Targets of Activity

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).

Two related transcription factors (Escargot and Snail) that are both expressed in the embryonic wing disc, function as intrinsic determinants of the wing cell fate. In escargot or snail mutant embryos, wing-specific expression of snail, vestigial and escargot is the same as in wild-type embryos. However, in escargot/snail double mutant embryos, wing development proceeds until stage 13, but the marker expression is not maintained in later stages, and the invagination of the wing primordium is absent. From such analyses, it has been concluded that escargot and snail expression in the wing disc are maintained by their auto- and cross-activation. Either ubiquitous escargot or snail expression induced from the hsp70 promoter rescues the escargot/snail double mutant phenotype with the effects confined to the prospective wing cells. The similar DNA binding specificities of Escargot and Snail suggest that they control the same set of genes required for wing development. It is proposed that prospective wing cells turn on escargot and snail transcription, and become competent for regulation by Escargot and Snail. The sustained escargot and snail expression then activates vestigial and other target genes, essential for wing development (Fuse, 1996).

Subsets of differentiating muscles in the Drosophila embryo express putative transcription factors, such as NK1/S59 and vestigial. These genes may control the development of specific muscle properties. In embryos mutant for wingless, myogenesis is grossly deranged. Mesodermal expression of S59 is lost, whereas some vestigial-expressing muscles develop. wingless dependence and independence of specific muscle subsets correlates with an early derangement of twist expression in wingless mutants (Bate, 1993).

Wing margin formation in Drosophila requires the Notch receptor and, in the dorsal compartment, Serrate, a Notch ligand. Delta, the other known ligand for Notch, is also essential for this process. Delta is required in ventral cells at the dorsal/ventral compartment boundary, where its expression is specifically elevated in second-instar wing discs during wing margin formation. Moreover, ectopic Delta expression induces wingless, vestigial, and cut and causes adult wing tissue outgrowth in the dorsal compartment. The effect is mediated by Notch, because loss of Notch activity suppresses Delta-induced ectopic wing outgrowth. Whereas either ectopic expression of Notch or the truncated activated Notch induces cut in both dorsal and ventral compartments, ectopic Delta expression induces cut only in the dorsal compartment and ectopic Serrate induces cut only in the ventral compartment. These observations indicate that Notch-expressing cells in a given compartment have different responses to Delta and Serrate. It is proposed that Delta and Serrate function as compartment-specific signals in the wing disc, to activate Notch and induce downstream genes required for wing formation (Doherty, 1996).

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. sd and vg regulate one another. Although a low level of vg expression is retained, all elevated vg expression is eliminated in both early and late mutant sd discs. Since embryonic vg is unaltered in sd mutants, it appears that the observed dependence of vg expression upon sd is a later acting wing-specific requirement. Conversely, sd expression is not detected in discs from a vg null mutant (Williams, 1993).

vestigial and scalloped, both expressed along the normal wing margin, are overexpressed at margin-like levels in shaggy-zeste white 3 clones, resulting in a margin-like transformation. This transformation 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, indicating a cell-nonautonomous effect. Since 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 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 (Blair, 1994).

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

Two lines of evidence suggest that Distal-less and vestigial expression at the wing margin, as well as bristle specification, are organized by Wingless. First, using a temperature-sensitive mutation of wg, it is possible to remove wg activity at chosen times during wing development. Dll expression is abolished within 48 hours following a shift to the nonpermissive temperature, and vg expression is eliminated except for a thin stripe of cells straddling the D/V boundary compartment boundary. Second, ectopic expression of wg as well as ectopic activation of the Wg-signal transduction pathway, caused by eliminating the Shaggy/Zeste-white 3 activity, up-regulates the expression of Dll and Vg within the wing-blade primordium. Expression of a teathered WG protein, genetically engineered to be attached to the cell surface and thus unable to diffuse like normal WG protein, drastically alters the up-regulation of vg and Dll in surrounding cells. Normal WG upregulates Dll in wild-type cells up to 10 or more cell diameters away from the WG source, while teathered WG up-regulates these genes only in their immediate wild-type neighbors. Thus normal WG can act as a long range morphogen, exerting a graded influence on vg and Dll in surrounding cells, while teatherd WG exerts only a short-range, all-or-non influence on surrounding cells (Zecca, 1996).

Armadillo is required autonomously and continuously to mediate the response of wing cells to WG-Secreting cells located at a distance. Clones of arm mutant cells were generated in wing discs. These cells stop dividing and either die or are actively eliminated from the disc epithelium. When stained for either VG or DLL expression 36 hours after mitotic recombination is induced, none of the cells within such clones express either protein (Zecca, 1996).

Short-range interaction between dorsal and ventral (D and V) cells establishes an organizing center at the DV compartment boundary that controls growth and specifies cell fate along the dorsal-ventral axis of the Drosophila wing. The secreted signaling molecule Wingless (WG) is expressed by cells at the DV compartment boundary and has been implicated in mediating its long-range patterning activities. Does WG acts directly at a long-range to specify cell fates in the wing? To investigate this question, mutant clones of two components of the WG transduction pathway, dishevelled and armadillo were examined. Cells mutant for dsh show reduced levels of Dll and vg expression. Cells mutant for a temperature sensitive hypomorphic allele of arm, likewise show loss of expression of Dll and vg when larvae are shifted to non-permissive temperatures. Reducing WG levels at the margin reduces both the maximum level of Dll expression near the DV boundary and the distance from the DV boundary at which Dll can be activated. An intermediate level of WG activity is not sufficient to support the specification of wing margin bristles, suggesting that WG has fallen below a critical threshold for activation of AS-C gene expression, while remaining above the respective thresholds of activation for both Dll and vg. Thus, WG acts directly, at long range, to define the expression domains of its target genes, Distal-less and vestigial. Expression of the Achaete-scute genes, Distal-less and vestigial at different distances from the DV boundary is controlled by WG in a concentration-dependent manner, with AS-C requiring the highest levels of WG. Dll, expressed in a wider range, requires the next highest level, and vg, which is expressed across the entire wing pouch, requires the lowest levels. It is proposed that WG acts as a morphogen in patterning the D/V axis of the wing (Neumann, 1997).

The gene homothorax is required for the nuclear import of Extradenticle, The functions of exd/hth and of the Hh/Wg/Dpp pathway are mutually antagonistic: exd blocks the response of Hh/Wg/Dpp target genes such as optomotor-blind and dachshund; high levels of Wg and Dpp eliminate exd function by repressing hth. This repression is mediated by the activity of Dll and dac. One prerequisite for appendage development is the inactivation of the exd/hth genes (Azpiazu, 2000 and references therein).

vg is one of the factors involved in the downregulation of hth: the elimination of vg activity in the wing pouch results in hth activation and its ectopic expression in the hinge region represses the normal activity of hth. Since a principal role of vg is to specify wing development, it appears that a component of this function is to eliminate hth activity and therefore exd function. Since vg is a target gene of both the Hh and the Wg pathways in the wing, it seems that the downregulation of hth by both pathways is mediated by vg. One question that is not fully understood about the role of vg is that, although it is able to repress hth, there is some vg activity normally in the wing hinge that coincides with that of hth. The levels of the Vg protein appear to be similar in the pouch and the hinge regions so that different levels of product do not seem to be a likely reason. It is believed that there may be other factors in the hinge; tsh is a likely candidate that counteracts the repression by Vg (Aspiazu, 2000).

