driven overgrowth is highly specific, since the same sd mutation had no effect on overgrowth driven by the activated Ras oncogene. The observation that Yki, but not Sd, can be overexpressed or mutated to elicit tissue overgrowth further suggests that Sd is normally present in excess, and the activity of the Sd-Yki complex is regulated through modulation of Yki activity effected by the Hpo kinase cascade (Wu, 2008).

The founding member of the TEAD family transcription factors, TEAD-1/TEF-1, was initially identified based on its binding to the GTIIC motif of the simian virus 40 (SV40) enhancer. The TEAD family transcription factors have been mostly studied in the context of muscle-specific gene transcription, and their roles in cell proliferation and cell survival are poorly understood. The observation that TEAD-2 and YAP have similar activity to Sd and Yki, respectively, suggests that the growth-regulatory activity of the Sd-Yki complex is likely conserved in the mammalian Hpo pathway. It is also worth noting that besides growth regulation, the Hpo pathway has also been implicated in controlling other biological processes such as rhodopsin gene expression in mature photoreceptors and dendrite morphogenesis in postmitotic neurons. It remains to be determined whether Yki partners with Sd or other (unknown) DNA-binding factors in such nongrowth contexts (Wu, 2008).

Despite their elevated transcription upon inactivation of Hpo pathway tumor suppressors or activation of the Yki oncoprotein, it was previously unknown whether Yki regulates the known Hpo pathway target genes directly or indirectly through intermediary transcriptional regulators. This study has taken an unbiased approach to this question by isolating a HRE for diap1. This DNA element provided several important insights into how Hpo signaling activity is converted into transcriptional output. First, the Sd protein directly binds to the HRE and activates an HRE-luciferase reporter in cell culture in conjunction with Yki, supporting the notion that Yki directly regulates the transcription of the diap1 gene. Second, the minimal diap1 HRE contains non-Sd-binding sequence that is indispensable for HRE activity, suggesting that the HRE likely binds to additional transcription factors besides Sd in vivo. This latter characteristic is not unique to the HRE, but is a general feature that has been observed for many signaling pathways in Drosophila. For example, Notch-regulated enhancers contain not only binding sites for the signal-regulated transcription factor Suppressor of Hairless, but also binding sites for additional cofactors whose activity is Notch independent. It will be important to identify the factors that bind to the non-Sd sequence in the diap1 HRE, and to investigate whether such factors play a general role in mediating the Hpo responsiveness of other target genes (Wu, 2008).

The identification of a minimal HRE makes possible several new avenues of investigation to better understand the Hpo signaling pathway. The minimal HRE revealed in this study should facilitate a comprehensive cataloguing of Hpo pathway target genes, many of which remain to be identified. It also provides a useful tool for constructing reporters that can be used to monitor the specific activity of the Hpo pathway in vivo. Furthermore, this work will facilitate cell-based RNAi screens for components or modulators of the Hpo pathway, as illustrated by the successful use of pathway-specific luciferase reporters for interrogating other signaling pathway (Wu, 2008).

An interesting and somewhat unexpected finding from this study concerns the differential requirement of yki for the basal expression of diap1 and expanded (ex), with the former being yki dependent and the latter being yki independent, respectively. Thus, different Hpo target genes, and by inference different enhancer elements or their combinations, can respond to different threshold levels of Yki activity. It is suggest the basal expression of ex is mediated by non-HRE sequence in the ex locus and is therefore independent of yki. Excessive yki activity, either directly via an HRE in the ex locus or indirectly by turning on another factor, promotes ex transcription above the basal level. In contrast, Yki (through Sd, non-Sd DNA-binding factors, or both), regulates the basal level transcription of diap1. It is noted that the basal transcription of diap1 is not necessarily regulated through the HRE, which was identified by virtue of reporter expression under hyperactive Yki activities. Indeed, it was found that although the HRE is responsive to yki overexpression, it is largely unresponsive to loss of yki. Thus, the diap1 HRE revealed in this study is uniquely sensitive to unrestrained Yki activity (Wu, 2008).

The exquisite sensitivity of yki-induced overgrowth to sd dosage suggests that Sd/TEAD could be specifically targeted to ablate certain unwanted tissue growth, such as that caused by aberrant Hpo signaling, with minimal effect on normal growth. Thus, Sd/TEAD belongs to a growing list of genes that cause 'non-oncogene addition' -- genes that cannot be mutated or overexpressed to an extent that directly promotes tumorigenesis, but are still rate limiting to their specific signaling pathways. The requirement of such non-oncogenes in tumor cells makes them excellent targets for the development of new cancer therapeutics (Wu, 2008).

