vestigial and development of drosophila wing · vestigial and development of drosophila wing 915...

INTRODUCTION

The development and patterning of structures such as teeth,limbs, heart or liver are complex processes that rely on thespatial and temporal orchestration of cell interactions. These inturn rely on signalling systems but, in the last few years, it hasbecome clear that a specific structure is not the outcome ofspecific signalling events. On the contrary, the availableinformation suggests that there is a limited number ofsignalling systems which often work through a small numberof effectors to elicit a great variety of responses. The specificityof the outcome resides on the effectors available to thesignalling system through the history of the cell. For example,in vertebrates, Sonic Hedgehog (Shh) is involved in thedevelopment and patterning of the limb, the specification of thefloor plate, the determination of neuronal cell fates and otherprocesses (Riddle et al., 1993; Echelard et al., 1993). For eachof these functions, Shh controls specific sets of genes.However, it always does it through the same signal transductionpathway and therefore something has to confer a particularinterpretation of these signals in a specific manner: tooth, limb,heart or liver.

As a group of cells develop into a particular structure,something has to keep the ‘group identity’ constant. The wingof Drosophila provides a useful system to unravel themolecular basis of this notion of identity. Developmental

studies have shown that the vestigial(vg) gene encodes anuclear protein that plays a central role in the development ofthe wings in Drosophila: loss of vg results in the failure ofwings to develop (Lindsley and Zimm, 1992) and ectopicexpression of vg leads to the development of ectopic wingtissue (Kim et al., 1996). These experiments suggest thatvestigialcan be viewed as a ‘wing selector’ gene i.e. a genethat enforces wing developmental pathway on cells (Kim et al.,1996).This notion is supported by the observation that ectopicexpression of vestigial can rescue the loss of wing caused bymutations in genes involved in the regulation of wingdevelopment such as apterous, Suppressor of Hairlessorwingless(Klein and Martinez Arias, 1998a).

The expression of vg during wing development is regulatedthrough two enhancers called the second intron or boundaryenhancer (vgBE), and the quadrant enhancer (vgQE) (Williamset al., 1994; Kim et al., 1996; and Fig. 1). These names reflectthe patterns of expression directed by these regulatory regions:the vestigial Boundary Enhancer (vgBE), a thin stripe over theprospective wing margin, and vestigial quadrant enhancer(vgQE), a pattern in four quadrants that are complementary tothe vgBE and which fill in the developing wing blade. Both,vgBE and vgQE, act as integrators of signalling systems thatdrive wing development and, in this manner, these regulatoryregions determine the tempo and the mode of wingdevelopment (Williams et al., 1994; Kim et al., 1996).

913Development 126, 913-925 (1999)Printed in Great Britain © The Company of Biologists Limited 1999DEV1380

The vestigial(vg) gene of Drosophilaplays a central role inthe development and patterning of the wing: loss of vestigialresults in failures in wing development and ectopicexpression of vestigial leads to the development of ectopicwings. The wing-specific regulation of vestigial is mediatedthrough two enhancers: (1) the Boundary Enhancer (vgBE)is early acting and becomes restricted to the wing margin,and (2) the Quadrant Enhancer (vgQE), acts later and isresponsible for the expression of vestigial in the developingwing blade. These enhancers receive regulatory inputsfrom three signalling pathways: wingless, decapentaplegicand Notch/Suppressor of Hairless. Our experiments showthat the vestigial gene product is also an input in theregulation of vestigial expression. In particular, Vestigial

provides an important input for the regulation of theactivity of the vgQE acting in concert with Wingless andDecapentaplegic. Our results suggest how interactionsbetween vgBE and the vgQE mediated by Vestigial canexplain the interactions between the wing margin and thewing blade during the growth of the wing. We further showthat Vestigial and Notch collaborate with Wingless tosubdivide and pattern the wing blade. These results lead usto propose a general role for Wingless during developmentin which it stabilizes cell fate decisions that have beenimplemented by other molecules.

