Table of contents

Wingless and determination of wing cell fate

An investigation has been carried out of how Drosophila imaginal disc cells establish and maintain their appendage-specific determined states. Ectopic expression of wingless (wg) induces leg disc cells to activate expression of the wing marker Vestigial (Vg) and to transdetermine to wing cells. Ectopic wg expression non-cell-autonomously induces Vg expression in leg discs; activated Armadillo, a cytosolic transducer of the Wg signal, cell-autonomously induces Vg expression in leg discs, indicating that this Vg expression is directly activated by Wg signaling. Ubiquitous expression of wg in leg discs can induce only dorsal leg disc cells to express Vg and transdetermine to wing. Dorsal leg disc cells normally express high levels of decapentaplegic (dpp) and its downstream target, optomotor-blind (omb). Ectopic omb expression is sufficient to dorsalize leg cells but is not sufficient to induce transdetermination to wing. Dorsalization of ventral leg disc cells, through targeted expression of either dpp or omb, is sufficient to allow wg to induce Vg expression and wing fate. Leg cells dorsalized by omb are competent to transdetermine to wing, as shown when wingless is expressed ubiquitously. Under these circumstances Vg is expressed in both dorsal and ventral regions. A non-autonomous effect of omb was observed on wg-induced Vg expression, suggesting that omb induces the expression of another signal that acts with wg to induce Vg expression (Maves, 1998).

High levels of dpp expression, which are both necessary and sufficient for dorsal leg development, are required for wg-induced transdetermination. Thus, dpp and omb promote both dorsal leg cell fate as well as transdetermination-competent leg disc cells. In leg discs, antagonist interactions between Wg and Dpp normally prevent wg expression from overlapping with high levels of dpp expresssion and with omb expression. It is thought that the interaction between Wg and Dpp in transdetermination mimics the interaction between Wg and Dpp normally used to establish the wing disc primordium. It is suggested that Wg signaling directly activates the vg boundary enhancer during wing disc development, presumably in conjunction with Notch signaling through Suppressor of Hairless. Taken together, these results show that the Wg and Dpp signaling pathways cooperate to induce Vg expression and leg-to-wing transdetermination. A specific vg regulatory element, the vg boundary enhancer, is required for transdetermination. It is proposed that an interaction between Wg and Dpp signaling can explain why leg disc cells transdetermine to wing and that these results have implications for normal leg and wing development (Maves, 1998).

The expression of vestigial during wing development is regulated through two enhancers: the second intron or boundary enhancer (vgBE), and the fourth intron quadrant enhancer (vgQE). These names reflect the patterns of expression directed by these regulatory regions: vgBE produces a thin stripe over the prospective wing margin, and vgQE produces a pattern in four quadrants that are complementary to the vgBE and which fill in the developing wing blade. Both, vgBE and vgQE, act as integrators of signaling systems that drive wing development and, in this manner, these regulatory regions determine the tempo and the mode of wing development (Klein, 1999 and references).

Wingless is shown to act synergistically with Vestigial to promote the activity of the vgQE. It is clear that the activity of the vgBE is required for the activation of the vgQE, but little is known about how this interaction takes place. The observation that vgQE is activated in Ser mutant discs in which vg is expressed ectopically suggests that the activation of the vgQE is mediated by Notch signaling through the activity of Vestigial on the vgQE. However, in this experiment, expression of wg is also restored and this raises the possibility that activation of the vgQE is mediated through the presumed organizing activity of Wingless. This is probably not the case since ectopic expression of wg alone does not lead to the activation of the vgQE in Ser mutants. The inability of Wingless to activate the vgQE in this experiment is not due to a general insensitivity of the cells to Wingless signaling, since ectopic wg is always capable of inducing hinge fate ectopically. These results clearly demonstrate a requirement for vg in the activation of the vgQE. However, clonal analysis has shown that vg acts cell autonomously and therefore, in the wild type, the non-autonomous effects of Notch on the vgQE must be mediated by another, diffusible molecule(s), which is under control of Notch signaling. A number of studies suggest that Wingless has an influence on the expression of vg in the wing pouch and that its expression at the wing margin is under control of Notch signaling. Therefore, it is possible that Wingless is mediating the non-autonomous effect of Notch on the vgQE. It might be that Wingless acts by acting on the vgQE to elevate and maintain the levels of vg expression that had been induced by Vestigial through the vgBE. In agreement with this proposal, it is found that the activity of the vgQE is elevated in response to the ectopic expression of wg and that expression of a dominant negative Wingless molecule suppresses the activity of the vgQE and reduces the size of the wing pouch. Altogether these results suggest that the upregulation of vg expression in response to wg is mediated by the vgQE. This conclusion is supported by the existence of several putative TCF-1 binding sites in the vgQE. However, the effects of Wingless are always restricted to the normal domain of vg expression, in agreement with the results presented above that Wingless alone is not sufficient to initiate ectopic expression of vg through the vgQE. These effects are likely to be mediated by Vestigial itself. The role of Wingless on this regulation is to maintain and modulate the levels of activity of the vgQE. Consistent with the conclusion that Wingless enhances the effects of Vestigial, coexpression of Wingless and Vestigial, which leads to the ectopic induction of pouch and hinge fate in the notal regions, triggers a widespread and stable expression of the vgQE throughout the wing disc (Klein, 1999).

The interactions between Dpp and Wingless during wing development take place at the level of the vgQE and require the vg gene product. Ectopic expression of wg alone leads to ectopic expression of the vgQE in the posterior region of the domain of vgBE expression, over a region in which dpp is normally expressed. Expression of dpp under the same conditions leads to an enlargement of the wing blade along the AP axis, with the vgBE as a reference but without extending it into the notum. Coexpression of wg and dpp leads to a pronounced extension of the wing blade toward the notum. In some instances, the development of up to eight wing blades occurs, all with a common origin over a point ventral to the notum. All these wings express the vgQE and each of them has a defined margin. It is possible that these multiple wings are produced by the splitting of an initial primordium. In the wild type, the activity of the vgQE is initiated at the intersection of wg and dpp expression and radiates from this focus. The results presented here indicate that this overlap determines important parameters of the morphogenesis of the wing. For example, it is possible that the distance from this focus -- perhaps defined by the range of diffusion of the two molecules -- defines a threshold that contributes significantly to the determination of the size and shape of the wing blade. By tampering with these overlaps, one can alter the shape of the wing or, by generating a series of them, trigger the development of multiple wings from one primordium (Klein, 1999).