Altogether, the results presented here suggest the subdivision of the non-thoracic part of the wing disc into two major domains: the wing hinge, where hth is expressed and Exd is functional (nuclear), and the wing pouch where hth is not expressed, and Exd is cytoplasmic and therefore inactive. By homology with the leg disc, the latter would be the genuine appendage part of the disc. These two regions are formed by two antagonistic genetic systems: in the hinge, the high levels of hth, inherited from the embryo and probably maintained by wg, tsh and maybe other regulators, prevent wg response to Notch signaling, which is necessary for the development of the wing pouch. In the wing pouch, the activities of the Wg and Dpp pathways suppress hth so that Notch may induce wg activity and the appendage is formed. In addition to its role in preventing excessive proliferation, hth may also contribute, together with tsh, wg and nub, to the partition of the wing hinge into two regions that correspond to the outer and inner rings of hth expression. The outer ring domain expresses tsh, wg and hth; has nuclear Exd and does not express vg and nub. The inner domain expresses wg, nub and hth, has nuclear Exd and does not express tsh. The individual role of these genes is not yet established, but it is possible that they function in some combinatorial manner (Aspiazu, 2000).

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).

Temporally dynamic response to Wingless directs the sequential elaboration of the proximodistal axis of the Drosophila wing

The Drosophila wing imaginal disc gives rise to three main regions along the proximodistal axis of the dorsal mesothoracic segment: the notum, proximal wing, and wing blade. Development of the wing blade requires the Notch and wingless signalling pathways to activate vestigial at the dorsoventral boundary. However, in the proximal wing, Wingless activates a different subset of genes, e.g., homothorax. This raises the question of how the downstream response to Wingless signalling differentiates between proximal and distal fate specification. A temporally dynamic response to Wingless signalling is shown to sequentially elaborate the proximodistal axis. In the second instar, Wingless activates genes involved in proximal wing development; later in the third instar, Wingless acts to direct the differentiation of the distal wing blade. The expression of a novel marker for proximal wing fate, Zn finger homeodomain 2 (zfh-2), is initially activated by Wingless throughout the 'wing primordium,' but later is repressed by the activity of Vestigial and Nubbin, which together define a more distal domain. Thus, activation of a distal developmental program is antagonistic to previously established proximal fate. In addition, Wingless is required early to establish proximal fate, but later when Wingless activates distal differentiation, development of proximal fate becomes independent of Wingless signalling. Since P-element insertions in the zfh-2 gene result in a revertable proximal wing deletion phenotype, it appears that zfh-2 activity is required for correct proximal wing development. These data are consistent with a model in which Wingless first establishes a proximal appendage fate over notum, then the downstream response changes to direct the differentiation of a more distal fate over proximal. Thus, the proximodistal domains are patterned in sequence and show a distal dominance (Whitworth, 2003).

The Drosophila wing imaginal disc gives rise to the structures of the dorsal mesothoracic segment. This is subdivided into three main regions: the notum, the wing blade, and the proximal wing and hinge. The wing is attached to the thorax via a complex joint comprising a small portion of the appendage, the hinge, which consists of several interlocking sclerites and plates. The wing blade tapers toward the body, forming a short, narrow region that is attached at the hinge. This region shall be referred to as the proximal wing since it is morphologically and mechanically distinct from the hinge itself. Fate mapping of the late third instar imaginal disc has determined that the central portion, the wing pouch, develops as wing blade, a ring surrounding the wing pouch develops as proximal wing and hinge, and the large dorsal territory and a narrow ventral domain form the notum and ventral pleura (Whitworth, 2003).

Previous studies have attempted to follow the development of the proximal part of the wing by analysis of genes that have some expression in the proximal region of the wing disc, e.g., wg or nub, or by the exclusion of markers for notum and wing fates, e.g., teashirt (tsh) and vg, respectively. The identification and analysis is described of a novel marker for proximal wing fate that specifically demarcates the whole of the developing proximal wing tissue, the zinc-finger homeodomain gene zfh-2. In third larval instar (L3) wing discs, Wg is expressed in a stripe along the D/V boundary, forming the wing margin, and in two concentric rings around the wing pouch. In the adult wing, expression of a wg-lacZ reporter indicates that the two rings of wg delimit the proximal wing. The inner (distal) ring runs from the medial costa, through the humeral crossvein to the alula, and the outer (proximal) ring runs from the proximal end of the proximal costa to the axillary cord. A GAL4 insertion within the zfh-2 transcription unit, MS209, (zfh-2MS209) and antisera against Zfh-2 have been used to monitor the expression of zfh-2. In both L3 wing discs and adult wings, Zfh-2 is expressed in a domain that completely overlaps the rings of Wg expression. In L3 wing discs, Zfh-2 does not extend either proximally into the notum or distally into the wing pouch. These observations indicate that, in late stages, Zfh-2 is specifically expressed throughout the developing proximal wing and therefore may be used as a useful marker for proximal wing fate (Whitworth, 2003).

Ectopic expression of Wg can induce zfh-2 only in regions outside of the wing pouch. This suggests that some factor has a repressive effect on zfh-2 in the pouch that cannot be overcome by Wg activation. Genes fundamental to wing blade development may be responsible for this repression. Since Vg expression is restricted to the presumptive wing blade and is required for wing blade development, the effects of ectopic expression of vg on the proximal wing region were examined. Using dpp-GAL4 to direct expression of vg along the A/P boundary represses zfh-2 in the proximal wing region. Endogenous wg expression, monitored with the wg-lacZ reporter, also shows complete repression at the point of intersection. Conversely, in vg1 mutant discs, the Zfh-2 expression domain is expanded into the remnant of the wing pouch and shows a greater overlap with Nub expression than in the wild type. In vg1 discs, much of the wing pouch anlagen fails to develop, and this is accompanied by complete loss of Wg expression at the wing margin; however, the two rings of Wg delimiting the proximal wing are maintained. This suggests that derepression of the zfh-2 domain into the pouch region is not caused by ectopic Wg activity (Whitworth, 2003).

Since the loss of vg does not result in complete derepression of zfh-2, it suggests that another repressor must be acting with vg. Nub is also required for wing blade development. Hypomorphic nub alleles display a severely reduced wing phenotype and a transformation of distal structures into proximal ones. nub2 discs show a complete loss of the inner ring of Wg and an expansion of Wg expression at the wing margin. In nub2 mutant discs, Zfh-2 expression is expanded into the wing pouch, along the line of the wing margin. This indicates two things: (1) that Nub normally acts to repress zfh-2 expression, and thus proximal wing fate, within the wing pouch, and (2) that ectopic zfh-2 is induced where Wg is expressed. Therefore, in an environment of reduced Nub, it can be predicted that ectopic Wg would be able to induce ectopic Zfh-2. To test this, ectopic Wg was expressed in a nub mutant background. As in the nub2 background, Zfh-2 is ectopically induced in the wing pouch along the wing margin . In addition, Zfh-2 can now be detected in the wing pouch along the line of dpp-GAL4, where high levels of Wg are ectopically expressed. This demonstrates that, in an environment of reduced Nub, Zfh-2 expression can be induced wherever Wg is expressed and is no longer restricted from the pouch. It is noted that, whereas Wg expression is expanded at the wing margin in nub discs, where ectopic Wg is induced in a nub background, endogenous Wg is expressed normally at the wing margin; however, the reason for this is unknown (Whitworth, 2003).