Key words: Drosophila, Vestigial, Wing, Boundary Enhancer,Quadrant Enhancer, Notch, Wingless, Decapentaplegic

SUMMARY

The Vestigial gene product provides a molecular context for the interpretation

of signals during the development of the wing in Drosophila

Thomas Klein and Alfonso Martinez Arias

Department of Zoology, University of Cambridge, Cambridge CB2 3EJ, UKCorrespondence (e-mail: [email protected] and [email protected])

Accepted 8 December 1998; published on WWW 2 February 1999

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However, the function of vgand, more specifically, how itendows cells with identity is not clear. One possibility is thatVestigial regulates patterns of proliferation and geneexpression that are characteristic of wing tissue. However,there is no direct evidence for this.

Here we study the regulation of the vg gene. We show thatVestigial is required for its own expression and that the threesignalling systems known to play a major role in wingdevelopment, Notch, Wingless and decapentaplegic (dpp), notonly act through the known enhancers of vg, but also showsynergistic interactions with the vg gene product. Theseobservations lead us to suggest that vestigial specifies identityin part by providing a molecular context in which differentsignalling events act to trigger the development of a wing.Perhaps by responding to this signalling system, Vestigialtargets factors onto specific genes required for the proliferationand patterning of the wing. Our results further show that vgplays an important role together with, rather than simplydownstream of, winglessin the patterning of the wing bladealong the dorsoventral axis. The experiments suggest that themain function of wg is to enforce geneexpression rather than initiate it.

MATERIALS AND METHODS

Drosophila stocksThe Sermutants used in this study are SerRX106

(Speicher et al., 1994) and Ser94C (Couso et al.,1995). Expression of vg at the DV boundarywas detected using the vestigial BoundaryEnhancer described in Williams et al. (1994)and referred to here as vgBE (for Boundaryenhancer). The vg-quadrant enhancer isdescribed in Kim et al. (1996, 1997b) and isreferred to here as vgQE. For the expression ofwg in the developing discs, we sometimes useda P-lacZ insertion in the wg gene on a CyOchromosome (Kassis et al., 1992).

Lines allowing us to overexpress thedominant negative UAS-wg carry a winglessgene with a stop codon at codon 398 of thewinglesscoding region (Zecchini and A. M. A.,unpublished data). This C-terminal deletionmimics a mutation in XenopusWnt (Hoppler etal., 1996) and causes dominant negative effectsin the embryo and the adult; details of theconstruct and the flies themselves are availableupon request.

Ectopic expression of the different genes wasachieved through the GAL4/UAS system ofBrand and Perrimon (1993). The UAS-vgconstructs were kindly provided by SeanCarroll (Kim et al., 1996); UAS-wg by K.Gieseler and J. Pradel; UAS-Nintra and UAS-Dl by L. Seugnet and M. Haenlin. The UAS-GFP is described in Yeh et al. (1995).

The expression of the different UASconstructs was driven in the imaginal discs withvarious GAL4 inserts.patched Gal4 (ptcGAL4) expresses UAS-X in a stripe along theAP boundary of the discs (Speicher et al.,1994). In the third instar, decapentaplegicGal4(dpp GAL4) (Wilder and Perrimon, 1995) is

expressed in a similar pattern to ptcGAL4, although the expressionis weaker over the ventral side (Klein and Martinez Arias, 1998a). ThevgBEGal4 is described in Neuman and Cohen (1996a).

Stocks carrying various GAL4 and UAS combinations in wild typeand mutants were generated. All stocks were balanced over the SM6a-TM6b compound balancer, which allowed the identification of larvaeof the correct genotype because of the dominant larval marker Tb(Lindsley and Zimm, 1992). Details of the stocks as well as the stocksthemselves are available upon request. In the case of ap mutations,stocks were established with a CyO balancer carrying a P-lacZinsertion in wgand the mutant discs were checked for the absence ofthe wg expression pattern. In the case of some experiments involvingap mutants, mutant discs were identified by the aberrant morphologyof the wing disc and the absence of the CyO wg-lacZbalancer.

Immunohistochemistry and in situ hybridizationThe Distalless antibody was a gift from S. Cohen and S. Bray andstainings were performed according to standard protocols. In situhybridizations were performed as described in Tautz and Pfeifle(1989). The X-Gal staining is described in Ashburner (1989). Thefluorescence of the green fluorescent protein (GFP) was detected byusing the FITC-filter sets on a Zeiss Axiophot microscope.