The contributions of the DV and AP axes to the patterning of the wing are becoming clear. However, little is still known about what triggers the foci of proliferation that drive the growth of the wing blade or how the interactions described here control the final size of the wing. The results presented here suggest a more indirect role of wg and vg in cell proliferation than previously suggested. Although the loss of function of each of these genes has a significant influence on the cell proliferation in the wing, the overexpression of both does not lead to the increase of the wing pouch of the late third instar. This suggests a more permissive role for the two genes in cell proliferation, probably to maintain the identity of the wing pouch. Studies that suggest that the proliferation is induced by local cues and is a cell autonomous property support this conclusion (Klein, 1999 and references).

The wing imaginal disc comprises the primordia of the adult wing and the dorsal thoracic body wall. During second larval instar, the wing disc is subdivided into distinct domains that correspond to the presumptive wing and body wall. Early activity of the signaling protein Wingless has been implicated in the specification of the wing primordium. Wingless mutants can produce animals in which the wing is replaced by a duplication of thoracic structures. Specification of wing fate has been visualized by expression of the POU-homeodomain protein Nubbin in the presumptive wing territory and by repression of the homeodomain protein Homothorax. Repression of the zinc-finger transcription factor Teashirt (Tsh) is the earliest event in wing specification. Repression of Tsh by the combined action of Wingless and Decapentaplegic is required for wing pouch formation and for subsequent repression of Hth. Thus, repression of Tsh defines the presumptive wing earlier in development than repression of Hth, which must therefore be considered a secondary event (Wu, 2002).

To understand the early specification of the wing field within the imaginal disc the patterns of expression of Wg, Tsh, Hth, Vestigial and Nubbin were examined at very early stages. Vestigial is expressed in every cell of the wing disc primordium in the embryo. In wing discs from early and mid second instar larvae, Vestigial expression has begun to retract from the presumptive notum, but is expressed in both cell layers in the ventral part of the disc. At this stage, Hth is expressed in every cell. By contrast, Tsh is expressed in the presumptive body wall but has already begun to be repressed in the future wing pouch (Wu, 2002).

These early gene expression patterns are best visualized by comparing the horizontal optical sections with optical cross sections that cross the two cell layers. Until mid third instar the epithelium is cuboidal. Later, the peripodial layer becomes a very thin squamous epithelium and the other layer becomes a thick pseudostratified epithelium, which is highly folded. By mid second instar, the patterns have resolved further and the subdivisions become more clear. Tsh is repressed in the presumptive wing territory. Vestigial is expressed in a larger area, foreshadowing its expression along the DV boundary in the body wall, as well as in the wing. Hth continues to be expressed in all cells. These observations indicate that repression of Tsh and restriction of Vestigial expression are the earliest markers of wing specification. These changes occur well before Hth repression is evident. Although Tsh and Vestigial end up in approximately reciprocal patterns by late second instar, the dynamics of their expression does not suggest that they regulate each other's expression. At the earliest stages they overlap considerably. Tsh then begins to be repressed in a small subset of the region where Vestigial is robustly expressed (Wu, 2002).

Wg activity has been implicated in specification of the wing field in the disc. The levels of Wg protein expression are too low to be detected by antibody labeling during the second instar, therefore, use was made of a wg-lacZ reporter gene to visualize wg gene expression. wg-lacZ is expressed in the region of the wing disc where Tsh is repressed, during mid second instar. Repression of Tsh occurs before expression of Nubbin can be detected. By late second instar, signaling between dorsal and ventral compartments induces Wg and Vestigial expression in cells adjacent to the DV boundary. At this stage Nubbin begins to be faintly detectable within the domain of Tsh repression. By early third instar, Vestigial is expressed in a band centered on the DV boundary that extends from the wing primordium into the body wall. Repression of Hth begins in the presumptive wing and Nubbin expression broadens. Hth and Nubbin still overlap at this stage. Nubbin expression becomes stronger in the presumptive wing pouch during mid and late third instar (Wu, 2002).

As Hth expression retracts from the wing pouch, it resolves into three distinct domains in the proximal region. Two of the Hth domains overlap with the proximal rings of Wg expression that are observed in the presumptive wing hinge region. Hth is regulated by Wg at these late stages. Both rings of Wg expression are distal to the Tsh expression domain. The most proximal ring of Hth, which is regulated by secreted Wg, overlaps the edge of the Tsh domain. At this stage, Vestigial and Nubbin expression are centered on the stripe of Wg expression at the DV boundary. Vestigial expression is limited to the distal wing pouch and does not extend as far as the first ring of Hth expression. Nubbin extends more proximally, overlapping the first and second rings of Hth and the first ring of Wg expression. Tsh expression is proximal to the outer ring of Wg expression, which runs through the base of the wing hinge. Thus, the border of Tsh expression coincides with border between wing and the body wall, whereas Hth is expressed in rings in the wing hinge as well as more proximally in the notum (Wu, 2002).

Wing patterning can be subdivided into at least three discrete stages. The earliest observable changes in gene expression patterns that indicate specification of the wing field are repression of Tsh and retraction of Vestigial expression. Wg signaling represses Tsh expression at this stage. Dpp contributes to repression of Tsh. At present, the time at which Dpp acts can only be addressed directly by comparing the effects of clones induced at different stages. Clones induced in late second or early third instar are ineffective, whereas clones induced in early second instar are able to repress Tsh. Interestingly, Wg and Dpp cooperate to repress Hth in the wing pouch, even though this occurs somewhat later that repression of Tsh. These observations support the view that Wg and Dpp act in conjunction to specify the wing field in a manner analogous to the way they cooperate in leg patterning (Wu, 2002).

The combined actions of Wg and Dpp are responsible for both dorsoventral and proximodistal patterning of the leg. The wing makes use of a different strategy from the leg to control DV patterning and outgrowth from the body wall. After the wing field is established, interaction between D and V cells leads to localized activation of Notch signaling, initially in ventral cells. Subsequently Notch is activated in cells on both sides of the DV boundary. Three separate mechanisms have been implicated in limiting Notch activation to cells adjacent to the boundary. Localized Notch activity turns on the vestigial boundary enhancer and Wg expression in cells adjacent to the DV boundary. Subsequently, the long-range Wg morphogen gradient regulates downstream genes, including Achaete-scute, Dll, the vestigial quadrant enhancer, the Wg receptor Fz2 and possibly other genes, to control wing development. Wingless also acts at this stage to regulate growth in the wing hinge and in the wing pouch. This is mediated in part through Vestigial and its co-factor Scalloped, that are both required for cell survival in the wing pouch (Wu, 2002 and references therein).

