Expression of scalloped is first detected in the cephalic neuroblasts of embryos during germ-band elongation in stage 9. Expression in the PNS is first seen during germ-band retraction [Images](late stage 11). By stage 14, scalloped is observed in the sense organs of the trunk and antennomaxillary complex. sd transcripts accumulate in the supraesophageal ganglion, and later are also seen in a dorsal cluster of peripheral sense organs and in sensory organs of the gnathal buds (Campbell, 1992).


scalloped transcripts are found in the larval CNS, probably in glia. Expression is also found in wing imaginal discs, where scalloped is required for proper differentiation. Still later, expression in the adult brain is resticted to subsets of cells, some in regions involved in the processing of gustatory information (Campbell, 1992). scalloped is essential for normal taste behavior in Drosophila (Inamdar, 1993).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Effects of Mutation or Deletion

Viable mutants of the scalloped gene exhibit defects that can include gapping of the wing margin and ectopic bristle formation on the wing (Campbell, 1991).

Chip may encode an enhancer-facilitator, acting to facilitate the activity of distal enhancers. The mechanisms allowing remote enhancers to regulate promoters several kilobase pairs away are unknown but are blocked by the Drosophila suppressor of Hairy-wing protein [su(Hw)] that binds to gypsy retrovirus insertions between enhancers and promoters. su(Hw) bound to a gypsy insertion in the cut gene also appears to act interchromosomally to antagonize enhancer-promoter interactions on the homologous chromosome when activity of the Chip gene is reduced. Chip is needed for the wing margin enhancer of cut. The Chip mutation dominantly enhances the mutant phenotypes displayed by partially suppressed gypsy insertions in both cut and Ultrabithorax and is a homozygous larval lethal, indicating that Chip regulates multiple genes. Chip is normally required for wing margin enhancer function of cut because Chip mutations also enhance the cut wing phenotype of a cut mutation and heterozygotes for Chip display cut wing phenotypes when either scalloped or mastermind (mam) are also heterozygous mutant. Both Sc and Mam are known to regulate the cut distal enhancer, but in contrast to sd and mam mutants, Chip mutants display stronger genetic interactions with gypsy insertions than with wing margin enhancer deletions. Thus, in a heterozygous Chip mutant, a heterozygous gypsy insertion in cut displays a cut wing phenotype, whereas a heterozygous enhancer deletion does not. Dependence on the nature of the heterozygous lesion in the regulatory region strongly suggests that Chip directly regulates cut. More strikingly, it indicates that in a Chip heterozygote, a gypsy insertion is more deleterious to enhancer function than deletion of the enhancer. The simplest explanation is that su(Hw) bound to gypsy in one cut allele acts in a transvection-like manner (interchromosomally) to block the wing enhancer in the wild-type cut allele on a second chromosome. This implicates Chip in enhancer-promoter communication (Morcillo, 1997 and references).

Chip was cloned and found to encode a homolog of the recently discovered mouse Nli/Ldb1/Clim-2 and Xenopus Xldb1 proteins, which bind nuclear LIM domain proteins. Chip protein interacts with the LIM domains in the Apterous homeodomain protein, and Chip interacts genetically with apterous, showing that these interactions are important for Apterous function in vivo. Importantly, Chip also appears to have broad functions beyond interactions with LIM domain proteins. Chip is a ubiquitous chromosomal factor required for normal expression of diverse genes at many stages of development. It is suggested that Chip cooperates with different LIM domain proteins and other factors to structurally support remote enhancer-promoter interactions (Morcillo, 1997).

The mechanisms that allow enhancers to activate promoters from thousands of base pairs away are disrupted by the Drosophila Suppressor of Hairy-wing protein (Su[Hw]). Su[Hw] binds a DNA sequence in the gypsy retrotransposon and prevents activation of promoter-enhancers that are distal to a gypsy insertion in a gene without affecting proximal promoter-enhancers. Several observations indicate that SUHW does not affect enhancer-binding activators. Instead, SUHW may interfere with factors that structurally facilitate interactions between an enhancer and promoter. To identify putative enhancer facilitators, a screen for mutations that reduce activity of the remote wing margin enhancer in the cut gene was performed. Mutations in scalloped, mastermind, and a previously unknown gene, Chip, were isolated. A TEA DNA-binding domain in the Scalloped protein binds the wing margin enhancer. Interactions among scalloped, mastermind and Chip mutations indicate that Mastermind and Chip act synergistically with Scalloped to regulate the wing margin enhancer. Chip is essential and also affects expression of a gypsy insertion in Ultrabithorax. Relative to mutations in either scalloped or mastermind, a Chip mutation hypersensitizes the wing margin enhancer in cut to gypsy insertions. Therefore, Chip might encode a target of su(Hw) enhancer-blocking activity (Morcillo, 1996).