The TEAD/TEF family of transcription factor Scalloped mediates Hippo signaling in organ size control

The Hippo (Hpo) signaling pathway governs cell growth, proliferation, and apoptosis by controlling key regulatory genes that execute these processes; however, the transcription factor of the pathway has remained elusive. This study provides evidence that the TEAD/TEF family transcription factor Scalloped (Sd) acts together with the coactivator Yorkie (Yki) to regulate Hpo pathway-responsive genes. Sd and Yki form a transcriptional complex whose activity is inhibited by Hpo signaling. Sd overexpression enhances, whereas its inactivation suppresses, tissue overgrowth caused by Yki overexpression or tumor suppressor mutations in the Hpo pathway. Inactivation of Sd diminishes Hpo target gene expression and reduces organ size, whereas a constitutively active Sd promotes tissue overgrowth. Sd promotes Yki nuclear localization, whereas Hpo signaling retains Yki in the cytoplasm by phosphorylating Yki at S168. Finally, Sd recruits Yki to the enhancer of the pathway-responsive gene diap1, suggesting that diap1 is a direct transcriptional target of the Hpo pathway (Zhang, 2008).

The Hpo pathway has emerged as a conserved signaling pathway that plays a critical role in controlling tissue growth and organ size. Despite the growing recognition of the importance of this pathway in development and cancer, the transcription factor that links the cytoplasmic components to the nuclear events has remained elusive and thus represents a major gap in the pathway. This study demonstrates Sd is the missing transcription factor of the Hpo pathway based on several lines of genetic and biochemical evidence. (1) Sd and Yki form a transcriptional complex to activate a reporter gene in S2 cells and this transcriptional activity is inhibited by Hpo signaling. Furthermore, Sd and Yki synergize in vivo to promote Hpo target gene expression and tissue overgrowth. (2) More importantly, loss of Sd function suppresses tissue overgrowth induced by Yki overexpression or loss-of-function mutations in hpo, sav, and wts. In addition, Sd inactivation either by RNAi or a genetic mutation blocks the ectopic expression of Hpo responsive genes induced by excessive Yki activity. (3) RNAi knockdown of Sd phenocopies knockdown of Yki, which is manifested by reduced organ size and diminished expression of Hpo pathway-responsive genes. (4) A constitutively active form of Sd activates multiple Hpo pathway-responsive genes and promotes tissue overgrowth. (5) Sd promotes Yki nuclear translocation and recruited Yki to the diap1 enhancer (Zhang, 2008).

Several sd null alleles were generated to further explore the consequence of loss of Sd. sd null clones located in the wing pouch region were found to exhibit growth deficit such that early-induced clones (48-72 hrs AEL) were eliminated by the end of late third instar. However, late-induced clones (72-96 hrs AEL) survived and exhibited diminished expression of diap1. In contrast, early-induced clones were recovered in the notal region of wing discs and in eye discs without showing discernible change in Diap1 levels. However, a previous study showed that yki mutant clones exhibited reduced diap1 expression in eye discs. It is possible that low levels of residual Sd activity persist in sd mutant clones, which are sufficient to support the basal expression of the Hpo target genes. Alternatively, Yki may act through another transcription factor to maintain the basal expression of Hpo target genes. Nevertheless, sd null mutation suppresses the overgrowth phenotype and ectopic cycE expression induced by excessive Yki activity, suggesting the residual Sd in sd mutant clones is insufficient to support the elevated Yki activity (Zhang, 2008).

The identification of Hpo pathway transcription factor provided an opportunity to assess direct transcriptional targets of the pathway. To this end, the diap1 enhancer was characterized, and a 1.8 kb enhancer element critical for diap1 expression was identified. This region contains a total of seventeen predicted Sd binding sites. Using the ChIP assay, it was demonstrated that both Sd and Yki physically interact with the 1.8 kb diap1 enhancer and the association of Yki with the diap1 enhancer is mediated by Sd. These results suggest that Sd recruits Yki to the diap1 enhancer to activate its transcription (Zhang, 2008).