Evidence has been presented that repression of Tsh in the earliest phase of wing specification appears to be required for subsequent Notch-dependent induction of Wg at the DV boundary. Clones of cells lacking Hth activity cause outgrowth of extra wing tissue along the DV boundary. The results presented here suggest that this is unlikely to be due to an early role of Hth in specification of the size of the wing field because Hth is expressed in the early presumptive wing well after Tsh is repressed. Instead, Hth appears to act in the second stage in conjunction with Tsh to limit the region in which Notch can activate Wg at the DV boundary. Wg expression at the DV boundary extends proximally into the domain of Hth expression in the anterior wing hinge but does not extend into the Tsh domain. In ectopic expression experiments, Hth has a limited ability to repress Notch-dependent activation of Wg on its own, but is able to do so when co-expressed with Tsh. These observations support the view that Hth cooperates with Tsh during later stages to repress Wg activation. This does not exclude a role for Hth as a co-factor in conjunction with Tsh at earlier stages, but if so, the positional information would seem to derive from regulation of Tsh expression (Wu, 2002).

Interestingly, Hth and Tsh can also repress the vestigial quadrant enhancer, which depends on Wg and Dpp signaling in phase 3. Homothorax, Tsh and Vestigial appear to form a loop of mutual repression at this stage, since Vestigial also represses expression of Hth. Together, these observations suggest that Wg and Dpp have a complex regulatory interaction with Hth. Their activities repress it in the pouch, perhaps through activation of Vestigial and Scalloped. At the same time, the outer rings of Wg expression are required for Hth expression in the wing hinge. It is suggested that regulation of Hth may be secondary to regulation of Tsh in specification of the wing field (Wu, 2002).

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 (Fortini, 1991). 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).

wg expression, monitored by a lacZ reporter, is initiated in the early second instar (L2) in an approximately anterior-ventral domain. A lacZ reporter driven by zfh-2MS209 is expressed in a very similar pattern. To determine the extent of coexpression of Zfh-2 and Wg, early L2 discs were examined with anti- Zfh-2 and anti-Wg antisera. Zfh-2 is expressed at this stage in a pattern that directly overlaps with Wg. It is apparent that, although the wg-lacZ reporter gene shows wg expression induced in a narrow domain, the protein can be detected at some distance outside of this region. This is a measure of the mobility of Wg protein. Consistent with this, Zfh-2 nuclear expression is at high levels in the wedge-like domain of wg-lacZ, but is also detectable away from this region at lower levels. As development proceeds, Zfh-2 quickly expands to cover the whole of the ventral portion of the wing disc, accompanying the expansion of the Wg domain. The expression of Wg at this stage is proposed to determine the differentiation of the presumptive 'wing primordium' as opposed to notum. However, since Zfh-2 is also widely expressed at this time, it suggests that the 'wing primordium' has not been further subdivided into proximal or distal domains. At the onset of L3, Zfh-2 begins to decline in the center of the disc, suggesting differentiation of more distal fates here. At this time, Wg is still expressed throughout the 'wing primordium' but becomes upregulated at the D/V boundary, where it plays a central role in defining the wing margin. This is also the time when the vg quadrant enhancer (vgQE) is activated on either side of the D/V boundary, marking the establishment of the wing pouch. During mid-L3, the pattern of Wg expression is refined further, becoming upregulated at the periphery of the wing pouch and at the D/V boundary, the presumptive wing margin. Zfh-2 is also refined and is now only present in a ring around the wing pouch overlapping the rings of Wg (Whitworth, 2003).

Taken together, these observations suggest that, at the beginning of L2, the wing imaginal disc is divided into the presumptive notum and the appendage or 'wing' primordium, and that the wing primordium is undifferentiated with respect to the proximodistal axis. This is supported by reports that tsh is also expressed throughout L2 wing discs, but is later restricted to the presumptive notum and hinge regions, and also that vgQE is not yet activated to differentiate the wing pouch. From the dynamic expression pattern of the proximal wing marker zfh-2, it appears that the elaboration of distal elements within the disc, marked by the disappearance of Zfh-2 and concomitant activation of the vgQE, is initiated at the start of L3 at the center of the wing disc where the A/P and D/V boundaries intersect. This suggests that the proximal wing and wing pouch differentiate sequentially, the distal wing pouch being induced later than the already established proximal wing. The early expression of zfh-2 indicates that it is a specific marker for proximal fate (Whitworth, 2003).

zfh-2MS209 homozygotes, while poorly viable, display a recessive proximal wing phenotype. The phenotype consists of deletion of both anterior and posterior wing structures, including the medial costa, parts of the radius, and the alula. The P-element insertion in zfh-2MS209 was mapped to the first intron of the zfh-2 transcription unit. Evidence that the insertion causes the wing phenotype is twofold; the phenotype can be reverted by loss of the P-element, and independently isolated Pelement insertions in the same region of the zfh-2 gene have similar phenotypes. M390.R and M707.R were isolated in a fourth chromosome P-element screen and, like zfh-2MS209, these insertions are poorly viable. Homozygous escapers and transheterozygotes with zfh-2MS209 display similar proximal wing phenotypes (Whitworth, 2003).

When examined for wg expression, the L3 wing discs of zfh-2MS209 homozygotes show a loss of tissue between the rings of wg expression that demarcate the proximal wing; there are no effects on the expression of wing pouch markers, such as nub or vg, or the notum marker tsh. Although null mutations in zfh-2 have not been isolated, the fact that at least three independently isolated P-element insertions show similar phenotypes strongly suggests that, consistent with its expression pattern, zfh-2 is required for the correct development of the proximal wing (Whitworth, 2003).

The overlap between Zfh-2 and Wg throughout the larval stages suggests that zfh-2 may be activated by Wg signalling. In order to test this, the effect of ectopic Wg expression on zfh-2 expression was analyzed. dpp-GAL4 was used to drive the expression of a UAS-wg construct along the A/P boundary in all domains along the P/D axis. Under these conditions, Zfh-2 shows a broad expansion into the presumptive notum region but no ectopic expression in the wing pouch. This indicates that ectopic Wg can activate zfh-2 at a distance from its site of expression (Whitworth, 2003).

In the wild type proximal wing disc, Nub overlaps the Zfh-2 domain at the inner ring of Wg. This situation is recapitulated when ectopic Wg is driven by dpp-GAL4, since ectopic Nub is only detected in regions of high Wg expression. This indicates that ectopic Wg is inducing a response similar to the inner ring and suggests that the region expressing ectopic Zfh-2 is now differentiating as proximal wing (Whitworth, 2003).