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

Clones of cells in imaginal tissues generally adopt very irregular shapes. Strikingly, however, clones of cells that are ectopically expressing Vg are more rounded and have smoother borders than control clones. Similar differences in clone behavior have been observed upon misexpression or mutation of a number of different genes in Drosophila and have been attributed to differences in the affinity of cells for their neighbors. This effect of Vg is evident in the notum, hinge, and proximal regions of the wing, as well as in other imaginal discs. Within the wing region of the disc, the influence of Vg overexpression on clone shape is graded: clones that are near the D-V wing border generally continue to have irregular shapes, while clones that are far from the D-V border are more circular. This effect was quantified by calculating the circularity of 117 Vg-expressing clones and then plotting the circularity of each clone against its relative distance from the D-V wing border. Circularity is a ratio of clone area to the square of the clone perimeter. The average circularity of VG-expressing clones increases with distance from the D-V border. The observation that the same, constitutive level of Vg expression induces graded changes in clone shape that depend upon clone location suggests that there are normally graded differences in Sd:Vg-dependent cell affinities. Three aspects of the behavior of sd clones in the wing blade are consistent with the hypothesis that reduction in normal Sd:Vg function also influences cell affinity. (1) In the few instances in which relatively large sd clones were recovered, they tended to be more rounded than their wild-type twins. (2) Over a quarter of the sd clones recovered in the wing pouch were associated with multiple wild-type 'twin' clones. Although in some cases this may occur fortuitously, it is also suggested that affinity differences with surrounding wild-type cells could force independent mutant clones into a coherent patch. (3) sd clones tend to be located farther from the D-V boundary than their wild-type twins. Differential location between mutant and wild-type twins has also been observed in the wing for shaggy mutant clones and in the abdomen for patched and smoothened mutant clones, and in both cases it has been hypothesized to derive from differences in cell affinity (Liu, 2000).

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

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

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

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::sd TEA 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).

When the sd gene is mutated, the phenotype includes not only the wing margins but also the sensory organs that are found at the wing margins. In addition to the loss of wing margin bristles, there is also a reduction in the number of cells, which results in notching of the wings. This reduction in the number of cells is thought to be a result of apoptosis. In addition, overexpression of sd is also associated with apoptotic cell death. Lyra (Ly) mutations, in contrast, result in the loss of the anterior and posterior margin bristles and this is not associated with apoptotic cell death. However, there is a reduction in the number of cells in the wing margin that manifests itself by erosion of the wing margin. Ly mutations have been shown to be dominant gain of function alleles of sens, in that in a Ly background sens is ectopically expressed. To see if Ly and sd interact genetically, wings were examined from sdETX4 males that were also heterozygous for Ly. Flies harboring mutants of both genes show a significant enhancement of the wing phenotype compared to flies with either mutant alone. In the transheterozygous fly, the margin bristles are completely absent, suggesting that these two genes work through a common pathway (Srivastiva, 2003).

