Gene name - vestigial
Cytological map position - 49D2-E1
Function - Presumptive transcription factor
Symbol - vg
Genetic map position - 2-67.0
Classification - novel protein
Cellular location - nuclear and cytoplasmic
|Recent literature||Pimmett, V.L., Deng, H., Haskins, J.A., Mercier,
R.J., LaPointe, P. and Simmonds, A.J. (2017). The
activity of the Drosophila Vestigial protein is modified by
Scalloped-dependent phosphorylation. Dev Biol [Epub ahead of
print]. PubMed ID: 28322734
The Drosophila vestigial gene is required for proliferation and differentiation of the adult wing and for differentiation of larval and adult muscle identity. Vestigial is part of a multi-protein transcription factor complex, which includes Scalloped, a TEAD-class DNA binding protein. Binding Scalloped is necessary for translocation of Vestigial into the nucleus. This study shows that Vestigial is extensively post-translationally modified and at least one of these modifications is required for proper function during development. There is p38-dependent phosphorylation of Serine 215 in the carboxyl-terminal region of Vestigial. Phosphorylation of Serine 215 occurs in the nucleus and requires the presence of Scalloped. Comparison of a phosphomimetic and non-phosphorylatable mutant forms of Vestigial shows differences in the ability to rescue the wing and muscle phenotypes associated with a null vestigial allele.
Vestigial is a novel nuclear protein with no known homologs, except for an N-terminal domain resembling that of Paired. vestigial expression is evident in thoracic and abdominal segments, in the embryonic primordia of the wing and haltere discs, in discrete cells in the ventral nerve cord, and possibly in progenitors of sense organs of the peripheral nervous system (Williams, 1991). There are no phenotypes associated with vestigial mutation, either in segmentation or neural development, but mutant flies manifest a complete lack of balance organs of the third thoracic segment, as well as a lack of development of wings and halteres.
Cells lacking vestigial undergo extensive cell death in the presumptive wing region of the third-larval instar imaginal discs. This results in the complete elimination of wing structures in adults. Several observations link vestigial function with cell survival and proliferation. Combining vestigial mutation with mutations that cause cell hypertrophy (too many cells) results in a partial reversal of the vestigial phenotype. Mutations in Drosophila tumor suppressor genes giant discs (lgd) and fat (ft) cause epithelial hypertrophy in all imaginal discs. Combining either lgd or ft mutations with vg increases the size of the wing disc and partially restores the bristle pattern (Agrawal, 1995).
vestigial has also been linked to regulation of cell proliferation. Treatment of flies with drugs that inhibit the enzyme dihydrofolate reductase gives rise to a defect that resembles vestigial mutation: nicks in the wings of wild-type flies and a strong vg phenotype in flies heterozygous for a deficiency of the vg locus. The effect of these drugs is to reduce the level of vestigial transcription, suggesting that the level of vestigial transcription is regulated by metabolites of dihydrofolate reductase. Specific mutations in the vestigial locus result in a diminished response to these foliate inhibitors, restoring normal development. These mutations do not knock out vestigial but result in an altered protein (Zider, 1996). The nicking phenomenon induced by drug treatment and its reversal by genetic modifications in vestigial, suggest a role for vestigial in cell proliferation. Thus future research in vestigial biology may reveal an interesting interaction with genes involved in regulation of cell cycle.
The wing margin represents a site of complex developmental interactions resulting in formation of a boundary separating dorsal and ventral cells and induction of cell proliferation generating the familiar adult structure (see the segment polarity and fringe sites for further discussion of events at the wing margin). An intronic enhancer, located between the first and second exons of vestigial, is responsible for vestigial transcription at the wing margin, assuring the presence of vestigial at the margin. Transcription at the margin requires an interaction between Notch and its ligands Serrate and Delta, and appears to be downstream of wingless (Blair, 1994 and Doherty, 1996).