It has been shown that Sd acts in conjunction with Vg to promote wing development by directly regulating the expression of wing patterning genes. This study has demonstrated that Sd acts in conjunction with Yki to control organ size by regulating the expression of genes involved in cell proliferation, cell growth, and apoptosis. These observations raise an important question of how Yki-Sd and Vg-Sd transcriptional complexes specifically select their targets. One possibility is that Vg-Sd and Yki-Sd prefer to interact with distinct Sd binding sites. Indeed, a previous study showed that binding of Vg to Sd modulates the DNA binding selectivity of Sd. Another possibility is that target selectivity could be influenced by cofactors that bind in the vicinity of Sd binding sites. In support of this notion, previous studies have shown that wing specific enhancers contain both Sd binding sites and binding sites for transcription factors that mediate specific signaling pathways. It is also possible that Vg-Sd and Yki-Sd may share common targets. For example, diap1 could be activated by Vg-Sd in the wing pouch, which might explain why sd mutant clones in this region exhibits diminished diap1 expression (Zhang, 2008).

In principle, the Hpo pathway could regulate the activity of Yki-Sd transcriptional complex at several levels. For example, Hpo signaling could regulate the formation Yki-Sd complex or the recruitment of other factor(s) to the Yki-Sd transcriptional complex. Alternatively, Hpo signaling could regulate the nuclear-cytoplasmic transport of Yki. In support of the latter possibility, Yki exhibits elevated nuclear localization in wts or hpo mutant clones. In addition, coexpression of Hpo with Yki depletes nuclear Yki in S2 cells, suggesting that Hpo signaling impedes nuclear localization of Yki and thereby limits the amount of active Yki-Sd transcriptional complex (Zhang, 2008).

Mutating Yki S168 to Ala increases nuclear localization and growth promoting activity of Yki. In addition, it has been demonstrated that phosphorylation of Yki S168 was stimulated by Hpo. Phosphorylation of Yki by Hpo signaling increases their association with 14-3-3, which is abolished by mutating Yki S168 to Ala. Since 14-3-3 often regulates nuclear-cytoplasmic shuttling of its interacting proteins, these observations suggest that Hpo signaling inhibits Yki at least in part by phosphorylating Yki S168, which promotes 14-3-3 binding and cytoplasmic sequestration of Yki (Zhang, 2008).

The Hpo pathway appears to restrict cell growth and control organ size in mammals. The finding that Sd is critical for Yki-induced tissue growth has raised the interesting possibility that the effect of YAP in promoting tissue growth may rely on the TEAD/TEF family of transcription factors. Corroborating this hypothesis, TEAD-2/TEF-4 protein purified from mouse cells was associated predominantly with YAP (Vassilev, 2001). Furthermore, YAP can bind to and stimulate the trans-activating activity of all four TEAD/TEF family members (Vassilev, 2001). The TEAD/TEF family members exhibit overlapping but distinct spatiotemporal expression patterns and thus may have redundant but unique roles during development . It will be important to determine which TEAD/TEF family members are involved in the mammalian Hpo pathway and whether YAP employs distinct sets of TEAD/TEF transcription factors in different tissues. Since abnormal activation of YAP is associated with multiple types of cancer, disrupting YAP-TEAD/TEF interaction may provide a new strategy for cancer therapeutics (Zhang, 2008).

TEAD mediates YAP-dependent gene induction and growth control

The YAP transcription coactivator has been implicated as an oncogene and is amplified in human cancers. Recent studies have established that YAP is phosphorylated and inhibited by the Hippo tumor suppressor pathway. This study demonstrates that the TEAD family transcription factors are essential in mediating YAP-dependent gene expression. TEAD is also required for YAP-induced cell growth, oncogenic transformation, and epithelial-mesenchymal transition. CTGF is identified as a direct YAP target gene important for cell growth. Moreover, the functional relationship between YAP and TEAD is conserved in Drosophila Yki (the YAP homolog) and Scalloped (the TEAD homolog). This study reveals TEAD as a new component in the Hippo pathway playing essential roles in mediating biological functions of YAP (Zhao, 2008).