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

sens has been shown to be both necessary and sufficient for the formation of organs of the peripheral nervous system (PNS). Ectopic expression of sens can result in the formation of extrasensory bristles on the wing and thorax. This ectopic formation of sensory bristles can also happen in the absence of genes of the achaete-scute complex, though to a lesser extent. To see if sd has any role in formation of sensory bristles by ectopic expression of sens, and to confirm that sens is necessary and sufficient for formation of the sensory bristles, sens was expressed in a sd mutant background. The UAS-sens transgene was expressed in both sdETX4 and sd58 mutant backgrounds using a vg-Gal4 driver and expression from the UAS-sens transgene was determined by staining wing discs with the anti-Sens antibody as a control. If sd has no role in ectopic bristle formation by sens, then expression of sens should result in formation of the sensory bristles missing in the margin of the sd mutants. However, sens expression is unable to restore the margin-specific bristles in sd mutants, suggesting that sens may need sd function for formation of bristles and for proper SOP differentiation. Instead of the formation of ectopic bristles, expression of sens in sdETX4 enhances the wing phenotype to resemble the result of the enhancement of sdETX4 caused by a Ly mutant. To test this further, UAS-sens was also expressed under the control of a dpp-Gal4 construct that drives expression at the A/P compartment border away from the margin. Wild-type wings expressing sens at the A/P border fail to inflate properly upon eclosion but exhibit numerous ectopic bristles at the position of the A/P border as well as numerous ectopic bristles on the thorax. Expression of sens in a sd58 mutant background, however, results in very little to no ectopic bristle formation at the A/P border, again suggesting that sens possibly needs sd function for formation of SOPs (Srivastiva, 2003).

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

Molecular and functional analysis of scalloped recessive lethal alleles in Drosophila

The Drosophila scalloped (sd) gene is a homolog of the human TEF-1 gene and is a member of the TEA/ATTS domain-containing family of transcription factors. In Drosophila, sd is involved in wing development as well as neural development. Data are presented from a molecular analysis of five recessive lethal sd alleles. Only one of these alleles complements a viable allele associated with an sd mutant wing phenotype, suggesting that functions important for wing development are compromised by the noncomplementing alleles. Two of the wing noncomplementing alleles have mutations that help to define a Vg-binding domain for the Sd protein in vivo, and another noncomplementing allele has a lesion within the TEA DNA-binding domain. The Vg-binding domain overlaps with a domain important for viability of the fly, since two of the sd lethal lesions are located there. The fifth lethal affects a yet undefined motif lying just outside the Vg-binding domain in the C-terminal direction that affects both wing phenotype and viability. This is the first example linking mutations affecting specific amino acids in the Sd protein with phenotypic consequences for the organism (Srivastava, 2004).

Four of the five lethal alleles studied affect the wing phenotype: the physical lesions associated with three of these four are within the C-terminal half of SD and are localized between amino acids 232 and 355. Two of these mutations help define a Vestigial binding domain (VBD) in vivo that overlaps a domain previously predicted by in vitro experiments to be responsible for binding Vg. The sd3L and sd47M lesions are within this predicted domain and, due to the molecular nature of these mutations, are predicted to abolish the VBD completely. The sd68L lesion is located just outside and 3' to this domain but also affects Vg localization in vivo to some extent. This conclusion is supported by the observation that the wing phenotype produced from these two alleles when heterozygous with sdETX4 is more severe than the sd68L/sdETX4 phenotype. Since the lesions in sd3L and sd47M would also be expected to abolish all aspects of Sd function C terminal to the respective lesion, this could also account for the early recessive lethal phenotype of these two alleles. The sd68L lesion is only a missense mutation so it would not be surprising if some sd function is retained. Although the sd68L lesion is located just outside and 3' to the predicted VBD, it is associated with the mutation of tyrosine, an amino acid that is often subject to phosphorylation and dephosphorylation. Phosphorylation-based mechanisms are known to play a role in a great many interactions between proteins. For example, the sd68L lesion is in the vicinity of a domain where phosphorylation is known to modulate RTEF-1 function in cardiac muscle. Therefore, it initially seemed that the simplest interpretation of the results for the sd68L allele was that it also directly affected the VBD. However, this now seems unlikely. The partial mislocalization of Vg could also result from reduced Sd levels due to protein instability or even from mislocalization of Sd itself. It is also possible that other protein factors present in vivo are important in regulating the kinetics of Sd binding to Vg or play a vital role in Sd stability. In an sd68L/+ heterozygote, regulation of this binding or Sd stability could be inefficient and result in the observed variability of the wing complementation phenotype with this allele. The wing phenotype of sd68L in trans with the more severe but phenotypically stable sd58 allele is nonvariable, but still less severe than that produced by sd3L or sd47M over sd58d. Thus, it appears that the sd68L allele provides some wild-type function, with respect to wing development, in a genetic background shared with the sdETX4 or sd58 alleles. However, since sd68L is a recessive lethal, the lesion also compromises some as yet unknown vital function as well. The most likely reason for complementation of the wing phenotype with sd11L is that the lesion does not affect the VBD because it is more distally located: 78 amino acids from the sd68L mutation and only eight residues from the C-terminal end of Sd. Moreover, it has been reported that the TEF-1 sequence (Sd homolog) from residue 329 to the C terminus is dispensable with respect to its ability to interact with the TDU protein (Vg homolog) even though high sequence conservation exists throughout this region. Further support for the mutations in sd3L and sd47M affecting the VBD and sd11L not affecting this domain comes from observing Vg localization data from wing discs derived from sd11L hemizygous larvae as well as from Vg localization in mitotic clones of the sd47M allele. Vg localization in sd68L wing discs and in sd mutant clones harboring the sd47M allele is diffuse rather than nuclear. This is a clear indication that the VBD (in sd47L) or a related role (as in sd68L) must not be fully functional, even though in vitro data indicate that sd68L is not defective in binding Vg. However, Vg in sd11L wing discs is entirely nuclear, supporting the conclusion that in this allele the VBD is unaffected, while still implicating the region in an alternative function that is essential for viability (Srivastava, 2004).