To assess whether the cells expressing ectopic Zfh-2 have altered their fate, the expression of the notum marker Tsh was analyzed. Tsh is completely repressed throughout the region of ectopic Zfh-2, indicating that cells are no longer fated as notum. Since Wg is an important factor in the development of wing blade, the expression of a wing blade marker, Vg, was also examined. Vg shows no expansion into more proximal regions and is still restricted to the wing pouch (Whitworth, 2003).

These observations support the idea that Wg is able to direct the differentiation of proximal wing fate at the expense of notum. This can also be inferred from an examination of the phenotype of pharate adults of genotype dpp-GAL4/UAS-wg. An outgrowth of tissue is seen with characteristic proximal wing sclerites and a concomitant loss of macrochaete and scutellum normally associated with the notum (Whitworth, 2003).

This experiment to induce ectopic proximal wing was carried out in such a way that the ectopic Wg was expressed in a pattern that intersects the endogenous domain of Wg expression and results in a continuous region of Wg expression. The observed effects could therefore be interpreted as directed overgrowth of the endogenous proximal wing and not differentiation of proximal wing de novo. This is supported by observations that ectopic expression of Wg in the proximal wing anlagen causes disc overgrowth and consequently overgrows proximal wing tissue. In view of this, attempts were made to reproduce the effects of ectopic Wg in a manner that was discontinuous with the endogenous wg domain. To achieve this, clones of wg-expressing cells were induced that were contained entirely within the notal region, outside of the endogenous proximal wing. To prevent diffusion of ectopic Wg, a construct (UAS-Nrt-flu-wg) was used that directs the expression of a membrane-tethered form of Wg marked with a Flu epitope tag. The colocalization of Nrt-Wg bound to the cell surface and GAL4-expressing cells confirms two things: (1) that the only cells expressing GAL4 induce expression of the UAS construct; (2) that the Flu epitope marker is not detectable beyond the site of expression, indicating that the Flu/Wg hybrid molecule is membrane-bound and not detectably diffusible. When stained to reveal Zfh-2, it can be clearly see that Zfh-2 is induced at a distance of several cell diameters from the site of Nrt- Flu-Wg expression, producing a large zone of Zfh-2-expressing cells surrounded by an epithelial fold. This observation is surprising considering that Wg protein is believed to be tethered to the cell membrane. In the wing pouch, the same construct elicits a Wg signal response only in the expressing cell and its immediate neighbors. Although the nature of the long-range induction cannot be explained at present, this does confirm that ectopically expressed Wg is able to induce the expression of Zfh-2 and therefore drive differentiation of proximal wing fate (Whitworth, 2003).

Taken together, these results show Wg is sufficient to direct the differentiation of proximal wing fate. Furthermore, Wg can only induce ectopic Zfh-2, and thereby proximal wing fate, in the more proximal notum tissue and not in the more distal wing pouch (Whitworth, 2003).

To determine whether Wg is required for zfh-2 expression and how this changes through development, a number of methods were employed to remove Wg function at different developmental stages. The temperature-sensitive allele wgIL114 was used in trans to a wg-lacZ insertion line to create a conditional null mutant. When larvae were moved to the restrictive temperature just prior to L2, Zfh-2 expression was no longer detected in the wing primordium. This indicates that Wg function is required at least for initiation of zfh-2 expression in the L2 wing disc. Wg signal transduction can also be antagonized by the expression of a dominant negative TCF (DN-TCF), a component of the Wg signalling pathway. dpp-GAL4 was used to drive expression of DNTCF along the A/P boundary from early larval stages. zfh-2 fails to be activated in the presence of DN-TCF, even into L3. This further supports the findings that Wg signal transduction is absolutely required for initiation of zfh-2 expression during L2 (Whitworth, 2003).

However, when Wg signalling is removed later in L3, under all experimental conditions tested, no effect on zfh-2 expression is seen. Large wg null clones or the expression of a dominant negative form of wg during L3 shows no detectable reduction in Zfh-2 levels. Similarly, no loss of Zfh-2 is observed with clonal expression of DN-TCF. This shows that, after activation in the L2, Wg activity is no longer required during L3 for the maintenance of zfh-2 expression (Whitworth, 2003).

Taken together, these results show that the regulation of zfh-2 by Wg is temporally dynamic. Although Wg is required early to activate zfh-2, when both are extensively coexpressed, Wg appears not to be required later to maintain zfh-2 expression. This raises the possibility that, once activated, zfh-2 might regulate its own expression by an unknown mechanism. This interpretation would also mean that the downstream response to Wg signal is temporally dynamic, since it appears that one set of genes, e.g., those required to determine proximal wing fate, is activated early and later becomes independent of Wg, and then another set of genes is in turn activated, e.g., those delimiting the wing blade (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).

Recent work has indicated that the homeobox gene homothorax (hth) is required for the correct development of the proximal wing by both upregulating Wg expression in the proximal wing and limiting the area of wing blade differentiation. Since loss of Hth function in the proximal wing leads to a dramatic reduction in the level of Wg expression, attempts were made to determine whether Hth is also required for regulation of Zfh-2 expression. In hth- clones, neither the expression pattern nor the level of Zfh-2 is altered compared with neighboring wild type tissue. This is consistent with the observation that late removal of wg does not affect the expression of zfh-2. Similarly, ectopic expression of Hth shows no effect on zfh-2 expression. These data suggest that Hth does not play a role in establishing or regulating the determination of proximal wing fate, since no change in the expression of Zfh-2 was observed. Thus, it appears that the prime functions of Hth in the proximal wing are to maintain Wg expression and define the limits of the wing pouch (Whitworth, 2003).

Thus the expression domain of zfh-2 is a discrete marker for proximal wing tissue throughout larval development. The zfh-2 gene is located on the fourth chromosome and encodes a large Zinc Finger Homeodomain protein. It is expressed in the CNS throughout embryonic and larval life (Lai, 1991; Lundell and Hirsh, 1992) and specifically in the wing imaginal disc. A set of P-elements inserted in the 5' region of the gene has been identified; one of these, zfh-2MS209, expresses GAL4 in the wing imaginal disc in a pattern indistinguishable from anti-Zfh-2 antisera. Significantly, zfh-2MS209 homozygotes and transheterozygotes between zfh-2MS209 and two other independently isolated P-elements (M390.R and M707.R) have a proximal wing deletion phenotype, suggesting that it is required for proximal wing development. Using zfh-2 as a specific marker for proximal wing fate, it has been shown that the P/D axis of the wing imaginal disc is sequentially elaborated from proximal notum to distal wing blade in a temporal sequence that is mediated by a set of differential responses to the signalling molecule Wg (Whitworth, 2003).