To investigate the function of TEAD in YAP-induced growth control, transgenic flies were generated that express human YAP-S127A (an active form) or YAP-S94A/S127A in developing eyes. YAP-S127A overexpression significantly increased eye size and the number of interommatidial cells. Mutation of S94A dramatically decreased the activity of YAP-S127A in promoting tissue growth. Scalloped (Sd) is the only TEAD homolog in Drosophila. Yki was found to directly interacted with Sd in an in vitro binding assay. Furthermore, Yki S97A mutation (equivalent to YAP-S94A) diminished its interaction with Sd. Moreover, this Sd-binding-defective Yki-S97A mutant was less potent in stimulating growth in vivo compared with wild-type Yki. The functional defect of the TEAD-binding-deficient YAP/Yki was further confirmed by generating overexpression flip-out clones in the Drosophila larval wing discs as labeled by positive GFP expression. Both YAP-S127A and Yki are potent in stimulating tissue growth as individual clones, and the whole discs were generally larger than wild-type clones or discs. However, neither YAP-S94A/S127A nor Yki-S97A showed a similar level of growth-promoting effect. These data indicate that TEAD/Sd binding is important for the physiological function of YAP/Yki (Zhao, 2008).

The genetic interaction between Yki and Sd was tested. A strong loss-of-function allele of sd dominantly suppressed the enlarged and rough eye phenotypes caused by Yki overexpression. Thus, the level of Sd is critical for Yki to promote tissue growth. Overexpression of Sd caused small eyes, presumably due to a dominant-negative effect, but it did not result in lethality. This phenotype was strongly enhanced by reduction of yki levels, such that all of these flies died at the late pupal stage and had no eyes. Furthermore, coexpression of Yki with Sd suppressed the reduced eye phenotype caused by Sd overexpression. In fact, the eyes of animals overexpressing both Yki and Sd were enlarged more than those of animals that only expressed Yki. Therefore, Sd overexpression enhanced the Yki overexpression phenotypes. Together, these results indicate that Sd is a critical functional partner of Yki, a conclusion consistent with TEAD as a critical downstream target transcription factor of YAP (Zhao, 2008).

Mask proteins are cofactors of Yorkie/YAP in the Hippo pathway

The Hippo signaling pathway acts via the Yorkie (Yki)/Yes-associated protein (YAP) transcriptional coactivator family to control tissue growth in both Drosophila and mammals. Yki/YAP drives tissue growth by activating target gene transcription, but how it does so remains unclear. This study identified Mask as a novel cofactor for Yki/YAP. Drosophila Mask forms a complex with Yki and its binding partner, Scalloped (Sd), on target-gene promoters and is essential for Yki to drive transcription of target genes and tissue growth. Furthermore, the stability and subcellular localization of both Mask and Yki is coregulated in response to various stimuli. Finally, Mask proteins are functionally conserved between Drosophila and humans and are coexpressed with YAP in a wide variety of human stem/progenitor cells and tumors (Sidor, 2013).

The Hippo signaling pathway is an emerging tumor suppressor pathway in Drosophila and mice. The key effector of the Hippo pathway is the Drosophila Yorkie (Yki)/mammalian Yes-associated protein (YAP). Yki/YAP is a transcriptional coactivator whose nuclear localization and activity is inhibited upon phosphorylation by the Wts kinase. Yki/YAP is an oncogenic component of the pathway that is sufficient to drive tissue overgrowth when overexpressed in either Drosophila tissues or mouse tissues such as liver and intestine. In addition, YAP is nuclear in human cancer cell lines but translocates to the cytoplasm upon contact inhibition of cell proliferation at confluence. Drosophila Yki acts by binding to transcription factors, such as Scalloped (Sd), Homothorax, and Teashirt, to activate the expression of genes promoting cell proliferation and survival, such as cyclin E, myc, DIAP1, and bantam, as well as genes encoding upstream components of the Hippo pathway, such as expanded, merlin, kibra, and four-jointed . Thus, Yki promotes tissue growth by regulating the transcription of target genes, but how it does so is poorly understood (Sidor, 2013).

In an in vivo RNAi screen based on the Vienna Drosophila RNAi Center library, the mask (CG33106) gene was shown to have a strong undergrowth phenotype in the wing and eye when silenced by expression of an inverted-repeat (IR) hairpin RNAi transgene (mask-IR), as opposed to the effect of wts-IR, which causes tissue overgrowth. To confirm these results, a null mutation in the mask gene mask10.22 was used to generate homozygous mutant eyes with the eyeless.flp FRT Minute system. mask10.22 mutant eyes were much smaller than controls. In the developing larval wing imaginal disc, mask10.22 mutant clones proliferated poorly, growing to only 10% of the size of their wild-type 'twin-spot' clones. These data show that mask is essential for cell proliferation and tissue growth. The mask loss-of-function phenotype is similar to that caused by mutants in the Hippo pathway component yki but distinct from that of other signaling pathways such as the Ras pathway (Sidor, 2013).