To date, knowledge about the TEA DNA-binding domain has been based primarily on in vitro mutational analysis. However, extrapolation from in vitro observations to in vivo functions is not always valid. A mutation has been identified within the conserved TEA DNA-binding domain that affects both the essential and the wing-specific functions of sd. The TEA DNA-binding domain has been predicted to have three alpha-helices. However, the limits of the third helix within the domain are not very well defined. The mutation associated with the sd31H allele (Arg to Lys) is located in the TEA DNA-binding domain between the second and third predicted helices. This lesion also lies between two putative phosphorylation sites, and the role of phosphorylation in regulation of DNA binding by the TEA domain from organisms other than Drosophila has been well documented. The cause of the observed heterozygous wing and homozygous lethal phenotypes associated with this allele can be explained in at least two ways. The phenotypes could simply be the result of a defect induced by this mutation in the regulation of DNA binding by phosphorylation. The second and the third helices of the TEA DNA-binding domain may actually be responsible for contacting the DNA so this mutation could directly affect the ability of Sd to contact DNA, thereby preventing transcription of essential and wing-specific genes controlled by sd. Alternatively, it is also possible that the mutation in sd31H affects the nuclear localization signal that overlaps the TEA DNA-binding domain of Sd, so that Sd is prevented from entering the nucleus. In the absence of an Sd antibody, it cannot be determined if the mutation prevents Sd from entering the nucleus or simply results in inefficient binding of the protein to its targets in the nucleus. Either of the above putative defects could explain the recessive lethality caused by sd31H. The late pupal lethality associated with this allele is consistent with an argument that this mutation results in inefficient transport of Sd to the nucleus. The mutant animal is able to survive until the pupal stage, beyond which the level of Sd in the nucleus would be unable to sustain the level of transcription needed for survival. However, Vg localization data from sd31H mutant discs argue against a defect in nuclear localization of Sd. Because Sd is needed for maintenance of vg and sd expression, one would expect to see some Vg in the wing pouch of the mutant discs if the mutation was simply causing inefficient nuclear localization of Sd. The absence of any noticeable Vg in the wing pouch favors the hypothesis that the mutation affects the DNA-binding ability of the TEA domain. So, this mutant Sd would then not bind its cognate regulatory DNA elements and, as a result, Vg expression in the wing pouch would not be maintained. It is also possible that the absence of Vg in the wing pouch area of the sd31H disc is merely a consequence of autoregulation in this system (Srivastava, 2004).

While specific lesions have been identified in each of the sd lethal alleles and these have been correlated with phenotypic consequences for the organism, the molecular reason for lethality has not been solved in every case. Since sd31H has a lesion in the TEA domain, it is relatively easy to understand why this may result in lethality. Similarly, the lethality associated with sd3L and sd47M is explicable because of the molecular nature of the lesions: all Sd function downstream of the respective lesion would be abolished. The reasons for the lethality associated with sd68L and sd11L are still not obvious. Evidence is provided that the lesion in sd68L affects wing development and also compromises a vital function, while sd11L does not appear to affect wing development but does compromise a vital function. The current hypothesis is that these two sd lethal alleles likely affect residues within a domain that is necessary for binding cofactors involved in other critical developmental functions of Sd. Future efforts will concentrate on attempting to identify these putative cofactors (Srivastava, 2004).