In nub discs, vg expression is unaffected, but vg is upregulated by high levels of ectopic Wg. Thus, it appears that the increased levels of Vg are not sufficient to repress Zfh-2 in the absence of Nub when Wg is present at high levels. However, further from the source of ectopic Wg, Zfh-2 is not induced in the nub background, and presumably here, Vg alone can repress Zfh-2. Taken together, these data suggest that zfh-2 expression is regulated by a balance between activation by Wg and repression by a combination of Nub and Vg, acting together or independently. The loss of either Nub or Vg is enough to cause only a partial derepression of zfh-2 in the wing pouch, indicating that alone neither Nub nor Vg is sufficient to completely repress proximal wing fate. However, their combined action, as is the case in the wild type, is able to completely repress zfh-2 expression in the wing pouch. Thus, these factors act to restrict zfh-2 expression to the periphery of the wing disc, thereby defining the distal limit of the proximal wing primordium (Whitworth, 2003).

Differing strategies for the establishment and maintenance of teashirt and homothorax repression in the Drosophila wing

Secreted signaling molecules such as Wingless (Wg) and Decapentaplegic (Dpp) organize positional information along the proximodistal (PD) axis of the Drosophila wing imaginal disc. Responding cells activate different downstream targets depending on the combination and level of these signals and other factors present at the time of signal transduction. Two such factors, teashirt (tsh) and homothorax (hth), are initially co-expressed throughout the entire wing disc, but are later repressed in distal cells, permitting the subsequent elaboration of distal fates. Control of tsh and hth repression is, therefore, crucial for wing development, and plays a role in shaping and sizing the adult appendage. Although both Wg and Dpp participate in this control, their specific contributions remain unclear. In this report, tsh and hth regulation were analyzed in the wing disc; Wg and Dpp act independently as the primary signals for the repression of tsh and hth, respectively. In cells that receive low levels of Dpp, hth repression also requires Vestigial (Vg). Furthermore, although Dpp is required continuously for hth repression throughout development, Wg is only required for the initiation of tsh repression. Instead, the maintenance of tsh repression requires Polycomb group (PcG) mediated gene silencing, which is dispensable for hth repression. Thus, despite their overall similar expression patterns, tsh and hth repression in the wing disc is controlled by two very different mechanisms (Zirin, 2004).

The results suggest that repression of hth in the wing disc occurs only in cells with a history of vg expression and continuous Dpp input. Consistent with this, ectopic vg expression in the medial DH and loss of brk in the lateral DH both result in hth repression. The requirement for vg can be separated into two distinct stages. The first stage occurs in the second instar, when vg expressed at the DV compartment boundary determines which cells are competent to repress hth in response to Dpp signaling. Thus, both vg or Dpp-pathway mutant clones induced at this early stage fail to repress hth (Zirin, 2004).

In the third instar, vg expression is required for hth repression only at the lateral edges of the wing pouch, whereas Dpp signaling is required at all positions along the AP axis. Accordingly, the boundary between the lateral hinge and pouch is dictated by the threshold of Dpp activity that permits the Vg-dependent repression of hth. Wg signal transduction is also required to repress hth in pouch cells far from the AP boundary. However, the requirement for Wg signaling in this part of the wing pouch could be due to its role in vg activation. Alternatively, it is possible that Wg and Vg are independently required to repress hth in these cells (Zirin, 2004).

A model that encompasses these observations is that Vg and Dpp activate another factor that directly represses hth. This factor would be activated in Vg-positive cells by Dpp signaling beginning in the late second instar. By the third instar, high levels of Dpp signaling would be sufficient to maintain its activation, with additional input by Vg and Wg required only at the lateral regions of the pouch. Even further from the source of Dpp, in the lateral hinge, high levels of Brk would prevent expresssion of this factor, thus allowing hth expression despite the presence of Vg. This model is consistent with the idea that Brk is a transcriptional repressor and Vg is a transcriptional activator. There is also precedent for the idea that early vg expression predisposes cells to a particular Dpp response, which was also proposed for the activation of the vgQE (Zirin, 2004).

The above model does not apply to PH cells, which have a distinct response to Dpp signaling. For example, Medadro clones located near the AP boundary of the PH ectopically expressed vg. tsh is an attractive candidate for mediating this switch in response to Dpp signaling, since it is expressed in the PH but not the DH, and is reported to bind Brk in vitro. However, the absence of reagents to readily examine tsh loss-of-function clones prevents this idea from being tested (Zirin, 2004).

Effect of folate inhibitors on vestigial expression

Depletion of the dTMP (deoxythymidine monophosphate) pool by aminopterin, an inhibitor of the enzyme dihydrofolate reductase, or by fluorodeoxyuridine, an inhibitor of thymidylate synthetase, induces nicks in the wings of wild-type flies and a strong vg phenotype in flies heterozygous for a deficiency of the vg locus. Furthermore, specific alterations of the vg locus, caused by intronic insertions, are associated with resistance to these drugs. Depletion of the dTMP pool by aminopterin leads to a decrease in the amount of vg transcript. The insertion of the retrotransposon 412 (giving rise to the vgBG mutant), results in resistance to aminopterin, leading to the formation of a truncated VG transcript that is prematurely terminated in the long terminal repeat of the transposable element; aminopterin also affects the level of this truncated transcript. These results indicate that alterations of the wing by inhibitors of dTMP synthesis are caused by an effect of these drugs on level of the VG transcript. The resistance to such agents observed for the vgBG strain is not due to a qualitatively different effect of this drug on the vg transcript; rather, it is related to the expression of a modified VG protein encoded by a truncated transcript. These results are also compatible with a role for vestigial in modulating cell proliferation (Zider, 1996).

Resistance or sensitivity to aminopterin seems to be associated with a specific alteration in vg gene function. Wild-type and vg mutant strains selected for growth on increasing concentrations of aminopterin display changes in physiological and biochemical parameters such as viability on normal and aminopterin-containing media, duration of development, wing phenotype, dihydrofolate reductase activity, and cross-resistance to fluorodeoxyuridine (FUdR) and to methotrexate. Mechanisms of resistance differ in the wild-type and mutant strains. The vg83b27 mutant, in which the major part of intron 2 of the vg gene is deleted, is associated with a high rate of resistance to FUdR, an inhibitor of thymidylate synthetase. Moreover, vg83b27/vgBG heterozygotes, which are wild type when grown on normal medium, display a strong vg phenotype when grown on aminopterin (Silber, 1993).

Different mechanisms initiate and maintain wingless expression in the Drosophila wing hinge

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

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

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

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

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

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

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

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

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

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

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

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

Actin capping protein α maintains vestigial-expressing cells within the Drosophila wing disc epithelium

Tissue patterning must be translated into morphogenesis through cell shape changes mediated by remodeling of the actin cytoskeleton. <Capping protein α (Cpa) and Capping protein β (Cpb), which prevent extension of the barbed ends of actin filaments, are specifically required in the wing blade primordium of the Drosophila wing disc. cpa or cpb mutant cells in this region, but not in the remainder of the wing disc, are extruded from the epithelium and undergo apoptosis. Excessive actin filament polymerization is not sufficient to explain this phenotype, since loss of Cofilin or Cyclase-associated protein does not cause cell extrusion or death. Misexpression of Vestigial, the transcription factor that specifies the wing blade, both increases cpa transcription and makes cells dependent on cpa for their maintenance in the epithelium. These results suggest that Vestigial specifies the cytoskeletal changes that lead to morphogenesis of the adult wing (Janody, 2006; full text of article).