Is vestigial regulated directly by Notch signaling or only via an intervening wingless activity? Notch-dependent activation of wg, cut and vestigial at the wing margin depends on the activity of Suppressor of Hairless. Su(H)-mutant cells lose expression of the vestigial early enhancer, of wingless and of cut in a cell autonomous manner. Clones of Su(H)-mutant cells cause loss of wing tissue and scalloping of the wing, but only in Notch mutant clones at the D/V boundary. vestigial expression at the D/V boundary does not depend on wingless, since misexpression of wild-type wg cDNA, which results in wing margin bristles, does not cause an expansion of vestigial expression. Likewise, wingless expression does not depend on an early function of vestigial. Both Notch and wingless cooperate to activate cut at the D/V boundary. Later expression of vestigial in the wing pouch is, however, wingless dependent. vestigial is expressed in a broad domain throughout the wing. Removing of Wg activity in late second instar larvae leads to almost complete loss of the secondary expression of vestigial in the wing pouch without affecting expression at the D/V boundary. Taken together with the observation that clones of cells lacking shaggy activity show a cell-autonomous increase of Vestigial expression, these results suggest the vestigial is a direct target of the Wg pathway (Neumann, 1996).
A small number of major regulatory (selector) genes have been identified in animals that control the development of particular organs or complex structures. In Drosophila, the vestigial gene is required for wing formation and is able to induce wing-like outgrowths on other structures. Because ectopic expression of Vg in many imaginal discs induces the outgrowth of wing tissue, the expression of various wing patterning genes was examined to see if they are induced in ectopic growths. Vg is expressed in the entire developing wing pouch whereas Spalt and Serum response factor (SRF) have specific expression patterns within this domain but are not expressed in wild-type leg discs. Targeted expression of Vg with the Gal4-UAS system induces ectopic expression of Sal and SRF in developing leg imaginal discs. Similarly, the nubbin (nub) gene (which is also expressed and required during wing development ) is ectopically induced in leg discs by Vg expression. In each case, only a subset of the cells expressing Vg activate the target gene, which suggests that additional factors control the expression pattern of each gene. In a first step toward elucidating the molecular mechanism by which Vg regulates gene expression, the response of wing-specific enhancers to ectopic Vg expression was examined. Attention was focused on both the boundary and quadrant enhancers of the vg gene and the enhancer from the SRF gene that drives expression specifically in the intervein region between veins three and four. All three enhancers are induced by ectopic Vg expression in leg and other imaginal discs. Importantly, ectopic expression of Vg in clones of cells induces the enhancers only within the clones. However, gene expression is not induced in all cells within clones nor in all clones. In addition, each individual enhancer is expressed in different regions of these discs that appear to correlate with the spatial distribution of the different signaling inputs known to be required for activation of these enhancers (Halder, 1998).
Scalloped is required for Vg function. In the notum primordia of the wing disc, the vg enhancers, as well as the sal, SRF, and nub genes are not induced by ectopic Vg even though the known required extracellular signals are present. Target gene activation could depend then on the function of another gene(s). One candidate for such a factor is the product of the sd gene, which is expressed in a pattern similar to Vg in the wing disc and is required for wing formation and the proper expression of Vg and other genes. In other discs, such as the leg and eye discs, sd is endogenously expressed and is upregulated wherever ectopic Vg is able to induce wing-specific gene expression and trigger wing development. It is noted, however, that a sd enhancer trap line and the SRF-intervein C enhancer transgene are also ectopically induced by Vg in the presumptive notum, although at levels lower than those observed in the wing pouch. This is consistent with the inability of Vg to trigger wing development and induce other wing patterning genes in the developing notum. Indeed, mis-expression studies show that Sd function is required in parallel with Vg in order for Vg to exert its wing inducing activity. The three wing-specific enhancers from the SRF and vg genes are activated synergistically when Sd and Vg are coexpressed in Drosophila S2 cells. Although each individual protein has some effect on reporter gene expression, this is significantly less than that observed in the presence of both Vg and Sd. Titration of the amounts of transfected Vg and Sd plasmids with all enhancers shows that the relative concentration of the two factors is critical and, at any given Vg concentration, high levels of Sd reduce activation (Halder, 1998).