The data presented here are relevant to several aspects of Sd function. This study reports the molecular characterization of lethal alleles of sd and this analysis has enabled the association of specific conserved residues within the Sd protein sequence with specific mutant phenotypes. The results have helped to define a VBD in Sd by in vivo criteria. A mutation within the Sd-TEA DNA-binding domain has been shown to be important for both wing development and viability of the fly. Because the residues affected in the sd lethal alleles are conserved across species and phyla, this study could also have important implications for understanding the properties of the vertebrate homolog TEF-1 (Srivastava, 2004).

Yki/YAP, Sd/TEAD and Hth/MEIS control tissue specification in the Drosophila eye disc epithelium

During animal development, accurate control of tissue specification and growth are critical to generate organisms of reproducible shape and size. The eye-antennal disc epithelium of Drosophila is a powerful model system to identify the signaling pathway and transcription factors that mediate and coordinate these processes. This study shows that the Yorkie (Yki) pathway plays a major role in tissue specification within the developing fly eye disc, in squamous peripodial portion of the epithelium (PE), at a time when organ primordia and regional identity domains are specified. RNAi-mediated inactivation of Yki, or its partner Scalloped (Sd), or increased activity of the upstream negative regulators of Yki cause a dramatic reorganization of the eye disc fate map leading to specification of the entire disc epithelium into retina. On the contrary, constitutive expression of Yki suppresses eye formation in a Sd-dependent fashion. It was also showm that knockdown of the transcription factor Homothorax (Hth), known to partner Yki in some developmental contexts, also induces an ectopic retina domain, that Yki and Scalloped regulate Hth expression, and that the gain-of-function activity of Yki is partially dependent on Hth. These results support a critical role for Yki - and its partners Sd and Hth - in shaping the fate map of the eye epithelium independently of its universal role as a regulator of proliferation and survival (Zhang, 2011).

The process of regional specification in the eye-antennal disc of Drosophila involves a number of signaling pathways that regulate the expression of identity-defining transcriptional regulators, often referred to as selector factors. This study shows that the Hippo signaling pathway and the transcription factors Yki, Sd, and Hth play critical roles in the regional specification of this disc. Specifically, all three factors are required for the establishment of the peripodial cell layer of the eye disc. Yki function is required in the PE to maintain tissue identity and appears to works in concert with Sd and Hth. Moreover, negative regulation of Yki through the Hippo/Warts genetic pathway is required in the disc proper (DP) to modulate Yki activity such that it promotes proliferation and survival of retina progenitor cells without interfering with their specification. The specification role of Yki and its partners in the eye epithelium is central to proper disc development, occurs in the early stages of regional specification within this disc, and appears to be distinct from its more general role in proliferation and survival (Zhang, 2011).

Yki and Sd have been shown to form a complex and regulate the anti-apoptotic gene Diap1 in the wing and eye discs of Drosophila. In the eye, Yki is also strongly required for proliferation and survival, whereas Sd makes a lesser contribution to these processes (Zhang, 2011).

Does Yki function in a complex with Sd in the context of regional specification within the eye disc and how? The evidence presented in this study shows that (1) the RNAi-induced knock-down of either gene induces essentially identical PE-to-DP transformations and (2) sd-RNAi can suppress the gain-of-function, anti-retina effect of Yki over-expression. These findings, together with the biochemical evidence of protein-protein interactions, suggest that Yki and Sd may indeed control transcription together in a complex during PE specification (Zhang, 2011).

Surprisingly, a comparison of loss-of-function analyses in mosaic discs versus RNAi-mediated disc-wide knock-downs has uncovered a discrepancy in the induced phenotypes. Specifically, loss-of-function clones of either yki or sd do not show signs of cell fate transformation. This is the case for traditional flip-FRT loss-of-function clones generated using mutant alleles as well as for knock-down MARCM clones induced by RNAi expression. Differences in reagents cannot explain this discrepancy, because the same yki-RNAi or sd-RNAi lines were used in disc-wide versus clonal analyses. Hence, this discrepancy must depend on the two different loss-of-function approaches employed. The two approaches differ significantly in two ways: (1) the amount of mutant tissue induced, (2) and the timing of loss-of-gene function (Zhang, 2011).