Functional CPs are a highly conserved αβ heterodimer that bind the barbed ends of actin filaments through the C-terminal regions of both subunits. CPs and the Arp2/3 complex, which promotes filament branching, favor formation of the short highly branched actin filaments required to generate protrusive force at the leading edge of migrating cells. Ena/VASP proteins have the opposite activity, promoting formation of long unbranched parallel bundles of actin filaments. In mouse or Dictyostelium cells, depletion of CPs can cause extensive formation of filopodia and increase the length and bundling of actin filaments, reducing cell motility. Another function of CPs is to cap a short filament of the actin-related protein Arp1 in the Dynactin complex, which is required for Dynein-mediated transport along microtubules (Janody, 2006 and references therein).

Wing blade cells lacking either cpa or cpb are extruded from the epithelium and subsequently die. A number of possible mechanisms might account for this loss of CP mutant cells. As extrusion of cpa mutant cells still occurs in the presence of the apoptotic inhibitors p35 or th, apoptosis is likely to be a secondary consequence of extrusion; extruded cells might undergo apoptosis because they are deprived of anti-apoptotic signals present in their normal niche. In addition, JNK activity is not essential for extrusion; cpa mutant clones were not rescued by expression of a dominant-negative form of basket (bsk), which encodes JNK. However, the possibility cannot be exclude that the p35 or Th inhibitors block apoptosis too late to prevent release of an extrusion signal (Janody, 2006).

The function of capping proteins (CPs) in organelle or vesicle transport is unlikely to explain the extrusion phenotype. CPs are thought to stabilize the barbed end of the Arp1 microfilament in the Dynactin complex, which is required for transport along microtubules. cpa and cpb, like other Dynactin complex subunits, are required to maintain the position of nuclei in Drosophila photoreceptor neurons (Whited, 2004). However, removal of kinesin heavy chain (khc), which counteracts Dynein/Dynactin-based transport, failed to rescue extrusion of cpa mutant cells in the wing disc (Janody, 2006).

The possibility is considered that the cpa phenotype is due to its effect on monomeric G-actin levels rather than on the filamentous actin cytoskeleton. G-actin has been shown to negatively regulate the nuclear localization and activity of Mal, a transcriptional cofactor for SRF, and overexpression of Mal or of its activator diaphanous can cause extrusion and death of wing epithelial cells. However, overactivity of the MAL/dSRF pathway is unlikely to be responsible for extrusion of cpa mutant cells in the wing blade; clones mutant for both cpa and blistered (bs), which encodes Drosophila SRF, are still extruded from the wing epithelium (Janody, 2006).

Extrusion of cpa or cpb mutant cells might be a direct result of defects in the actin cytoskeleton. Consistent with the requirement for CPs to inhibit addition of actin monomers to the fast-growing end of actin filaments, a strong accumulation of actin filaments was observed in cpa mutant clones. However, tsr and capt mutations also induce excessive actin filament polymerization but do not cause cell extrusion. The major function of Tsr (Cofilin) is to promote dissociation of ADP-actin from the pointed end of the filament, while Cpa prevents elongation of the barbed end of each branch and Capt sequesters actin monomers. The phenotypic differences between cpa, tsr and capt might therefore be due to different degrees of branching of the actin network formed in mutant cells. Possibly long unbranched filaments do not provide a framework of sufficient strength to withstand forces that place tension on the cell within the epithelium (Janody, 2006).

Extrusion is associated with dispersion of the adherens junction components Arm and DE-Cad along the lateral membranes. However, this defect is also observed in tsr mutant clones, and mislocalization of adherens junction components caused by overexpression of a dominant form of the polarity gene crumbs does not lead to cell extrusion. Therefore, mislocalization of AJ components is unlikely to be sufficient to cause extrusion of cpa mutant cells. By contrast, expression of dominant-negative Rac1, which prevents actin localization to adherens junctions, induces cell extrusion and death. Thus, another possibility is that CPs may be crucial for linking actin filaments to the membrane. Loss of cpb displaces actin bundles from the cell membrane in Drosophila bristles by increasing the concentration of non-bundle actin snarls and CPs may specify actin filament position in the sarcomere. In the Drosophila wing blade epithelium, loss of CPs might disrupt attachment of the actin cytoskeleton to the adherens junctions, breaking the connection between cells and inducing cell extrusion. The localization of HA-Cpa to apical junctions and the mislocalization of actin filaments throughout cpa mutant cells in the wing blade are consistent with this possibility. Such a role would be restricted to the wing blade, as cpa mutant cells within the notum epithelium accumulate actin filaments only at the apical cell membrane (Janody, 2006).

Surprisingly, it was found that cpa and cpb are required to prevent cell extrusion and death only in the region of the wing disc giving rise to the wing blade, but not in the primordia of the hinge or notum, or in the eye or leg discs. The requirement for cpa depends on the wing blade selector gene Vg; expression of Vg in notum cells is sufficient to induce their extrusion in the absence of cpa. Vg also enhances the transcription of cpa in the wing blade primordium. Taken together, these results imply that patterning genes regulate cytoskeletal properties in order to achieve distinct morphological outcomes (Janody, 2006).

The molecular mechanism that makes Vg-expressing cells dependent on CPs for their maintenance in the epithelium is unknown, although the data support a cell-autonomous target of Vg. One possibility is that Vg promotes the expression or recruitment of an actin filament polymerizing factor. The role of CPs might be to restrict its activity at barbed ends, preventing the formation of a specific actin-based structure that induces loss of cell-cell contacts and extrusion. For example, Vg activates the expression of the type II transmembrane protein Four jointed (Fj), which regulates the activity of the cadherin Fat. Mammalian Fat1 can recruit Ena/VASP proteins, which promote actin polymerization at cell-cell contacts by antagonizing CPs. However, misexpression of fj does not induce extrusion of either wild-type or cpa mutant cells in the notum. DE-cadherin levels are also higher in the wing pouch, but increasing them in the hinge or notum by activating Wg signaling does not cause extrusion of cpa mutant cells. Alternatively, Vg might control the expression of factors that promote the remodeling of cell junctions required for morphogenesis of the wing. Cpa could be required to maintain the connection between cells in the epithelium during these morphogenetic movements (Janody, 2006).

The non-uniform distribution of and requirement for cpa suggests that cytoskeletal organization varies in different regions of the wing disc. It has been observed that lateral wing disc cells had moderately reduced levels of basolateral cortical F-actin. In addition, filopodial extensions called cytonemes are oriented towards the AP and/or DV boundary within the wing pouch, while hinge cells do not extend cytonemes and notum cells radiate short cytonemes in all directions. Changes in cytoskeletal organization have been shown to establish cell affinity boundaries, to control the subcellular localization of transcription factors and to modulate the transport of signaling molecules. Investigating the control of cpa by Vg may help understanding of how and why patterning genes regulate cell architecture. In addition, identifying additional target genes of Vg may illuminate how actin dynamics and changes in intercellular adhesion control the formation of the wing blade (Janody, 2006).