To define the sequences of the enhancers that respond to Vg/Sd, the activation of smaller fragments from the 704-bp SRF intervein C enhancer, the 806-bp vg quadrant enhancer, and the 754-bp vg boundary enhancer in tissue culture were analyzed. A 125-bp fragment (SRF-A) derived from the 5' end of the SRF enhancer is activated, whereas an adjacent 131-bp fragment (SRF-B) is not activated. A 65-bp fragment from the vg quadrant enhancer (MD2) has been identified that, when multimerized, produces an expression pattern very similar to the full-length enhancer in wing discs. When assayed in tissue culture, MD2 is activated by Vg and Sd. Within the vg boundary enhancer, a 120-bp fragment sufficient to drive reporter gene expression in the wing pouch (vg-A) as well as a nonoverlapping 90-bp fragment (vg-B) are also activated synergistically by cotransfection of Vg and Sd. Sd was shown, using mobility shift and DNase I footprinting assays, to bind specifically to essential sites for target gene activation (Halder, 1998).
One possible reason for the importance of the concentration of Sd on Vg function concerns the localization of the Vg protein. It was observed that in S2 cells transfected with the vg expression plasmid alone, the Vg protein appears to be localized to both the cytoplasm and the nucleus. In contrast, in cells cotransfected with the Vg and Sd expression plasmids, Vg is clearly localized to nuclei. Vg localization is more diffuse in sd mutant clones than in sd+ cells; this is also true of ectopic Vg localization in regions of imaginal discs that lack endogenous Sd expression. Furthermore, deletion of the Sd interaction domain of Vg results in cytoplasmic accumulation of Vg in vivo. Thus, Sd may facilitate the transport or retention of Vg protein in the nucleus and, coupled with the concentration-dependent, synergistic effects of Vg and Sd on target gene expression, these results suggest that the proteins form a complex in vivo (Halder, 1998).
These results demonstrate that the activation of several genes in the wing field requires Vg/Sd function. It is also known that for each of the cis-regulatory elements analyzed here, direct input(s) of particular signaling pathways are also required. Specifically, the activation of the SRF intervein C element requires both Vg/Sd and Hh signaling; the activation of the vg boundary enhancer requires Vg/Sd and N signaling, and the activation of the vg quadrant enhancer requires Vg/Sd and Dpp signaling. Because these regulatory elements are not expressed in all tissues in which the signals are active, nor in all wing cells in which Vg/Sd are active, it is deduced that neither the input of various signals nor of Vg/Sd alone are sufficient for gene activation in vivo. Rather, the results suggest that the various wing-specific cis-regulatory elements require a combination of direct inputs, comprising the Vg/Sd selector function, which restricts expression to the wing field, and at least one signal transducer that mediates signaling inputs and hence, the pattern of gene expression within the wing field. One prediction of this model is that gene expression patterns within the wing field may be changed by altering the signal-transducer binding sites within a cis-response element. To test this, the Suppressor of Hairless [Su(H)] binding site that mediates the N input in the vg boundary enhancer was changed to sites for the Cubitus interruptus (Ci) protein that transduces Hh signaling. This switches the pattern of gene expression from a N-induced dorsoventral stripe to a Hh-induced anteroposterior stripe while retaining the restriction of gene activation to the wing disc (Halder, 1998 and references).
These results demonstrate that the role of the Vg/Sd selector function is to directly regulate wing-specific cis-regulatory elements that also require particular signaling inputs. The patterns of gene expression induced in the wing disc are limited to cells in which both the selector genes and specific signaling pathways are active. The response of the SRF-A, vg boundary, and vg quadrant enhancers to Hh, N, and Dpp signaling are limited to the wing pouch by Vg/Sd and occur in different patterns because of their direct regulation by the Ci, Su(H), and Mad proteins, respectively. Furthermore, the finding that the changing of the Su(H) binding site into a Ci binding site in the vg boundary enhancer switches the pattern from a wing-specific dorsoventral N-regulated stripe to a wing-specific anteroposterior Hh-regulated stripe suggests that spatial expression patterns are determined by the sites for individual DNA-binding signal transducers. One corollary of this model is that for any given signaling protein, different selector proteins may be involved in directing tissue-specific responses in different organs and tissues. For example, other studies have shown that tissue-specific enhancers in the embryo that are regulated by Dpp also require the action of the Labial/Extradenticle or Tinman selector proteins to limit expression to the endoderm or mesoderm, respectively. It is suggested that, in general, combinatorial control by selector proteins and common signal transducers at a cis-regulatory level is required for the tissue- and organ-specific responses of target genes to widely deployed signaling systems (Halder, 1998 and references).