At the beginning of the second larval instar, the wing imaginal disc expresses markers of proximal fate, hth and tsh, in the entire anlage. During early L2, the expression of wg and zfh-2 is initiated in an anterior-ventral wedge pattern. The data indicate that Wg function is required to activate zfh-2 expression at this stage, since early removal of Wg function leads to a simultaneous loss of zfh-2 expression. As development proceeds, wg and zfh-2 expression rapidly expands filling the whole of the ventral portion of the wing disc by the end of the second instar. Concomitant with the expansion of wg and zfh-2, both hth and tsh become repressed in the ventral portion of the disc. This transition appears to mark the first P-D differentiation of the wing disc into appendage and notum. However, since zfh-2 is expressed in the entire wing anlage at this time, it is believed that the appendage has not differentiated proximal wing and blade. Around the L2-L3 transition, the wing blade markers nub and vgQE are activated by the combined activity of the Wg and N signalling pathways. Nub and Vg, acting together or independently, repress zfh-2 expression in the center of the disc. This marks the second phase of P-D elaboration where the appendage anlage is split into proximal wing and blade. It is noted that, at this time, hth and tsh remain coexpressed in the notum, where zfh-2 is not expressed. The pattern of zfh-2 expression at this stage suggests that it is still influenced by Wg signalling since it remains restricted to areas of high Wg expression. During L3, the division of the wing disc into three distinct domains is maintained and refined as the individual domains undergo their characteristic patterning. At this time, Hth and Wg are upregulated in the proximal wing anlage, where their activities are interdependent, while zfh-2 expression persists but becomes independent of Wg activity (Whitworth, 2003).

These data further support a qualitative difference in the activity of Wg in the proximal wing compared with wing blade. In addition to the activation of different effectors, previous investigations have shown that ectopic Wg expression in the proximal wing causes large overgrowth of proximal tissue, but similar overexpression in the wing blade produces no overgrowth. This indicates that a different mitogenic response to Wg signalling is activated in the wing pouch compared with proximal wing (Whitworth, 2003).

Therefore, these observations suggest a model in which the wing disc is sequentially partitioned in a proximal to distal direction: notum, proximal wing, and finally wing blade. This view of temporal specification of PD identities is supported by transplantation experiments where L2 wing disc fragments can only differentiate proximal wing structures, whereas L3 disc fragments can produce wing blade elements. In support of the more general applicability of these findings, a study of PD patterning in the Drosophila leg has shown that Wg and Dpp act early to establish the PD axis, but later are not required. These data appear strikingly similar to the results presented in this study and suggest an important common mechanism for PD axis elaboration that has previously been unappreciated. This investigation also serves to emphasize the importance of considering the development of the imaginal disc as an extremely dynamic field, with respect to rapid changes in both size and patterning (Whitworth, 2003).

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).

In the course of a screen for mutations affecting the PD axis of the wing, an allele of the Drosophila Smad4 homolog Med was isolated. Like other Dpp pathway mutations, Medadro clones located in the wing pouch cell autonomously de-repress hth. This is evident even in late-induced clones, demonstrating the continuous role of Dpp signaling in shaping the wing blade/hinge subdivision during larval development. By contrast, no de-repression of tsh was detected resulting from any manipulations of the Dpp pathway (Zirin, 2004).

The ability of ectopic Dpp activity to repress tsh in early-induced proximal clones was interpreted to suggest that Wg and Dpp cooperate to repress tsh in the early pouch. However, because Dpp is dispensable for tsh repression, this model must be an over-simplification. It is concluded that Wg, not Dpp, must be considered the primary repressor of tsh in the wing. The lack of synergy between the two pathways is reminiscent of the regulation of Dll, which is activated in the leg by the combined activities of Wg and Dpp, but requires only Wg for its expression in the wing pouch (Zirin, 2004).

In the absence of Dpp signaling, wing pouch cells co-express hth, nub and Dll, but not tsh. This combination of factors is normally found only in the distal hinge (DH), suggesting a transformation from pouch to DH when the Dpp pathway is compromised. The expression of the Iro-C genes, normally restricted to the notum, extends to the distal limit of the tsh domain in dpp mutant discs, leading to the hypothesis that Dpp signaling is essential for the separation of wing and body wall. However, because loss of Dpp signaling transforms wing pouch to DH, an alternative view is proposed in which Dpp further divides an already extant appendage/trunk subdivision by repression of hth in the pouch and Iro-C in the proximal hinge (PH). According to this proposal, the distal limit of tsh expression, initiated by early Wg expression and maintained by PcG silencing, denotes the boundary between the appendage and the body (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).

If tsh repression marks a fundamental subdivision along the PD axis, then the maintenance of tsh repression is crucial for the maintenance of this subdivision. Although Wg signaling is clearly required for the initiation of tsh repression, it is dispensable by the time the DV margin is established. The elbow-no ocelli (el-noc) gene complex has been identified as a target of both Dpp and Wg that is necessary for tsh repression in the wing. However, tsh de-repression is observed in el-noc loss-of-function clones induced only in first or early second instar larvae. tsh repression must therefore be maintained by a wg- and el-noc-independent mechanism. The possibility of redundant Wg- and Dpp-mediated tsh repression was ruled out by making clones doubly mutant for both signaling pathways. Such clones upregulate hth, and lose Dll expression, but show no ectopic tsh expression. Thus, neither of the two major long-range signaling systems of the wing pouch is involved in the maintenance of tsh repression (Zirin, 2004).

Instead, analysis of Su(z)12daed and Pc mutant clones indicates that the maintenance of tsh repression is mediated by a heritable silencing mechanism. By inducing Pc mutant clones in third instar discs, it was demonstrated that this ectopic tsh expression represents a failure to maintain rather than a failure to establish repression. The weak hth levels observed in some PcG mutant clones may be due to the ability of tsh to upregulate hth. This interpretation is supported by the fact that hth expression is seen only in large Pc mutant clones, and only in cells expressing the highest levels of tsh. The general absence of hth expression in PcG mutant clones, together with the ectopic hth expression resulting from late Dpp pathway disruption, points to the need for continuous signaling input to maintain hth repression. By contrast, tsh requires PcG gene activity, but not continuous Wg or Dpp input, to maintain its repression during the third instar (Zirin, 2004).