Whether mask is required for the expression of Yki target genes was examined. Silencing of mask in the posterior compartment of the wing disc by expression of mask-IR with hh.gal4 led to a significant reduction in the expression of four-jointed.lacZ and DIAP1.lacZ reporters. In mask10.22 mutant clones, the levels of expression of four-jointed.lacZ, DIAP1.lacZ, and expanded.lacZ were also reduced compared to their levels in wild-type surrounding cells. These results show that mask is required for the expression of Yki target genes (Sidor, 2013).

The mask gene encodes a very large protein of 4,001 amino acids containing two highly conserved ankyrin-repeat domains and a KH domain. The KH domain was first identified in the hnRNP K protein, which was originally found in association with RNA in ribonucleoprotein particles but later found to bind to DNA via its KH domain and to act as a transcriptional cofactor for p53 by facilitating assembly of the transcription factor complex on DNA. Similarly, the NF-κB transcription factor must form a complex with the KH-domain protein RPS3 on certain target genes to drive transcription. Whether Mask could interact with Yki on target-gene promoters was tested. In immunoprecipitation experiments, it was found that the ankyrin-repeat domains of Mask are able to bind to an EGFP-tagged form of Yki. In addition, a DNA pull-down experiment was performed with a biotin-tagged 583 bp fragment of the DIAP1 promoter that contains multiple Sd binding sites. When the DNA is pulled down with streptavidin-coated beads from S2 cells transfected with Yki and Sd, it was found that endogenous Mask binds with Yki and Sd to the DIAP1 promoter, but not to a negative control actin sequence. RNAi knockdown of Sd reduced the binding of Mask to the DIAP1 promoter. These results indicate that Mask is able to complex with Yki/Sd on target-gene promoters (Sidor, 2013).

Henetic epistasis experiments were performed. Overexpression of Yki in clones of cells is sufficient to cause a strong overproliferation phenotype. In contrast, when Yki is overexpressed in mask mutant clones, it is no longer able to induce overproliferation. Overexpressed Yki accumulates in the cytoplasm and nucleus of both wild-type and mask10.22 mutant cells, indicating that Mask is not essential for Yki to enter the nucleus. The overgrowth of clones mutant for wts or expressing nonphosphorylatable or nuclear-localized Yki is also partially suppressed by mutation of mask. Furthermore, the activation of Yki target-gene transcription in wts mutant clones is suppressed by mutation of mask. These results show that Mask is not required for activation or nuclear localization of Yki but is instead required for Yki to normally activate transcription of its target genes. However, Yki still retains some activity in the absence of Mask, as indicated by the fact that wts, mask double mutants grow larger than mask single mutant clones (Sidor, 2013).

Drosophila Mask has two human homologs, which have been named Mask1 (also called ANKHD1) and Mask2 (also called ANKRD17). Both Mask1 and Mask2 can coimmunoprecipitate with FLAG-tagged YAP. YAP5SA strongly induces CTGF expression in human cells, but not when the cells are cotransfected with siRNAs targeting either YAP or both Mask1 and Mask 2. Mask1 and Mask2 colocalize with YAP in the nucleus of sparsely plated HEK293 cells and cytoplasm of densely confluent HEK293 cells. Translocation of Mask1, Mask2, and YAP was also observed in Caco2 cells. These results show that human Mask proteins bind to and colocalize with YAP to promote target-gene expression. A similar coregulation of Mask and Yki localization and stability was observed in Drosophila. Finally, the expression of YAP, Mask1, and Mask2 was examined in human epithelial tissue sections. The three proteins are strongly expressed in the basal cell layer of epithelial tissues, where stem/progenitor cells are located. A systematic survey of YAP and Mask1 expression confirms that they are coexpressed in a wide variety of human tissues and tumors (Sidor, 2013).