The Drosophila wing primordium is defined by expression of the selector gene vestigial (vg) in a discrete subpopulation of cells within the wing imaginal disc. Following the early segregation of the disc into dorsal (D) and ventral (V) compartments, vg expression is governed by signals generated along the boundary between the two compartments. Short-range DSL (Delta/Serrate/LAG-2)-Notch signaling between D and V cells drives vg expression in 'border' cells that flank the boundary. It also induces these same cells to secrete the long-range morphogen Wingless (Wg), which drives vg expression in surrounding cells up to 25-30 cell diameters away. Wg signaling is not sufficient to activate vg expression away from the D-V boundary. Instead, Wg must act in combination with a short-range signal produced by cells that already express vg. Evidence that this vg-dependent, vg-inducing signal feeds forward from one cell to the next to entrain surrounding cells to join the growing wing primordium in response to Wg. It is proposed that Wg promotes the expansion of the wing primordium following the D-V segregation by fueling this non-autonomous autoregulatory mechanism (Zecca, 2007a; full text of article)
Following the D-V segregation, local DSL-Notch signaling across the compartment boundary induces the differentiation of specialized border cells that express vg, secrete Wg, and organize a dramatic ~200-fold expansion of the wing primordium. In ap0 wing discs, D-V segregation fails to occur, border cells are not specified, and the early expression of vg that initially defined the wing primordium fades away. This mutant condition was used to explore how vg and wg activity in border cells controls wing growth by asking what happens when the missing border cells were replaced with cells that ectopically express Wg, Vg or both (Zecca, 2007a).
The main finding of this study is that Wg is not sufficient to sustain or induce vg expression in ap0 discs, even when the morphogen is overexpressed, continuously, in all cells. Instead, Wg can only drive vg expression in these discs when the responding cells are near or next to cells that express exogenous Vg. The clearest demonstration of this is the experiment in which two types of clones were generated in the same ap0 disc: clones that express Nrt-Wg, a membrane tethered immobile form of Wg, and clones that express moderate levels of exogenous Vg. Neither type of clone, alone, can restore normal expression of the endogenous vg gene. However, ectopic Vg-expressing clones can induce neighboring Nrt-Wg-expressing clones to express vg, provided that they abut. Moreover, this vg expression can spread through the Nrt-Wg-expressing clone and extend to adjacent cells outside the clone (Zecca, 2007a).
These results indicate that vg-expressing cells send a short-range, possibly contact-dependent signal that is required to entrain neighboring cells to express vg in response to Wg. Furthermore, they indicate that once the responding cells express vg, they can in turn entrain their neighbors in the same way, propagating the recruitment of additional cells into the wing primordium. These findings establish the existence of a Wg-dependent feed-forward circuit of vg autoregulation and suggest that D-V border cells normally organize wing growth by providing Wg, as well as the initial Vg-dependent entraining signal that triggers reiteration of this autoregulatory circuit from one cell to the next (see feed-forward circuit of Wg-dependent vg autoregulation in Drosophila). Thus, feed-forward regulation in this context has a spatial component, mediating the expansion (in mass and cell number) of a developing primordium by a process of recruitment (Zecca, 2007a).
These results are concordant with previous reports that Wg signaling cannot drive vg expression in the wing imaginal disc in the absence of border cells, and that co-overexpression of Wg and Vg can synergize to drive vg expression in surrounding cells. However, the current findings advance these results in three significant ways. First, it was shown that vg-expressing cells provide a discrete second signal, required together with Wg, to induce vg expression in surrounding cells. Second, it was demonstrated that production of this signal can propagate from one cell to the next, establishing a feed-forward autoregulatory mechanism fueled by morphogen. Third, it was shown that physiologically normal levels of wg and vg activity are sufficient to initiate and propagate this feed-forward mechanism, establishing that it is a natural process and not an overexpression artifact (Zecca, 2007a).