At this stage the possibility that the affects of PcG mutant clones on tsh repression described here are indirectly due to the de-repression of another factor cannot be ruled out. It is suggested that this is unlikely, however, in part because the spatial distribution of tsh de-repression in PcG mutant clones differs significantly from reports of Hox gene de-repression. Additionally, the ectopic tsh expression in Pc mutant clones is repressible by Nrt-Wg, indicating that tsh is still subject to regulation by Wg signaling (Zirin, 2004).

In the embryo, Hox genes are repressed in some segments by the transient presence of the gap genes. This initial repression is then maintained by the PcG proteins through a heritable silencing mechanism. A model of tsh repression follows this general outline, whereby Wg signaling is required transiently to establish the limits of the tsh expression domain. PcG proteins subsequently maintain the tsh silenced state, while the appendage is further subdivided along the PD axis. Similar mechanisms may be important for tsh regulation in other tissues, as is suggested by tsh de-repression in PcG mutant clones in the eye disc (Zirin, 2004).

tsh and hth repression are distinct events during the development of the wing imaginal disc. The requirement for PcG activity in tsh, but not hth, repression points to the primacy of tsh repression in determining appendage versus trunk fate. PcG regulation ensures a strict and inflexible pattern of gene expression, ideal for defining the fundamental divisions of the disc. Within the specified appendage domain, Wg and Dpp signaling can then modify the shape and size of the hinge and wing blade through continuous input into transcription factors that control patterning and growth. In the absence of Tsh, Hth is an essential mediator of this process, since it promotes hinge development at the expense of wing pouch growth. The complexity of hth relative to tsh regulation may, therefore, reflect the greater need for plasticity in the response of hth to the Wg and Dpp morphogen gradients (Zirin, 2004).

Wingless targets Nemo, a Wingless antagonist

The cellular events that govern patterning during animal development must be precisely regulated. This is achieved by extrinsic factors and through the action of both positive and negative feedback loops. Wnt/Wg signals are crucial across species in many developmental patterning events. Drosophila nemo (nmo) acts as an intracellular feedback inhibitor of Wingless (Wg) and it is a novel Wg target gene. Nemo antagonizes the activity of the Wg signal, as evidenced by the finding that reduction of nmo rescues the phenotypic defects induced by misexpression of various Wg pathway components. In addition, the activation of Wg-dependent gene expression is suppressed in wing discs ectopically expressing nmo and enhanced cell autonomously in nmo mutant clones. nmo itself is a target of Wg signaling in the imaginal wing disc. nmo expression is induced upon high levels of Wg signaling and can be inhibited by interfering with Wg signaling. Finally, alterations are observed in Arm stabilization upon modulation of Nemo. These observations suggest that the patterning mechanism governed by Wg involves a negative feedback circuit in which Wg induces expression of its own antagonist Nemo (Zeng, 2004).

In Drosophila, several examples of Wg feedback inhibition have been identified. (1) It has been shown that Wg downregulates its own transcription in the wing pouch to narrow the RNA expression domain at the DV boundary. (2) Wg signaling can repress the expression of its receptor Dfz2 in the wg-expressing cells of the wing disc. Wg regulation of Dfz2 creates a negative feedback loop in which newly secreted Wg is stabilized only once it moves away from the DV boundary to cells expressing higher levels of Drosophila Fz2. (3) The Wg target gene naked cuticle (nkd) acts through Dsh to limit Wg activity. (4) Wingful (Wf), an extracellular inhibitor of Wg, is itself induced by Wg signaling (Zeng, 2004).

This research adds Nemo to this list of inducible antagonists participating in Wg signaling. Nemo antagonizes the Wg signal in wing development, as evidenced by phenotypic rescue, suppression of Wg-dependent gene expression in discs ectopically expressing nmo, and ectopic expression of a Wg-dependent gene in nmo mutant clones (Zeng, 2004).

Since both wf and nmo expression are positively regulated by Wg signaling in the wing, their expression patterns are relatively similar to that of Wg. Even though nkd also has a similar pattern to Wg in the larval wing disc, unexpectedly, it has no detectable role in wing development. As an intracellular antagonist, Nkd regulates embryonic Wg activity in a cell-autonomous manner by acting directly with Dsh to block accumulation of Arm in response to Wg signaling. Wf apparently has no role during embryogenesis, although both Wf and Nkd can inhibit Wg signaling throughout development when overexpressed. Wf is an extracellular protein that functions non-autonomously to regulate Wg signaling. This mechanism of inhibition parallels that of Argos, a secreted feedback antagonist in the EGFR pathway (Zeng, 2004).

The effect of Nemo on the Wg-dependent reporter gene Dll is confined to regions of endogenous gene expression. In the absence of nmo expression, ectopic Dll expression is only seen at elevated levels within the endogenous expression domain, thus being dependent on Wg activity. This is in contrast to inhibition of the Dpp pathway by Brinker. Brinker acts independently of Dpp in its repression of Dpp target genes, such that in the absence of both brk and Dpp the target genes are expressed ectopically. It is speculated that the role of Nemo in the Wg pathway is analogous to the role of Daughters against Dpp (Dad) in Dpp signaling. Dpp induces the expression of dad, which in turn antagonizes the pathway through an as yet undefined mechanism. These might include either interactions with the intracellular transducer Mothers against Dpp (Mad) or with TGFß receptors (Zeng, 2004).

In further support that Wg signaling regulates the transcription of nmo, several dTCF consensus binding sites have been found in the 5' region of the nmo gene that may represent enhancer elements. Indeed, two sites match 9 out of 11 bp (GCCTTTGAT) of the T1 site (GCCTTTGATCT) in the dpp BE enhancer that has been shown both in vitro and in vivo to bind and respond to dTCF. The presence of these sites suggests that the observed transcriptional regulation of nmo by Wg may involve direct binding to the nmo DNA sequence by dTCF (Zeng, 2004).