T. Klein and A. Martinez Arias

Fig. 1.Expression of lacZdirected by the vestigial boundary enhancer, vgBE (A-C), thevestigial quadrant enhancer, vgQE (E-G), both together (D) and the endogenous Vestigialprotein (H), throughout wing development. All discs shown with anterior to the left andventral to the bottom. (A) Activity of the vgBE is detected in the second instar in ahorseshoe pattern, with weaker expression in a region that fate maps to the anterior ventralregion of the disc. (B) At the beginning of the third instar, the vgBE is active in a stripe thatfate maps the wing margin and extends on either side of the wing disc into the flanks of thenotum. At this stage, the wing primordium is the domain of the vgBE in the center of thedisc. (C) The pattern defined at the beginning of the third instar is maintained throughoutthis stage, with the addition of a stripe along the AP boundary. (D) The patterns of the vgBEand vgQE, together, make up for the pattern of vgexpression (compare with H) during wingdevelopment (Kim et al., 1996). (E) The vgQE is not active in the second instar, but becomesactivated at the beginning of the third instar in a region around the intersection of the AP andDV boundaries (arrowhead). (F,G) Throughout later stages of the third instar, the activity ofthe vgQE outlines the developing wing pouch, is excluded from the wing margin and islower in the regions between the proteins. (H) Pattern of Vestigial protein expression in athird instar wing disc. Comparison with C, D and G indicates that vgBE and vgQE accountfor the complete pattern of vgexpression, except in the adepithelial cells underneath thenotal region.

915Vestigial and development of Drosophila wing

RESULTS

The expression of vgduring wing development is regulated bytwo enhancer sequences located in the second and fourthintrons of the gene (Williams et al., 1994; Kim et al., 1996).The second intron enhancer regulatesexpression of vg along the DV boundary(Williams et al., 1994) and is thereforecalled the boundary enhancer (vgBE). Theenhancer located in the fourth intron iscalled quadrant enhancer (vgQE), isactivated much later than the vgBE (seeFig. 1 and Kim et al., 1996) and regulatesthe expression of vg in the pouch awayfrom the DV boundary. This enhancer isonly active in the wing pouch of the wingdisc and not in any other imaginal disc.

The development and patterning of thewing depends on a series ofinterdependent regulatory loops thatdefine a sequence of spatial patterns ofgene expression. For this reason, it isimportant to determine both spatial andtemporal aspects of the inputs on differentgenes (Klein and Martinez-Arias, 1998a).We do this below by focusing on theregulation and function of the two mainenhancers of the vg gene.

Early activity of the vestigial DVboundary enhancer ( vgBE)The vgBE is activated first during thesecond instar and its expression pattern isvery similar to that of the Vestigial proteinat this stage (Klein and Martinez Arias,1998b) suggesting that, in these earlystages, the vgBE is responsible for thecomplete pattern of vg expression (alsosee below). Deletion analysis of theenhancer reveals two regions essential forits activity: a binding site for Suppressorof Hairless and sequences contained in thefirst 80 base pairs of the enhancer(Williams et al., 1994; Kim et al., 1996,1997b).

In an attempt to map the nature andtiming of the inputs into this enhancer, wehave compared during wing developmentthe activity of the wild-type vgBE and ofdeletions of the two essential regions (Fig.2). To do this, we used lacZfusions andmonitored their expression in thedeveloping wing disc (Williams et al.,1994; Kim et al., 1996, 1997b). We findthat at the end of the second instar, lacZexpression from the wild-type vgBEoutlines a horseshoe over the ventralregion of the wing disc, with weakexpression in the ventral anterior regionwhere it overlaps with the expression ofwingless(Figs 1A, 2A,L). lacZexpression

increases in this region at the beginning of the third instar (Figs1B, 2B,C). This increase does not occur in Su(H)mutants (datanot shown), or in wg mutants (Klein and Martinez Arias,1998a).