Thus far, two major functions of Hth have been uncovered in the DP cell layer: 1) Hth, together with Tsh, suppresses the expression of the late retinal determination (RD) genes eya and so, thus slowing down the conversion of early eye progenitors (Ey, Tsh and Hth positive) into more mature (Ey, Tsh, Eya and So positive) eye precursor cells, and 2) Hth together with Tsh and Yki enhances proliferation by inducing expression of the proliferation-promoting microRNA Bantam. The combined effect of these two activities results in the generation of an abundant pool of eye progenitor cells. Protein-protein interactions among these factors have been documented in vitro and/or in vivo, leading to the proposal that two complexes (inclusive of Hth/Tsh and Hth/Yki/Tsh) may perform these major functions (Zhang, 2011).

The data presented in this study uncover another critical function of Hth in the eye disc. In the PE cell layer, fated to give rise to portions of the head cuticle, Hth prevents conversion of this tissue into retina. As in the eye progenitors within the DP, this anti-retina activity of Hth involves suppression of late-RD factors (Eya, So, Dac). However, unlike its role in eye progenitors, it also entails down-regulation of the early factors Ey and Tsh. Thus, Hth suppresses late but not early RD factors in early eye disc progenitors in conjunction with Tsh, whereas it suppresses retina formation at the level if the early factors in the PE through a process that appears to involve Yki and Sd, but not Tsh (whose expression is normally absent from the PE). In agreement with this context dependent role of Hth, hth-RNAi reverses the anti-retina effect of Yki over-expression, at least in part, by relieving Yki suppression of the early retinal determination factor Ey, and expression of exogenous Tsh within the PE cell layer leads to the formation of an ectopic retina (Zhang, 2011).

Lastly, this study has shown that Hth expression is under the genetic, positive control of Yki and Sd, and that neither yki-RNAi nor sd-RNAi can suppress the gain-of-function activity of Hth. These findings, together with the reported association of the Hth and Yki proteins in S2 cells and on the regulatory region of Bantam, suggest a potentially more complex relationship between Hth and Yki in the development of the PE. The observation that Yki and Sd control Hth expression does not preclude the possibility that the Hth protein also functions in a complex with these factors. Indeed, a related example is offered by the retinal determination factor Eya, which first induces and then partners with the Sine oculis to regulate retina development. It is proposed therefore that Hth is one of the direct or indirect targets of a Yki/Sd complex in the PE. Thereafter, association with Hth may modify Yki/Sd activity eventually resulting in the transcriptional silencing of critical retinal determination genes, such as Tsh (Zhang, 2011).

The Hippo/Yki signaling pathway is a general regulator of cell proliferation and survival in metazoans. In a handful of cases, the Hippo pathway has been shown to regulate processes other than proliferation or cell death, such as the maintenance of the undifferentiated state of progenitor cell types in the neural tube and in the gut and, in two cases, aspects of neuronal differentiation. In one, the pathway controls the choice of opsin gene expressed in the R8 cells of the adult fly eye; in the other, it is required for maintenance of the dendritic harbors of body-wall sensory neurons in the Drosophila larva. While transcriptional regulation by Yki is thought to play a role in these non-proliferation-related processes, it is difficult to draw parallels between these cases and what is seen in the eye disc (Zhang, 2011).

Two more recent reports implicate Yki in processes related to tissue specification. In one case, a transient, expanding burst of Yki activity within rows of cells at the border of an already established wing field results in a marginal expansion of the wing primordium, ultimately ensuring proper wing size. In a second example, the YAP/Yki and TEAD4/Sd proteins specify the trophoectoderm as distinct from the inner cell mass (ICM) in mammalian embryos. It is thought that the circumferential cell-cell contacts experienced by ICM cells triggers Hpo activity and cytoplasmic retention of YAP, whereas the less extensive cell-cell contacts experienced by outer cells do not, thus allowing nuclear accumulation of YAP (Zhang, 2011).