As a result of comparing the endogenous expression pattern of nmo with stabilized Arm, it was noticed that the highest levels of Nemo exclude Arm stabilization, while high levels of Arm are present in cells in which nmo levels are lower. Since Arm protein stabilization is a direct consequence of Wg pathway activation, attempts were made to examine whether Nemo may function to inhibit Wg by promoting Arm destabilization and subsequent breakdown. Indeed, ectopic expression of Nemo can lead to cell-autonomous reduction in Arm protein levels. This preliminary result suggests a mechanism in which Nemo may contribute to the destabilization of Arm that involves the Axin/APC/GSK3 complex. One explanation to account for such a finding would concern the interaction with TCF in the nucleus and the role of dTCF as an anchor for Arm. Given what is known about NLKs, it is likely that Nemo may act on the ability of the dTCF/Arm complex to bind DNA and activate transcription. It has been proposed that dTCF acts as an anchor for Arm in the nucleus. It remains to be determined how efficient this anchor is and whether there are conditions in which the interaction may become compromised, such as is seen with elevated Nemo. NLKs have been shown to affect the DNA-binding ability of TCF/ß-catenin. Perhaps in the absence of DNA binding, this complex is less stable and Arm could be free to shuttle to the cytoplasm where it could associate with Axin or APC and become degraded. It is proposed that the ectopic nmo leads to destabilization of the dTCF/Arm/DNA complex, thus causing Arm to exit the nucleus and be degraded through interaction with Axin, APC and GSK3. The observation that ectopic expression of full-length Arm cannot induce any activated Wg phenotypes has been explained by the hypothesis that even these high levels of protein are not sufficient to overcome the degradation machinery. Thus, the finding that there is no elevated Arm in nmo clones is consistent with an inability to overcome the endogenous degradation machinery; even though less Nemo could lead to more stabilized DNA interactions, this would not lead to higher levels of stabilized Arm than are normally found (Zeng, 2004).

Wingless and wing blade development

The Drosophila wing imaginal disc gives rise to three body parts along the proximo-distal (P-D) axis: the wing blade, the wing hinge and the mesonotum. The more distal portion of the hinge is continuous with the wing blade, but contains three identifiable structures: the costa (Co), the radius (Ra) and the allula (Al). A second, more proximal part of the hinge (or axillary region), is morphologically demarcated from the rest of the wing and consists of several sclerites (Scl), which are mostly devoid of trichomes, and the axillary cord (aCrd). The tegula (Te), although positioned just anterior to the sclerites, fate maps in the wing disc to a distinct and more dorso-proximal region than these hinge structures, and therefore is not considered a part of the hinge. Correspondingly, the distalmost portion of third instar wing discs is referred to as the wing pouch, which will give rise to the wing blade. Surrounding the wing pouch is a region that will give rise to the hinge and, more proximally, there is a large dorsal territory that will give rise to the mesonotum (mnt) and a thin ventral region that gives rise to the pleura (pl) (Casares, 2000).

Several genes are known to be expressed in the wing pouch including vestigial (vg), scalloped (sd), nubbin (nub) and Distal-less (Dll), which encode transcription factors, and four-jointed (fj), which encodes a putative secreted factor. Development of the wing blade initiates along part of the dorsal/ventral (D/V) compartment boundary and requires input from both the Notch and wingless (wg) signal transduction pathways. wg is expressed along the D/V compartment boundary within the wing blade and in two concentric rings that surround the wing blade region. The rings of wg expression have been fate mapped to the adult hinge and, using a wg-lacZ reporter gene, they map within the hinge as follows: the outer wg ring (OR) maps to the proximal hinge, and the inner wg ring (IR) stains structures in the distal hinge, including the medial costa (mCo), distal radius (dRa) and part of the allula (Al). hth is also highly expressed in the wing hinge region of third instar wing discs, straddling both wg rings. Using a hth-lacZ reporter gene, hth expression maps to the same structures in the adult hinge as does wg. In late third instar wing discs, teashirt (tsh), which encodes a Zn-finger transcription factor, is strongly expressed in cells that are more proximal than hth-expressing cells, although low levels of tsh and hth overlap in the proximal hinge region. Consistent with this expression pattern, tsh-expressing cells fate map in the adult to the axillary sclerites and pleura (Casares, 2000).

In the wing blade, wg activates the gene vestigial (vg), which is required for the wing blade to grow. wg is also required for hinge development, but wg does not activate vg in the hinge, raising the question of what target genes are activated by wg to generate hinge structures. wg is shown to activate the gene homothorax (hth) in the hinge and hth is shown to be necessary for hinge development. Further, hth also limits where along the D/V compartment boundary wing blade development can initiate, thus helping to define the size and position of the wing blade within the disc epithelium. teashirt (tsh), which is coexpressed with hth throughout most of wing disc development, collaborates with hth to repress vg and block wing blade development. These results suggest that tsh and hth block wing blade development by repressing some of the activities of the Notch pathway at the D/V compartment boundary (Casares, 2000).

The overlap between wg and high levels of hth in the hinge region suggested that wg might play a role in activating hth in this region of the wing disc. Four experiments tested this idea. (1) hth expression was examined in discs in which the wg signaling cascade was compromised due to the expression of a dominant negative form of dTCF, a downstream transcription factor in the wg signal transduction pathway. Expression of dominant negative dTCF (dTCFDN) using ptc-Gal4 results in the repression of hth in the hinge. Similar results are obtained when flip-out clones expressing dTCFDN are generated in the hinge between 72 and 96 hours of development. (2) A second piece of evidence supporting a role for wg in the upregulation of hth is the finding that in wgspd-fg mutant discs, in which wg expression is specifically reduced in the IR of third instar discs, hth expression is no longer upregulated. (3) Clones of cells doubly mutant for frizzled1 and frizzled2 (fz1-;fz2-), which are required for the reception of the Wg signal, were generated. fz1-;fz2- clones show a cell-autonomous loss of hth expression. These findings suggest that wg signaling, mediated by the Frizzled family of receptors, is required for high levels of hth expression in the hinge region of wing discs. (4) A test was performed to see if ectopic expression of wg could trigger the expression of hth in more proximal regions of the wing disc, where hth levels are usually low and tsh levels are high. Based on the expression patterns of tsh and hth in third instar discs, it was predicted that, if induced by wg, hth would also repress tsh. Flip-out clones of wild-type (secreted) Wg or of a membrane-tethered, and therefore non-diffusable, form of Wg, Nrt-Wg were generated, and the expression of wg, hth and tsh were monitored. Expression of either Wg or Nrt-Wg in clones in the notum (just dorsal to the hinge region) non-autonomously induces high levels of hth and repressed tsh, recapitulating the situation found in the wild-type hinge. In contrast to clones induced in the notum portion of the disc, Nrt-Wg was never observed to activate hth in the wing pouch. Instead, activation of the Wg pathway in the wing pouch induces higher levels of vg expression. These data suggest that hth is a wg target gene in the wing hinge. In addition, they suggest that, in response to wg, cells in the wing pouch are biased in favor of activating vg, whereas in more proximal positions, induction of hth is favored (Casares, 2000).