Nab controls the activity of the zinc-finger transcription factors Squeeze and Rotund in Drosophila development. nab expression is regulated by vg

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

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

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

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

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

The Dorsocross T-box transcription factors promote tissue morphogenesis in the Drosophila wing imaginal disc

The Drosophila wing imaginal disc is subdivided into notum, hinge and blade territories during the third larval instar by formation of several deep apical folds. The molecular mechanisms of these subdivisions and the subsequent initiation of morphogenic processes during metamorphosis are poorly understood. This study demonstrates that the Dorsocross (Doc) T-box genes promote the progression of epithelial folds that not only separate the hinge and blade regions of the wing disc but also contribute to metamorphic development by changing cell shapes and bending the wing disc. Doc expression is restricted by two inhibitors, Vestigial and Homothorax, leading to two narrow Doc stripes where the folds separating hinge and blade are forming. Doc mutant clones prevent the lateral extension and deepening of these folds at the larval stage and delay wing disc bending in the early pupal stage. Ectopic Doc expression is sufficient to generate deep apical folds by causing a basolateral redistribution of the apical microtubule web and a shortening of cells. Cells of both the endogenous blade/hinge folds and of folds elicited by ectopic Doc expression express Matrix metalloproteinase 2 (Mmp2). In these folds, integrins and extracellular matrix proteins are depleted. Overexpression of Doc along the blade/hinge folds causes precocious wing disc bending, which can be suppressed by co-expressing MMP2RNAi (Sui, 2012).

Although the morphogenesis of the Drosophila wing disc epithelium has been studied intensively, the molecular mechanisms that tie wing disc subdivision to different fates and subsequent morphogenetic processes remain poorly understood. This study demonstrates that the T-box Doc genes take part in fold formation and promote the metamorphic development of the wing disc by controlling cell shape changes and tissue remodeling (Sui, 2012).

To study the role of Doc in wing disc development, its spatiotemporal expression pattern was analyzed by antibody staining. By simultaneous recording of Doc expression and fold formation, it was found that there was a correlation between Doc expression and blade/hinge (B/H) fold formation and progression. Doc was not activated until the initiation of the B/H folds, which appeared later than the hinge-internal fold in early L3. During early pupal development, the ventral compartment of the wing disc folds underneath the dorsal compartment, leading to a basal apposition of the ventral and dorsal B/H folds. This suggests that Doc plays an important role during these morphogenetic changes (Sui, 2012).

As in the embryo, Doc in the peripheral wing pouch is activated by Dpp. The formation of the double-crescent pattern requires, in addition, Doc repression. Previous studies established that the subdivision of the wing disc into notum, hinge and blade regions is attained by the action of Iro-C, Hth/Tsh and Vg in these territories, respectively. The subdivision of hinge and blade cell fates requires mutual repression between Hth and Vg. The current data revealed that Doc is expressed in the proximal region of low vg expression adjacent to the hth expression domain. Ectopic expression of either vg or hth was sufficient to repress Doc. As with the mutual repression between vg and hth, there was feedback repression between Doc and vg and between Doc and hth, ectopic Doc being sufficient to repress both vg and hth. Therefore, the mutual antagonism between Doc/Vg and Doc/Hth defines Doc expression at the two B/H folds (Sui, 2012).

Doc was not detectable in the wing imaginal disc until the initiation of the B/H folds at ~85 hours AEL. Doc expression coincided in time and space with B/H fold formation. Lack of Doc function inhibited B/H fold extension at the larval stage and caused a delay in wing disc bending at the early pupal stage. When Doc was ectopically expressed in the dpp-Gal4 domain, it was sufficient to generate an apical fold with the same characteristics as the endogenous B/H folds (Sui, 2012).

To explore the mechanism of Doc-controlled cell shape changes and collective cell movement, the distribution of cell adhesion, cytoskeletal and basal membrane proteins was examined. The cell adhesion molecule DE-cadherin is localized at adherens junctions, which maintain the polarized architecture of epithelial cells but limit their movement. Remodeling adhesion to neighboring cells contributes to cell shape changes and cell movement. No obvious effect of Doc overexpression was found on the distribution of the cell adhesion protein E-cadherin. Similarly, the ectopic fold formed at the anterior-posterior boundary of the wing pouch when expression of the Tbx gene omb (bifid -- FlyBase) is reduced, is not associated with an altered distribution of DE-cadherin. The actin and microtubule cytoskeletons coordinately control cell shape. The elongation of columnar epithelial cells requires the assembly of aligned microtubules that form a diffuse microtubule-organizing center at the apical surface. Failure of proper microtubule organization causes columnar cells to round up and shorten along their apicobasal axis. Both in endogenous folds and in folds triggered by ectopic Doc expression, the apical microtubule web was redistributed to a basolateral position, along with an expanded cell diameter and shortened cell height. It is proposed that one mechanism by which Doc causes cell shape changes is by reorganizing the microtubule web (Sui, 2012).

During development, morphogenesis requires the coordination of cell-cell and cell-ECM adhesions, and coordination between molecules involved in these processes is essential for tissue formation and morphogenesis. The degradation of basement membrane barriers is an essential step in cancer invasion. Basement membrane modulation also plays an important role during development. A role for extracellular proteolysis in imaginal disc eversion has long been recognized. Recently, it was shown that integrin-ECM interactions are necessary to maintain the columnar shape of wing disc epithelial cells. This study has shown that overexpression of Doc2 also causes cell shortening and widening of the cell diameter. The levels of integrin and main ECM components were downregulated in Doc-overexpressing cells and MMP expression was ectopically activated in these cells. The same changes were observed in wild-type B/H folds (Sui, 2012).

Members of the MMP family are able to degrade most ECM proteins. Therefore, the changes in the distribution of ECM proteins and integrins might be secondary to increased MMP activity. Mmp2 is downstream of Doc in cell shape control, as overexpressing Doc was sufficient to induce Mmp2 and repressing Doc induced a reduction of Mmp2 in B/H fold cells. Manipulation of the Mmp2 level mimicked the effects of Doc on microtubule web redistribution and fold progression. Repression of Mmp2 by expressing its inhibitor Timp or MMP2RNAi efficiently rescued the abnormal wing disc bending induced by 30A>Doc expression. Taken together, Doc-expressing cells loosen their contacts with the underlying ECM owing to enhanced MMP activity, and changes in integrin and the ECM promote cell shape changes and facilitate subsequent tissue remodeling (Sui, 2012).

In the wing pouch bending process, cells at the dorsal-ventral boundary detach from the basal membrane, shorten and acquire a wedge-shaped morphology. This process is likely to contribute to the force that causes the doubling-up of the flat pouch epithelium. The data show that Doc is required for proper hinge development by deepening the B/H folds of the wing disc. Doc mutant clones covering the B/H fold cells lead to the delay in wing disc bending at the early pupal stage. Overexpression of Doc along the B/H folds elicits precocious wing pouch bending. These data indicate that the B/H folds contribute to the bending process in either a passive or active way. The deepening of the B/H folds could provide more pliability during wing disc bending (Sui, 2012).

Members of the MMP family are involved in tissue remodeling and contribute to cell migration by destroying the ECM and the basement membrane barrier. Elevated MMP levels cause cell shape changes and promote cell mobility by disorganizing the normal tissue architecture. Doc-expressing B/H fold cells have increased MMP levels, causing enhanced ECM degradation. This might facilitate the concerted migration of fold cells. Ectopic Doc expression in the central wing pouch induced abnormal cell migration, both in the plane and out of the plane of the epithelium. Doc could contribute to the wing disc bending process by promoting the movement of B/H fold cells, possibly emulating its role in amnioserosa development (Sui, 2012).

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).