Based on similarities with the latter example, it is tempting to hypothesize that less extensive cell-cell contacts in the squamous cell layer, than in the columnar one, would promote a more efficient nuclear localization of Yki in the PE, than in the DP cells. At moderate activity levels in the DP, Yki would then promote proliferation and survival without interfering with retina specification, whereas at higher activity levels in the PE, Yki would be able to not only promote cell proliferation and survival but also PE-identity by suppressing retina formation. This scenario would be consistent with the observed need for down-regulation of Yki activity by Warts in the DP and the stronger expression of Diap1-2-lacZ in the PE. However, the observation that the two cell layers differ significantly in the availability of factors found in Yki-based complexes suggests that 'Yki-complex composition' plays a critical role in the PE versus DP/retina distinction in the developing fly eye (Zhang, 2011).

Yki association with specific co-factors would, therefore, modify the output of the Hpo-Yki pathway to bring about different outcomes in distinct regions of the eye epithelium. A Yki/Hth/Tsh complex would ensure maintenance and expansion of the retina progenitors pool, whereas a Yki/Sd, and possibly Hth, complex would contribute to regional specification within the eye disc by ensuring formation of PE-derived head structures. That Yki/YAP-based complexes can include a variety of different co-factors is supported by other recent examples, including the above mentioned role of Yki, Hth and Tsh in promoting eye progenitors' proliferation in the fly, and its interaction with IRS1 to promote proliferation of neural precursors or with Smad1 to enhance BMP-mediated suppression of neuronal differentiation of embryonic stem cells in the mouse (Zhang, 2011).

In the L2 eye disc, the Sd protein is believed to be available throughout, but does not appear to contribute greatly to Yki-induced proliferation in either cell layer. On the contrary, in the PE, it behaves as a critical co-factor in the control of tissue identity. Unlike Sd, Tsh expression has been shown to be restricted specifically to the DP cell layer. Thus, Yki must perform its PE-promoting tasks in transcriptional complexes that do not include Tsh. Interestingly, misexpression of Tsh within the PE has been shown to induce retina development in this tissue. Whether Tsh does so, at least in part, by diverting Yki activity away from 'anti-retina-fate' functions remains to be determined. Hth, also present broadly in both cells layer at the L2 stage, would play a more general role contributing to Yki's function in both proliferation and tissue specification (Zhang, 2011).

One possible scenario is that the tissue specification functions of Yki in the PE are critically dependent on its association with Sd, and that the availability of Tsh specifically in the DP interferes with the ability of Sd and Yki to associate or work together on fate-determining tasks, thereby preventing any interference with retina formation (Zhang, 2011).

As shown by the paucity of examples, this analysis is still in the early days of deciphering how Yki and its partners regulate cell fate. Nonetheless, this role is apparently separate from its contribution to cell proliferation and survival, and likely involves a number of distinct molecular mechanisms including co-factor specificity and differing levels of Yki activity (Zhang, 2011).

Causes and consequences of genetic background effects illuminated by integrative genomic analysis

The phenotypic consequences of individual mutations are modulated by the wild-type genetic background in which they occur. Although such background dependence is widely observed, whether general patterns across species and traits exist is not known, nor the mechanisms underlying it understood. Knowledge is also lacking on how mutations interact with genetic background to influence gene expression, and how this in turn mediates mutant phenotypes. Furthermore, how genetic background influences patterns of epistasis remains unclear. To investigate the genetic basis and genomic consequences of genetic background dependence of the scallopedE3 allele on the Drosophila melanogaster wing, multiple novel genome-level datasets were generated from a mapping-by-introgression experiment and a RNA gene expression dataset was tagged. In addition whole genome re-sequencing of the parental lines, two commonly used laboratory strains, were used to predict polymorphic transcription factor binding sites for SD. These data were integrated with previously published genomic datasets from expression microarrays and a modifier mutation screen. By searching for genes showing a congruent signal across multiple datasets, it was possible to identify a robust set of candidate loci contributing to the background-dependent effects of mutations in sd. It was also shown that the majority of background-dependent modifiers previously reported are caused by higher-order epistasis, not quantitative non-complementation. These findings provide a useful foundation for more detailed investigations of genetic background dependence in this system, and this approach is likely to prove useful in exploring the genetic basis of other traits as well (Chandler, 2014).


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

date revised: 25 March 2015 

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