Additional experiments presented here suggest that tsh collaborates with hth to interfere with Notch's ability to activate wg at the D/V boundary. During wild-type wing disc development, both hth and tsh are coexpressed in all non-wing blade cells and, at least in the posterior compartment, the D/V boundary expresses vg but not wg. Consistent with these wild-type expression patterns, the combination of Hth plus Tsh is sufficient to completely block wg expression at the D/V boundary in the wing blade. In contrast, vg is still expressed at the D/V boundary in the presence of both Hth and Tsh. It is suggested that the repression of wg by Hth and Tsh represents a normal function of these two proximally expressed transcription factors. The results further suggest that hth is necessary for this repression, because wg is derepressed in hth minus clones that straddle the D/V boundary (Casares, 2000).

Growth of tissues and organs during animal development involves careful coordination of the rates of cell proliferation and cell death. Cell proliferation depends on signals to stimulate cell growth and cell division. In addition, cells compete for intercellular survival signals which are required to prevent them from undergoing apoptosis in response to growth stimuli. How these cellular processes are coordinated with pattern formation during animal development is a challenging question in developmental biology. The bantam gene of Drosophila has been found to encode a 21 nucleotide microRNA (miRNA) that promotes tissue growth. bantam expression is temporally and spatially regulated in response to patterning cues. bantam microRNA simultaneously stimulates cell proliferation and prevents apoptosis. The pro-apoptotic gene hid has been identified as a target for regulation by bantam miRNA, providing an explanation for bantam's anti-apoptotic activity (Brennecke 2003).

The correlation between elevated sensor (expressing GFP under control of the tubulin promoter and two copies of the bantam target in the 3'UTR) expression and the zone of nonproliferating cells adjacent to the dorsoventral boundary (ZNC) suggests that Wingless might control cell proliferation in the ZNC by reducing bantam miRNA levels. To test this, use was made of a dominant-negative form of the Wingless receptor DFz2 to locally reduce Wingless activity. Expression of DFz2-GPI under engrailed-Gal4 control reduces bantam sensor levels in the ZNC of the posterior compartment, indicating that bantam miRNA expression increases when Wingless signaling is impaired. Consequently, cells in the posterior ZNC continue to undergo DNA synthesis and are labeled by BrdU incorporation. Comparable results were obtained by overexpression of the Wingless-pathway inhibitor naked. These observations indicate that Wnt signaling contributes to bantam miRNA expression to exert developmental control over cell proliferation in the ZNC. It is noted that the entire posterior compartment is small under these conditions because Wingless is required earlier to promote overall growth of the wing pouch, in addition to its later role in specifying the ZNC. A second area of reduced bantam sensor expression was noted immediately anterior to the AP boundary, where Hedgehog signaling has been shown to induce cell proliferation. These observations provide a link between signaling proteins that serve as morphogens to control spatial pattern and bantam, a regulator of cell proliferation (Brennecke, 2003).

Wingless and the subdivision of the notum

Aldaz, S., Morata, G. and Azpiazu, N. (2003). The Pax-homeobox gene eyegone is involved in the subdivision of the thorax of Drosophila. Development 130: 4473-4482. 12900462

The eyegone (eyg) gene is involved in the development of the eye structures of Drosophila. eyg and its related gene, twin of eyegone (toe), are also expressed in part of the anterior compartment of the adult mesothorax (notum). The anterior compartment is termed the scutum and consists of the part of the notum from the anterior border to the suture with the scutellum. In the absence of eyg function the anterior-central region of the notum does not develop, whereas ectopic activity of either eyg or toe induces the formation of the anterior-central pattern in the posterior or lateral region of the notum. These results demonstrate that eyg and toe play a role in the genetic subdivision of the notum, although the experiments indicate that eyg exerts the principal function. However, by itself the Eyg product cannot induce the formation of notum patterns; its thoracic function requires co-expression with the Iroquois (Iro) genes. The restriction of eyg activity to the anterior-central region of the wing disc is achieved by the antagonistic regulatory activities of the Iro and pnr genes, which promote eyg expression, and those of the Hh and Dpp pathways, which act as repressors. It is argued that eyg is a subordinate gene of the Iro genes, and that pnr mediates their thoracic patterning function. The activity of eyg gives rise to a new notum subdivision that acts upon the pre-extant one generated by the Iro genes and pnr. As a result the notum becomes subdivided into four distinct genetic domains (Aldaz, 2003).

Localized expression of eyg/toe is achieved by the activity of two antagonistic factors: the promoting activity induced by the Iro and pnr genes, and the repressing activities exerted by the Hh and the Dpp pathways. The latter are probably mediated by Hh and Dpp target genes that are yet to be identified (Aldaz, 2003).

The elimination of eyg activity from the scutellum and lateral notum is caused by the Hh and Dpp pathways. Because the AP compartment border is displaced posteriorly in the notum, these two pathways are active at high levels in the posterior region of the mesothorax. Assuming that the two signals behave as in the wing, Hh activity will be higher in the region close to the AP border, whereas the effect of Dpp will extend further anteriorly. Thus the repressive role of Hh will be greater in the proximity of the AP border and that of Dpp will be greater in more anterior positions. This is precisely what the results indicate. In the region close to the AP border the high Hh levels alone are sufficient to repress eyg. However. in more anterior positions, close to the border of the eyg domain, Hh levels are lower and, although Hh is still necessary, it is not sufficient to repress eyg. Here there is an additional requirement for Dpp activity (Aldaz, 2003).

Thus, the part of the notum that does not express eyg can be subdivided into two distinct zones according to the mode of eyg regulation: a region close to the AP border that requires only Hh, and a more anterior region that requires both Hh and Dpp. In the posterior compartment, the repression of eyg has to be achieved by a different mechanism because neither the inactivation of the Hh pathway in smo- clones, nor the inactivation of the Dpp pathway, induces ectopic eyg activity. A probable possibility is that en itself may act as repressor (Aldaz, 2003).

The result of the antagonistic activities of the Iro genes and pnr in one case and of the Hh and Dpp signalling pathways in the other, subdivides the notum into an eyg/toe expressing domain and a non-expressing domain. The localized expression of eyg/toe contributes to the morphological diversity of the thorax, as it distinguishes between an anterior-central region and a posterior-lateral one. It provides another example of a genetic subdivision of the body that is not based on lineage. It also provides an example of a patterning gene acting downstream of the combinatorial code of selector and selector-like genes. Its mode of action supports a model in which the genetic specification of complex patterns, such as the notum, is achieved by a stepwise process involving the activation of a cascade of regulatory genes (Aldaz, 2003).

Table of contents

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

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