The capacity of Wg to drive recruitment of new cells into the wing primordium by fueling vg feed-forward autoregulation provides one mechanism for promoting wing growth. However, it appears to operate within the context of other mechanisms for promoting wing growth, as well as for limiting where and when such growth occurs. At least three additional mechanisms for promoting wing growth, all dependent on Wg, an be distinguished. First, in addition to recruiting new cells into the wing primordium, Wg acts continuously to retain cells that were previously recruited: wing cells in which Wg transduction is abrogated rapidly lose vg expression and either die, or sort out. It is suggested that retention, like recruitment, depends on the same Wg-dependent vg autoregulatory circuit. Specifically, it is posited that the feed-forward signal is required both to induce vg expression in cells about to enter the primordiium, as well as to maintain vg expression in cells after they enter (Zecca, 2007a).
Second, independent of its role in fueling vg autoregulation, Wg also appears necessary for the survival and proliferation of vg-expressing wing cells. It is possible to bypass the requirement for Wg-dependent vg autoregulation by using a Tubalpha1>vg transgene to express exogenous Vg: nevertheless, such 'rescued' Tubalpha1>vg wing cells still require Wg input to survive, grow and proliferate (Zecca, 2007a).
Third, cells are normally recruited into the vg-expressing population from a surrounding population defined by detectable expression of rn but not vg. Accordingly, the 'rn-only' population must proliferate in conjunction with the growth of the wing primordium; otherwise, it would be depleted, limiting further recruitment and compromising the development of more proximal structures. In support, it was found that the rescue of the wing primordium by Wg-dependent vg autoregulation is associated with the rescue and expansion of the surrounding population of rn-only cells. Hence, once cells are recruited into the wing primordium in response to Wg, they may send an additional signal to sustain the source population of rn-only cells from which additional wing cells will be recruited (Zecca, 2007a).
Conversely, at least three mechanisms can be distinguised that appear to constrain operation of the feed-forward circuit, limiting expansion of the wing primordium in space and time. First, is the early segregation of the wing imaginal disc into distinct distal (pre-blade) and proximal (pre-hinge/notum) compartments, only one of which, the pre-blade, is competent to engage the feed-forward autoregulatory circuit. This event, which occurs before D-V compartmental segregation, appears to be governed by an early burst of Wg signaling that selectively and heritably represses tsh expression in the founder cells of the putative pre-blade (tshOFF) compartment. Although Wg-dependent vg autoregulation normally appears to operate only within the resulting pre-blade (tshOFF) compartment (which includes the rn-only domain, as well as the presumptive wing pouch), this limit can be exceeded if cells are exposed to ectopic Wg signal before they would otherwise segregate into the pre-hinge/notum (tshON) compartment. It is suggested that this ectopic Wg activity inappropriately blocks tsh activity in the prospective pre-hinge/notum, creating an ectopic pre-blade compartment in which feed-forward regulation can occur (Zecca, 2007a).
Second, is the availability of Dpp secreted by A compartment cells along the A-P compartment boundary. Dpp, like Wg, is essential for vg expression and wing growth. Hence, operation of the feed-forward mechanism might depend on the combined inputs of Wg and Dpp, centering the expanding domain of Wg-dependent vg expression on the intersection between the D-V and A-P compartment boundaries. In agreement, evidence for Wg-dependent feed-forward propagation is observed only in cells located within ~25 cell diameters of the A-P boundary, the expected range of Dpp emanating from A cells along the boundary (Zecca, 2007a).
Third, operation of the vg feed-forward circuit might be temporally constrained. It is striking that vg is initially expressed in ap-null discs up until the time the D-V compartmental segregation would normally occur; yet, flooding such discs with exogenous Wg signal is not sufficient to sustain and propagate this early vg expression. By contrast, clones of Tubalpha1-vg cells generated in these same discs are effective in triggering the propagation of vg expression in surrounding cells, suggesting that cells within the 'pre-blade' become competent to operate the feed-forward autoregulatory circuit only after the time at which the D-V segregation normally occurs, concomitant with the differentiation of wg- and vg-expressing border cells (Zecca, 2007a).