The activity of the enhancer deleted for the Su(H)-binding

Fig. 2. Expression of lacZdirected by the wild-type and mutated vgBE in wild-type (A-C,E-G,I-L) and Su(H)mutant wing discs (D,H). All discs shown with anterior to the left andventral to the bottom. (Inserts in A,E and I) Higher magnifications of the ventral regionwhere the wing primordium is established. (A-C) Pattern of activity of the vgBE in wild-typewing discs of second (A), early third (B) and late third (C) instar. (D) In Su(H)mutant discs,the activity of the vgBE is abolished from the wing region and most of the notum. (E-G) Activity of a vgBE deleted for the Su(H)binding site. (E) In the second instar, there isno activity over the center of the disc (arrowhead; compare with A and see insert). Alsoduring later stages no activity was detected in the region of the developing wing (F,G).Notice that, nevertheless, there is activity of the enhancer at the flanking regions of the wingprimordium from early on. (H) Expression of lacZ directed by the vgBE deleted for the first80 bp in a Su(H)mutant. The residual notal expression indicates that there are other inputs inthis enhancer. (I-K) Expression of lacZdirected by the vgBE deleted for the first 80 bp in thewild type. (I) In the second instar, the activity of the deleted vgBE is similar to that of thewild-type one (see insert; compare with A). However, as the wing grows, there is noenhancement over the developing wing pouch and faint activity can be detected over the DVboundary as the disc grows (J) and eventually disappears (K). Very faint expression cansometimes be observed, perhaps due to perdurance of the lacZ (K). (L) Coexpression of lacZdriven by the vgBE lacking the first 80 bp and wg in a second instar disc. The region of lowactivity is covered by the expression of wg (compare with I). Since wgexpression defines theregion of the wing primordium, the double staining reveals that the region of low expressionis in the region of the developing wing. Arrowheads in A, E and I are indicating the regionwhere the wing primordium develops.

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site is different from the wild type. The activity of this enhanceris never initiated over the ventral region of the disc, where thewing primordium is established and remains absent during laterstages (Fig. 2E-G). This result demonstrates that the activity ofNotchis required not only for the maintenance, but also for theinitiation of the expression of vg through the vgBE (Klein andMartinez-Arias, 1998a; Fig. 2).

The activity of the enhancer deleted from 0-80 is similar tothat of the wild-type enhancer early on (Fig. 2I), but it neveracquires the high levels of activity in the anterior ventral region.As the wing blade develops, a line of faint activity can bedetected over the DV boundary (Fig. 2J), but it fades quicklyand, by the end of the third instar, in most discs there is noactivity over the developing wing blade (Fig. 2K). This enhancerstill shows some activity in the flanking notal regions of the wingdisc in wild-type and Su(H) mutant discs (Fig. 2H,K). Thisindicates the existence of additional inputs in the regulation ofthe vgBE in the notal region (see also Kim et al., 1997a).

Our results (and see also Klein and Martinez Arias, 1998a)suggest that the cells in which the expression of the vgBE isupregulated at the end of the second instar represent the anlageof the wing and require Notch/Su(H) signalling. These cellsare located at the DV interface, on the domain of wgexpression and overlap the expression on nubbin. Oursuggestion that these cells represent the primordium of thewing pouch can explain why a deletion of the vgBE, as in thevg83b27 allele, results in the abolition of the development ofthe wing pouch since, in this mutant, the anlage would neverbe defined.

Wingless is an input on the DV Boundary Enhancer(vgBE)We have recently demonstrated that, in addition to the activityof Notch, the activity of wgis required for the initiation of vgexpression since, in second instar larval discs of wgmutants, theactivity of the vgBE is absent over the region of the wing anlage(Klein and Martinez Arias, 1998a). The possibility that Winglessacts on the vgBE is supported by the observation that a dominantnegative version of DFrizzled2, a receptor for Wingless, reducesthe activity of the vgBE (Zhang and Carthew, 1998). Altogetherthese results raise the possibility that Wingless has a direct inputon the vgBE. Consistent with this we find that the first 80 bp ofthe vgBE, which are required for the full activity of the enhancer,contain two putative TCF-1-binding sites that are associated withWingless signalling (Fig. 3A,B).