Protein Interactions

Molecular interactions between Vestigial and Scalloped promote wing formation in Drosophila

Scalloped (Sd) and Vestigial (Vg) are each needed for Drosophila wing development. Sd is required for Vg function: altering Sd and Vg cellular levels relative to one another inhibits wing formation. Whereas Vg expression is normally restricted to the wing and haltere imaginal discs, a subset of cells within almost all imaginal discs normally express sd. Thus, when Vg is ectopically produced a supply of Sd is already present in those tissues. Since Sd is required for formation of the normal wing, a test was performed to see if there is a similar requirement for Sd in the formation of Vg-induced ectopic wings. The induction of wing tissue overgrowths by ectopic Vg is partially suppressed in animals heterozygous for a strong viable allele of sd and is completely suppressed in hemizygotes for the same viable allele. These observations demonstrate that Vg requires Sd to transform cells to wing fates. This requirement does not appear to reflect a role for Sd as a downstream effector of Vg function, because the expression of Sd alone, whether under the control of dpp or other promoters, does not induce the formation of ectopic wing tissue. Instead, these observations suggest that Sd and Vg could act in parallel to induce wing cell fates (Simmonds, 1998a).

In vitro, Vg binds directly to both Sd and its human homolog, Transcription Enhancer Factor-1. The interaction domains map to a small region of Vg that is essential for Vg-mediated gene activation and to the carboxy-terminal half of Sd. To map Vg-Sd and Vg-TEF-1 interaction domains, a Far Western blotting assay was used to screen 15 deleted proteins that remove terminal or internal regions of Vg. Only Vg proteins that contain amino acids 279-335 have any significant affinity for Sd. The Vg-Sd interaction appears to be limited to this 56-amino-acid domain, since Sd does not bind to a deleted Vg protein missing only these amino acids, and a construct encoding only this portion of the protein will still bind to Sd. Significantly, a duplicate panel of Vg deletion proteins probed with TEF-1 shows that TEF-1 interacts with Vg via the same protein domain. Affinity columns containing this protein fragment of Vg bind Sd and TEF-1 protein as well as does full-length Vg. This Sd/TEF-1-binding domain of Vg is serine rich and includes putative phosphorylation sites. Phosphorylation of Vg at these sites may potentially modify the Vg-Sd interaction. This region is highly conserved in Vg proteins from Drosophila virilis and Aedes aegypti. Similar sequences also occur in mammalian genomic and expressed sequence tag databases. The amino- and carboxy-terminal portions of Sd were also tested to map which region of Sd interacts with Vg. Previous studies with TEF-1 have demonstrated that regions mediating interaction with cell-specific TIFs are separable from the DNA-binding TEA/ATTS domain. The Vg-binding region of Sd maps to the carboxy-terminal half of the protein, separable from the TEA/ATTS domain in the amino-terminal half. The carboxy-terminal portion of Sd is also highly similar to TEF-1, which is consistent with the observation that TEF-1 binds to Vg with the same affinity as does Sd. To confirm the direct protein-protein interaction between Vg and Sd in a cellular environment, a yeast two-hybrid assay was used. In yeast, Vg and Sd proteins show a specific and reciprocal interaction when fused to either Gal4-binding domain (pGBDU) or Gal4 activation domain (pACT) fusion constructs. Activation of target genes sd and cut by Vg requires the Sd-Vg interaction domain identified in vitro, implying that this activation is Sd dependent. Moreover, a UAS-vg construct with the Sd-binding domain deleted is unable to induce the formation of ectopic wing tissue, consistent with the observation that this induction is sd dependent (Simmonds, 1998a).

A wide variety of studies have suggested that TEA/ATTS domain proteins require tissue-specific transcriptional intermediary factors (TIFs), although relatively little progress has been made toward identifying and characterizing these TIFs . According to the definitions established by the analysis of TEF-1, a TIF for Sd would be expected to bind directly to Sd, to show a restricted pattern of expression, and to be required for Sd function in vivo. These observations presented here, together with the analysis of the coordinate regulation of downstream target genes by Sd and Vg, argue that Vg functions as a tissue-specific TIF for Sd. Although it is possible that Vg interacts with proteins other than Sd, genetic studies argue against this, because all vg mutant phenotypes are shared by sd. In contrast, sd is required for the development of other tissues in which vg is not required. Thus, it is likely that there are other trans-acting factors in Drosophila that interact with Sd. Although the DNA target sequence of Sd is as yet uncharacterized, one target of a yeast TEA/ATTS domain protein (TEC-1) is an element in the TEC-1 promoter. Likewise, in flies, one target of Sd-Vg is likely to be the sd promoter itself, since activation of sd during early development is dependent on Vg, and ectopic Vg induces elevated expression of sd. This suggests a model whereby low levels of Sd expression within wing imaginal discs are elevated in the presence of Vg by positive autoregulation. The dependence on sd of elevated levels of Vg in the wing disc suggests that vg is also a target of positive autoregulation, and direct evidence for this now been obtained. The TEA/ATTS domain protein family is involved in developmental processes as diverse as mammalian neuronal and cardiac muscle development to conidial formation in Aspergillus and pseudohyphal growth of Saccharomyces cerevisiae. Although Vg homologs have not yet been identified in these organisms, genes containing sequences related to the Sd/TEF-1 interaction domain of Vg are conserved in mammals; these genes are thus candidate TIFs for mammalian TEF-1-related proteins. One of these candidate TIFs is expressed in fetal heart tissue, which is intriguing, given that gene-targeted mutations in TEF-1 result in cardiac defects. Future challenges will be to determine whether genes encoding these putative Sd-interacting domains actually function as TIFs for TEF-1 or related proteins, and whether distinct regulatory TIFs have evolved that adapt the transcriptional activities of conserved Sd/TEF-1 homologs to specific functions in different tissues in their respective organisms (Simmonds, 1998a).

Specific interactions between vestigial and scalloped are required to promote wing tissue proliferation in Drosophila melanogaster

The two genes vestigial and scalloped are required for 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).

Binding of the Vestigial co-factor switches the DNA-target selectivity of the Scalloped selector protein

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).

A Vestigial:Scalloped TEA domain chimera rescues the wing phenotype of a scalloped mutation in Drosophila

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).

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

SUMO enhances Vestigial function during wing morphogenesis

The conjugation of the ubiquitin-like protein SUMO (SUMO) to lysine side chains plays widespread roles in the regulation of nuclear protein function. Since little information is available about the roles of SUMO in development, a screen was performed of a collection of chromosomal deficiencies to identify developmental processes regulated by SUMO. Flies heterozygous for a deficiency uncovering vestigial (vg) and mutations in any of several genes encoding components of the SUMO conjugation machinery exhibit severe wing notching. This phenotype is due to an interaction between sumo and vg since it is suppressed by expression of Vg from a transgene, and is also observed in flies doubly heterozygous for vg hypomorphic alleles and sumo. In addition, the ability of Vg to direct the formation of ectopic wings when misexpressed in the eye field is enhanced by simultaneous misexpression of SUMO. In S2 cell transient transfection assays, overexpression of SUMO and the SUMO conjugating enzyme Ubc9, but not a catalytically inactive form of Ubc9, results in sumoylation of Vg and augments the activation of a Vg-responsive reporter. These findings are consistent with the idea that sumoylation stimulates Vg function during wing morphogenesis (Takanaka, 2005).