Thus, it is proposed that following the D-V segregation, Wg drives wing growth by at least four distinct outputs: first, by recruiting new cells into the wing primordium; second, by maintaining the recruited cells and their descendents within the primordium; third, by sustaining the survival and proliferative growth of cells defined as 'wing' by the selector activity of Vg; and finally, by acting through the agency of newly recruited wing cells to induce the expansion of the surrounding population of rn-only cells from which additional wing cells will be recruited. Counterbalancing these effects would be a requirement for heritable repression of tsh, availability of Dpp, and transition to a discrete phase of wing disc development during which the feed-forward circuit can operate. Within these constraints, the size of the wing primordium at any point following the D-V segregation would reflect the increasing range of Wg emanating from the D-V border cells via its capacity to propagate and sustain the vg autoregulatory circuit and, separately, its capacity to promote the proliferative growth of vg- and rn-only-expressing cells (Zecca, 2007a).
Following segregation of the Drosophila wing imaginal disc into dorsal (D) and ventral (V) compartments, the wing primordium is specified by activity of the selector gene vestigial (vg). Evidence is presented that vg expression is itself driven by three distinct inputs: (1) short-range DSL (Delta/Serrate/LAG-2)-Notch signaling across the D-V compartment boundary; (2) long-range Wg signaling from cells abutting the D-V compartment boundary; and (3) a short-range signal sent by vg-expressing cells that entrains neighboring cells to upregulate vg in response to Wg. These inputs define a feed-forward mechanism of vg autoregulation that initiates in D-V border cells and propagates from cell to cell by reiterative cycles of vg upregulation. Evidence is provided that this feed-forward mechanism is required for normal wing growth and is mediated by two distinct enhancers in the vg gene. The first is a newly defined 'priming' enhancer (PE), that provides cryptic, low levels of Vg in most or all cells of the wing disc. The second is the previously defined quadrant enhancer (QE), which activates by the combined action of Wg and the short-range vg-dependent entraining signal, but only if the responding cells are already primed by low-level Vg activity. Thus, entrainment and priming constitute distinct signaling and responding events in the Wg-dependent feed-forward circuit of vg autoregulation mediated by the QE. It is posited that Wg controls the expansion of the wing primordium following D-V segregation by fueling this autoregulatory mechanism (Zecca, 2007b).
The dramatic expansion of the Drosophila wing primordium following the D-V compartmental segregation provides a valuable paradigm of organ growth. Growth in this context is manifest as a rapid ~200-fold expansion of the population of cells expressing the wing selector gene vg, under the control of the long-range morphogens Wg and Dpp. This system thus poses the fundamental question of how morphogens organize the increase in the mass and number of cells that express a given selector gene, to yield an adult appendage of appropriate size and shape (Zecca, 2007b).
A novel autoregulatory property of vg has been defined that appears crucial for this process. Evidence is presented that vg-expressing cells send a short-range feed-forward signal that neighboring cells must receive in order to express vg in response to Wg. This led to a hypothesis that Wg controls wing development by fueling this non-autonomous autoregulatory mechanism. This study establishes that the vg quadrant enhancer (QE) can mediate vg autoregulation in response to Wg and then uses a transgene that expresses Vg under QE control to provide a proof-in-principle that wing growth normally depends on the operation of the autoregulatory circuit (Zecca, 2007b).
Wing growth following D-V segregation is envisioned as an outcome of vg autoregulation, primed by cryptic, low-level Vg in all cells that is seeded by DSL-Notch-mediated induction of specialized D-V border cells that express high levels of vg and wg, and then propagated by the capacity of vg-expressing cells to induce and sustain vg expression in neighboring cells in response to Wg. In support, it has been possible to restore wing growth in vg0 discs in a step-wise manner by the sequential addition of transgenes that provide, first priming (rp49-vg), then initiation (BE-vgGFP), and finally feed-forward propagation (5XQE>vg). Priming is necessary but not sufficient for wing development, initiation provides local rescue of wing tissue, and propagation is responsible for the dramatic expansion in the size of the prospective wing (Zecca, 2007b).
Importantly, priming and feed-forward signaling are linked in a self-reinforcing autoregulatory circuit in which a gain in either input leads to an amplification of both. It is envisaged that the QE normally integrates both the priming and feed-forward inputs together with Wg in a way that is sensitive to the initial strength of each input and subject to autoamplification. For example, in the 'resting' state, cells have a low level of priming that falls beneath the minimal threshold necessary to specify the wing state or generate appreciable feed-forward signal. Upon receipt of sufficient Wg and feed-forward signal, the level of Vg expression rises, crossing the threshold defining the wing state and enhancing the capacity of the responding cell both to send and to receive the feed-forward signal. Amplification of this circuit then leads to the maximum output of Vg expression and feed-forward signaling that can be supported by the strength of the Wg signal received (Zecca, 2007b).