In conclusion, Mask proteins are novel cofactors for Yki/YAP whose function is conserved between Drosophila and humans. Mask acts in a similar fashion to other KH-domain cofactors for NF-κB and p53, being coregulated with their cognate transcription factor in response to signals and assembling with their cognate transcription factor on promoters to drive transcription. Thus, KH-domain cofactors represent a novel class of control mechanism for signal-regulated transcription factors. Given the essential role of Mask in Yki/YAP function and the increasing evidence implicating human YAP in stem cell proliferation and tumorigenesis, human Mask proteins are candidate targets for new cancer therapies (Sidor, 2013).

Notch inhibits yorkie activity in Drosophila wing discs

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Brahma regulates the Hippo pathway activity through forming complex with Yki-Sd and regulating the transcription of Crumbs

The Hippo signaling pathway restricts organ size by inactivating the Yorkie (Yki)/Yes-associated protein (YAP) family proteins. The oncogenic Yki/YAP transcriptional coactivator family promotes tissue growth by activating target gene transcription, but the regulation of Yki/YAP activation remains elusive. In mammalian cells, Brg1, a major subunit of chromatin-remodeling SWI/SNF family proteins, was identified that interacts with YAP. This finding led the authors to investigate the in vivo functional interaction of Yki and Brahma (Brm), the Drosophila homolog of Brg1. Brm was found to function at the downstream of Hippo pathway and interacts with Yki and Scalloped (Sd) to promotes Yki-dependent transcription and tissue growth. Furthermore, it was demonstrated that Brm is required for the Crumbs (Crb) dysregulation-induced Yki activation. Interestingly, it was also found that crb is a downstream target of Yki-Brm complex. Brm physically binds to the promoter of crb and regulates its transcription through Yki. Together, this study has shown that Brm functions as a critical regulator of Hippo signaling during tissue growth and plays an important role in the feedback loop between Crb and Yki (Zhu, 2014).

The core signaling cascade of Hippo pathway has been extensively studied. However, the regulatory mechanism of Yki/YAP activation remains largely elusive. This study found that Brm, a component of SWI/SNF complex, interacts with Yki and regulates organ growth in Drosophila. The findings indicated that Brm is indispensable for Yki activation to drive the expression of target genes. Interestingly, it was also found that the expression of crb is regulated by Yki-Brm complex. The ChIP assay showed that Brm and Yki physically bound to the promoter region of crb, and knockdown of Yki reduced the binding of Brm to crb promoter. Taken together, this study presents a novel feedback loop between Crb and the Yki-Brm complex. Thus, the mutual regulation between apical polarity and Hippo pathway could be critical for tissue growth and homeostasis (Zhu, 2014).

Considering the transcriptional co-dependence with RNA Polymerase II and broad localization in actively transcribed regions, the Brm complex may be present on multiple promoters and is required by global gene transcription. However, the expression of only 872 genes has been observed significantly altered by Brm knockdown. This suggests that there is a selectivity of Brm-mediated transcriptional regulation. In addition to Hippo pathway, morphogens, such as Wingless (Wg), Hedgehog (Hh) and Decapentaplegic (Dpp), also play fundamental roles in organ patterning and growth. Therefore, the readouts of Wg, Hh and Dpp pathway in wing discs with brm knockdown were examined. Interestingly, only Wg secretion was found to be altered, which argued that there was gene specificity of Brm-mediated transcription. Many other factors including the interaction of transcription factors and co-factors, histone modifications and DNA methylation have been also shown essential in this process. Therefore, how Brm regulates the specific targets transcription needs to be further investigated (Zhu, 2014).

These data demonstrate that the enhancement of Crb levels caused by yki overexpression can be suppressed by brm knockdown. However, the overexpression of Brm alone cannot alter the Crb levels. These results suggest that the regulatory function of Brm complex is indispensable for crb but relies on Yki. Together with the fact that Brm physically associate with Yki, it is argued that Brm complex is recruited to target gene promoters through Brm-Yki interaction. Accordingly, another Drosophila transcription factor Zeste has been reported that recruits the Brm complex to chromatin to initiate transcription. Beside of Brm, Moira (Mor), another subunit of the complex, has also been reported to directly interact with Yki and occupy on the promoters of Hpo target genes, which support the working model that Yki facilitates the recruitment of Brm complex onto crb promoters. Taken together, this study draws a conclusion that the specificity of Brm for regulating Hippo targets is determined by Yki recruitment. Given that both the Brm complex and Hippo pathway are conserved in mammals, these results shed a light on a conserved interaction, which might be critical for mammalian growth and tissue homeostasis (Zhu, 2014).

scalloped: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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