Thus, sumo loss-of-function mutations act as genetic enhancers of vg loss-of-function mutations. For example, flies doubly heterozygous for recessive hypomorphic vg alleles and recessive sumo or ubc9 alleles exhibit wing notching that is as severe as that exhibited by flies homozygous for the vg mutant alleles. In addition, co-overexpression of SUMO and Vg in the wing or eye significantly exacerbates the phenotype due to overexpression of Vg alone. These findings are consistent with the idea that the SUMO machinery acts to augment Vg function. However, attempts to further confirm this idea by generating homozygous SUMO loss-of-function clones in discs have failed, probably because SUMO is required for cell cycle progression or cell survival (Takanaka, 2005).

Transient transfection assays further support the idea that the sumoylation machinery can potentiate Vg/Sd transactivation. Specifically, cotransfection of Ubc9 and SUMO augments the Vg/Sd dependent activation of the VgQ-luciferase reporter. This effect requires a catalytically active form of Ubc9 strongly suggesting that it is dependent upon sumoylation (Takanaka, 2005).

Attempts were made to map the SUMO acceptor lysine in Vg. There is only a single lysine (Lys 180) that falls in a sequence context with any resemblance to the consensus sumoylation site. Lys 180 falls in the sequence TKEE, while the sumoylation consensus is ψKxE (with ψ signifying a hydrophobic residue). Surprisingly, however, mutagenesis of this lysine to arginine does not significantly reduce the ability of Vg to serve as a target for sumoylation in S2 cells. Apparently, sumoylation occurs at non-consensus sites in Vg. There are multiple precedents for such non-consensus sites in other sumoylation targets (Takanaka, 2005).

The mechanism by which sumoylation renders Vg a more potent activator appears to be distinct from the mechanism by which sumoylation regulates a number of transcription factors. There are numerous examples in which sumoylation of a transcription factor alters the subcellular localization of a factor by directing it to the PODs, resulting in either the activation or inhibition of the factor. However, immunofluorescence studies reveal no evidence for an effect of sumoylation on Vg subcellular localization. There are also numerous examples in which sumoylation upregulates a transcription factor by disrupting an interaction with a negative regulatory factor. Although the existence of a similar negatively acting factor in the case of Vg cannot be ruled out, there is no direct evidence for such a factor. An alternative intriguing possibility, which remains to be explored, is that the sumoylation of Vg enhances transcription by enhancing the interaction between Vg and Sd (Takanaka, 2005).

This study represents one of only a few efforts using genetic approaches to illuminate the biological role of SUMO conjugation in a multicellular organism. Previous genetic analyses have demonstrated a role for the sumoylation machinery in embryonic patterning. For example, in C. elegans embryos, loss of SUMO, Ubc9, or the SUMO activating enzyme results in homeotic transformations apparently due to a role for sumoylation in the function of the Polycomb group protein SOP-2. In Drosophila embryos, loss of Ubc9 results in the deletion of variable numbers of thoracic and anterior abdominal segments, but in this case the relevant sumoylation target is not known. Previous genetic analysis also suggests a role for sumoylation in immune system function as mutations in sumo or ubc9 compromise the Drosophila innate immune response by attenuating the LPS-induced expression of genes encoding anti-microbial peptides such as Cecropin A1. This is consistent with the finding that sumoylation significantly stimulates the function of the Drosophila rel family protein Dorsal since rel family proteins play critical roles in both vertebrate and invertebrate innate immunity. Finally, a recent yeast two-hybrid screen indicates that Dof, a cytoplasmic components of the FGF signaling pathway, interacts with multiple components of the SUMO conjugation pathway. This suggests possible roles for SUMO conjugation in the morphogenetic processes controlled by FGF receptors such as mesodermal and tracheal morphogenesis. Thus, the finding of a likely role for sumoylation in wing development adds to a growing body of evidence suggesting pleiotropic roles for sumoylation in the development and function of multicellular organisms (Takanaka, 2005).

Scalloped interacts with Yorkie, the nuclear effector of the hippo tumor-suppressor pathway in Drosophila: Yki function is required for Sd-Vg activity

In Drosophila, Scalloped (Sd) belongs to a family of evolutionarily conserved proteins characterized by the presence of a TEA/ATTS DNA-binding domain. Sd physically interacts with the product of the vestigial (vg) gene, where the dimer functions as a master gene controlling wing formation. The Vg-SD dimer activates the transcription of several specific wing genes, including sd and vg themselves. The dimer drives cell-cycle progression by inducing expression of the dE2F1 transcription factor, which regulates genes involved in DNA replication and cell-cycle progression. Yorkie (Yki) is a transcriptional coactivator that is the downstream effector of the Hippo signaling pathway, which controls cell proliferation and apoptosis in Drosophila. This study identified Sd as a partner for Yki. Interaction between Yki and Sd increases Sd transcriptional activity both ex vivo in Drosophila S2 cells and in vivo in Drosophila wing discs and promotes Yki nuclear localization. Yki overexpression induces vg and dE2F1 expression, and proliferation induced by Yki or by a dominant-negative form of Fat in wing disc is significantly reduced in a sd hypomorphic mutant context. Contrary to Yki, Sd is not required in all imaginal tissues. This indicates that Yki-Sd interaction acts in a tissue-specific fashion and that other Yki partners must exist (Goulev, 2008).

Yorkie (Yki) encodes a transcriptional coactivator that is the downstream effector of the Salvador-Warts-Hippo signaling pathway. This pathway consists of the two serine-threonine kinases, Hippo and Warts (Wts, also known as Large Tumor Suppressor), the adaptator molecules Salvador (Sav) and Mob as Tumor Suppressor (MATS), the FERM domain proteins Expanded (Ex) and Merlin (Mer), and the protocadherin Fat (Ft). In this pathway, the Hpo kinase phosphorylates Wts, which in turn phosphorylates and inactivates Yki by excluding it from the nucleus. Mer and Es colocalize at the cell cortex and act upstream of Hpo to regulate the activity of Yki. Fat acts upstream of the Hippo signaling cascade by recruiting Ex to the apical plasma membrane and modulating the abundance of Wts. Inactivation of ft, hpo, wts, and sav, inactivation of both ex and mer, and overexpression of yki all lead to increased proliferation and reduced apoptosis, This pathway acts through Yki to regulate the expression of cyclin E (cycE) and the Drosophila inhibitor of apoptosis 1 (diap-1), which are involved in cell-cycle progression and cell-death inhibition, respectively, and the microRNA bantam, which can promote growth and inhibit apoptosis. The Yki peptide shares significant homology with the mammalian Yes Associated Protein 65 (YAP) (31% identity with human YAP). A particular region near the amino terminus of Yki (amino acids 57-114) displays the highest homology with YAP (56.2% identity). This region in YAP corresponds to a domain that binds all mammalian transcription-enhancer factors (TEFs), which are the homologs of the Drosophila Sd protein (Goulev, 2008).

Conservation between mammalian YAP and Yki of the YAP domain that interacts with TEF suggests that Yki might interact with Sd. To investigate this possibility, glutathione S-transferase (GST)-pull-down experiments were conducted. 35S-labeled Yki specifically binds to GST-Sd beads (Goulev, 2008).