The self-reinforcing nature of this autoregulatory circuit, both between and within cells, helps explain how Wg spreading from D-V border cells normally fuels the expansion of the population of vg-expressing cells. It also helps account for the unexpected responses observed in experiments using the rp49-vg, BE-vgGFP and 5XQE>vg transgenes to mimic the normal priming, initiation and feed-forward inputs. All of these transgenes depend on heterologous promoters and potentially complex enhancer elements operating outside of their normal genomic contexts. Consequently, weak, inappropriate activities of any of these transgenes (e.g. cryptic priming by BE-vgGFP and 5XQE>vg transgenes, or faint QE activity of the BE-vgGFP transgene) could be amplified by the autoregulatory circuitry, yielding spatially inappropriate responses. Nevertheless, despite these experimental limitations, the results indicate that the major factor governing the expansion of the wing primordium is feed-forward autoregulation mediated by the QE (Zecca, 2007b).
Wing growth does not depend solely on the capacity of Wg to recruit and maintain cells in the wing primordium by fueling vg autoregulation. Instead, even when wing pouch cells are supplied constitutively with exogenous Vg (thus bypassing the requirement for vg autoregulation), they still depend on continuous Wg input to survive and grow within the context of the wing primordium. This is in contrast to cells in the more proximal hinge and notum primordia, which survive and grow without Wg input. Thus, Wg appears to promote wing growth via two distinct mechanisms: by continuously 'selecting' which cells enter and remain within the wing primordium, and by allowing the survival and growth of cells so selected. The relative contributions of these two mechanisms cannot be distinguished. However both appear essential, as cells fail to enter, or stay, within the wing primordium when either one is eliminated (Zecca, 2007b).
Wing growth depends not only on Wg emanating from D-V border cells, but also on Dpp secreted by A compartment cells along the A-P compartment boundary, suggesting that the QE might mediate feed-forward autoregulation in response to Dpp, as well as Wg. In support, the QE contains binding sites for the Dpp transducer Mad, and there is evidence that these sites, as well as Mad itself, contribute to QE activity. Moreover, clones of cells that cannot transduce Dpp behave like those that cannot transduce Wg: they cease to express Vg and are lost specifically from the wing primordium, in contrast to clones located in the more proximal hinge and notum primordia. Hence, it is likely that Dpp and Wg act together to fuel the feed-forward autoregulatory circuit, and by so doing, regulate the size and shape of the developing wing (Zecca, 2007b).
The ability of Wg, and potentially Dpp, to promote wing growth by fueling a non-autonomous autoregulatory circuit of vg expression is, to our knowledge, novel, and has implications for the control of organ growth by morphogens. As epitomized by the developing wing, a long-standing enigma is that gradient morphogens drive relatively uniform growth and proliferation across a tissue at the same time that they function in a concentration-dependent manner to organize complex patterns of gene expression and cell differentiation. It is suggested that a minimum threshold level of morphogen might be sufficient to fuel both feed-forward autoregulation of organ selector genes and the growth and proliferation of cells so selected. Accordingly, organ growth would be governed primarily by the progressive expansion in the range of morphogen (a process that might itself depend on the ability of morphogen to regulate expression of its receptors and other binding proteins) and by any boundary conditions that limit the availability and capacity of surrounding cells to respond (Zecca, 2007b).
Exons - 8
Base pairs in 3' UTR - 549
Vestigial is a novel protein with no known homologs. The protein regions flanking the exon 4 glycine repeat are probably functional, because strong protein sequence conservation between D. melanogaster and D. virilis is seen in the intervals flanking the exon 4 glycine-rich interval. Two regions containing alternating histidine residues are present in the amino-terminal domain and are similar to the paired or His-Pro repeat. Similar regions are found in paired, bicoid, daughterless, Deformed and E74. Homology in this region is most similar to Deformed (Williams, 1991).
date revised: 3 July 97
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