These results are in good accordance with a large two-hybrid screen in which Sd was identified as a binding partner for Yki. To refine the Sd domain that binds Yki and the region of Yki involved in the interaction with Sd, the binding of deleted Sd or Yki peptides was examined. Results that were obtained indicate a requirement of the N terminus of Yki and the C terminus domain of Sd. These results implicate Yki as an auxiliary protein that specifically interacts with Sd and demonstrate that this ability to interact is conserved between TEA factors (TEF-1 and Sd) and YAP factors (YAP and Yki) (Goulev, 2008).

To determine whether Sd, which possesses a putative nuclear localization signal (NLS) within the TEA domain, can modify Yki localization, transfection experiments were performed with plasmids expressing Sd fused to FLAG sequence and Yki fused to hemagglutinin (HA) sequence. In Drosophila S2 cells, Yki is mainly cytoplasmic. S2 cells were transfected with Yki and Sd and it was observed that Yki is exclusively nuclear. The nuclear translocation of Yki when Sd is expressed argues in favor of the idea that Sd and Yki interact ex vivo. Co-transfection experiments indicate that Yki interacts in a cellular context with Sd to stimulate the transcriptional activity conferred by Sd and additional experiments indicate that Vg and Yki do not compete for binding to Sd (Goulev, 2008).

It has been shown that the transcriptional activity of Yki is suppressed by coexpression of upstream components of the Hippo pathway. The transcriptional activity of Sd-Vg complex was analyzed when Hpo was overexpressed. Coexpression of GAL4db-Sd and Vg results in a powerful induction of luciferase activity. This induction was suppressed when Hpo was overexpressed. Although it cannot be excluded that Hpo used a mechanism other than Yki inhibition, the most likely expanation for these results is that the Hippo pathway, and thus Yki function, is required for Sd-Vg activity (Goulev, 2008).

It has been proposed that, in the Sd-Vg complex, Vg provides the activating component, and that the binding of Vg to Sd switches the target selectivity of Sd. If it is assumed that the Sd-Vg-Yki complex is able to form into a cell, one attractive explanation for the role of interaction between Yki and Sd is that Yki provides the main activating component and Vg undergoes a conformational change in Sd ensuring accurate DNA target selection (Goulev, 2008).

To explore the possibility that Yki function is evolutionarily conserved, the effects of Yki on TEF transcriptional activity was tested in a HeLa human cell line. Yki, similarly to YAP, stimulate TEF1 transcriptional activity. These results reveal an evolutionarily conserved regulatory mechanism. Interestingly, it has recently been shown that YAP is the effector of the mammalian Hippo pathway. It is amplified in mouse tumor models and has several oncogenic properties. Furthermore, YAP can functionally substitute for Yki in Drosophila. The relevance of the YAP-TEF interaction in this pathway, however, remains to be determined (Goulev, 2008).

To determine whether Yki-Sd interaction regulates Sd transcriptional activity in vivo in Drosophila, a sensor transgene, 'hsp70-GAL4db-sd', expressing a modified Sd protein was used, in which the TEA DNA-binding domain has been replaced by that of the yeast GAL4 DNA-binding domain, under the control of the hsp70 promoter. GAL4::Sd activity, monitored by the UAS-lacZ-responsive reporter transgene, is restricted to the wing pouch, where Sd dimerizes with Vg. A significant reduction in lacZ activity was observed in wing discs heterozygous for the amorphic ykiB5 allele compared to control wing discs. The fact that Sd activity is sensitive to yki dosage in wing imaginal discs supports the idea that Yki is required for Sd transcriptional activity in vivo (Goulev, 2008).

To better understand the functional relationship between Yki and Sd during wing development, genetic interactions between these genes was assessed. The results indicate that reduction of both vg and yki dosage enhances the sd phenotype and support the hypothesis that Yki works together with Sd-Vg during wing development (Goulev, 2008).

It has been shown that binding sites for Sd and Sd-Vg are necessary for regulation of vg expression and thus may regulate by a feedback mechanism vg expression. Results obtained in S2 cells indicate that Yki increases Sd transcriptional activity. This prompted an investigation of whether yki overexpression in imaginal discs enhances Vg-Sd activity, thus regulating vg expression and vgQE activity. yki overexpression was drived by using a UAS-GAL4 system or by flip-out (FLP-FRT) recombination in clones. In both cases, overexpression of yki was shown to induce vg and vgQE expression in a cell-autonomous manner in wing discs and in haltere discs (Goulev, 2008).

Several sets of data argue strongly in favor of an involvement of Vg-Sd in cell proliferation and cell-cycle progression in wing imaginal discs. vgnull cell clones do not proliferate in the wing pouch, whereas they can be recovered in the notum region, where vg is not expressed, indicating that vg is required for the proliferation of wing blade cells or for their survival. In contrast, vg ectopic expression induces wing outgrowths and dE2F1 expression. dE2F1 is the Drosophila homolog of the E2F transcription-factor family that plays a pivotal role during cell-cycle progression in controlling expression of different genes involved in G1-S transition (including cycE, a target gene of the Hippo pathway) and DNA replication. It was found that Yki overexpression with the ptc-GAL4 driver clearly upregulates expression of the dE2F1-LacZ reporter strain in a cell-autonomous manner. This indicates that Yki and Vg-Sd both activate dE2F1 expression and might work together in this process. It can be hypothesized either that Sd-Vg is required for dE2F1 induction by Yki or that Yki induces dE2F1 independently from Vg-Sd and reinforces dE2F1 expression through Sd-Vg induction (Goulev, 2008).

To determine more precisely the relationship between Sd and Yki in cell proliferation, the effect of Yki overexpression was tested in a mutant context for sd. Strong hypomorph alleles of sd are characterized by an absence of wing pouch cells. In contrast, sdETX4 corresponds to a weak allele of sd in which a large number of wing pouch cells are still present. This allele was used to exclude the possibility that the observed effect resulted from an absence of cell proliferation in the wing pouch because of a lack of sd expression. Wing discs overexpressing yki with the en-GAL4 driver exhibit clear increases in size of the posterior region, resulting in massive overgrowth. In contrast, the size of the posterior compartment was almost normal in wing discs from flies hemizygous for sdETX4 and overexpressing yki with the en-GAL4 driver. This result shows that Yki needs Sd to fully induce cell proliferation. Next, it was asked whether Sd-Yki interaction is acting downstream of the Hippo pathway. To test this, use was made of a dominant-negative form of Ft that lacks the intracellular domain (FTΔICD) . Indeed, Ft is an upstream component of the Hippo pathway, and overexpression of FTΔICD induces overgrowth and the expression of Hippo target genes. A significant reduction was observed of induced overgrowth in sdETX4 wing discs. This result strongly suggests that Sd-Yki interaction acts downstream of the Hippo pathway in wing discs (Goulev, 2008).

This paper has addressed the role of Sd-Yki interaction in the wing disc. The role of Sd in other imaginal tissues is poorly understood. Strong alleles of sd are associated with lethality in early larval stage. In the eye disc, clones homozygous for a strong allele of sd die or poorly survived, whereas they are associated with truncated legs when generated in the leg disc, suggesting that Sd should play a role in cell survival in these tissues. In the wing disc, sd clones die in the wing pouch but can be easily recovered in regions that will give rise to the notum, whereas Yki is required in the entire wing disc. This implies that other partners must interact with Yki to promote tissue growth in other structures. However, the induction of vg that was observed in cells overexpressing yki and the results showing that proliferation induced by Yki in the wing disc is significantly reduced in sd mutant context suggest that Sd-Vg is required for Yki function in the wing disc (Goulev, 2008).

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).

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

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