To test further the relationship between winglessand vgearly in wing development, we monitored the influence ofNotch and Wingless signalling on the activity of the vgBE. Incontrast with ectopic expression of wg, which never leads toectopic expression of the vgBE (Fig. 3C), ectopic expressionof Delta with ptcGAL4 leads to ectopic expression of thevgBE, but only within the developing wing blade and wherethe levels of ectopic expression of Deltaare high, near the APcompartment boundary (Fig. 3D). Interestingly, an effect ofWingless on this enhancer can be detected when Wingless iscoexpressed ectopically with Delta. Although Wingless alonehas no effect on this enhancer, coexpression of Delta withWingless extends the realm of activity of the vgBE intoregions where expression of Delta on its own has no detectableeffects regions (Fig. 3E and see also Klein and Martinez Arias,1998a).

These results suggest that the major effect of Wingless onthe vgBE is to collaborate with Notch/Su(H) signalling duringthe early stages of wing development. The failure to observeeffects of ectopic expression of Wingless on this enhancermight be related to the absolute requirement for Notchsignalling in this function of Wingless. Since, during the earlystages of wing development, Notch signalling is restricted tothe DV interface (see Klein and Martinez Arias, 1998b), theeffects of Wingless could only be observed in this region andare highlighted by the loss of wg function or the loss ofWingless responding sites in the enhancer. Extension of thisdomain by activating Notch signalling ectopically allows thevisualization of the effects of ectopic Wingless. It is likely thatthe residual activity of the vgBE∆0-80 observed along the DVboundary during early stages of wing development reflects the


A

Fig. 3. Influence of wg and Nsignalling on the vgBE. (A) The first120 bp of the vgBE with the putative Tcf-1 sites highlighted in boldand the Su(H)binding site highlighted in italics. (B) Comparisons ofthe two sites found in the first 80 bp of the vgBE with the Tcf-1consensus binding site. (C) Expression of UAS-wgwith dppGal4does not change the activity of the vgBE, although morphologicalchanges are visible in the wing disc, which are the result of theectopic induction of hinge fate in the notal area (Klein and Martinez-Arias, 1998a; T. K. and A. M. A., unpublished data). (D) Expressionof UAS-Dl is ectopically inducing the activity of the vgBE in thewing pouch perpendicular to the DV boundary. (E) The coexpressionof UAS-wg and UAS-Dl results in a stronger activity of the enhancerin the pouch and in the extension of the ectopic expression domaininto notal area (asterisk). Further, as we have reported before, thecoexpression of the two genes leads to the extension of the blade intothe peripodial membrane and the expression of the vgBE is alsoextended into this region. The arrow indicates the point where thepouch is extending into the region of the peripodial membrane. Sincethe peripodial membrane is covering the disc, the extension comes tolie underneath the normal pouch area and. consequently, theextension of the expression of the vgBE is not visible in thisphotograph (see Klein and Martinez Arias, 1998a and Fig. 5E forfurther details).


input of Notch signalling without the enhancement of Winglesssignalling.

Notch signalling is required for the initial activity ofthe Quadrant Enhancer ( vgQE)The activity of the vgQE can be detected first at the beginningof the third instar, several hours after the upregulation of thevgBE, when it closely outlines the realm of the growing wing(Fig. 1E-H and see Kim et al., 1996). This enhancer is onlyexpressed in the growing wing blade and thus provides aunique and most specific marker for wing blade tissue.

A variety of experiments have shown that the vgQE receivesa negative input from Notchsignalling and a positive one fromDpp (Kim et al., 1996 and see below). The presence of a E(spl)-binding site in the sequence of the vgQE led to the suggestionthat this suppression by Notchis mediated by the E(spl)protein. However, we do not find a strong suppression of theactivity of the vgQE if UAS-m8 (E(spl)) is ectopicallyexpressed with dppGal4 (data not shown), suggesting that theeffect of Notchrequires other mediators. In our experiments,we find that, although the vgQE is suppressed in the domainof Notch activity (Fig. 5D), Notchsignalling plays a non-autonomous role in its activation. For example, the vgQE isnever active in Serrate (Ser) mutants in which wingdevelopment initiates normally but is aborted very early (Kleinand Martinez-Arias, 1998b; Fig. 4A). Expression of UAS-Dlunder the control of dppGal4 in Sermutants rescues the wingpouch and leads to the activation of the vgQE (Fig. 4B).Interestingly, this activity arises in regions devoid of Notchsignalling (compare Fig. 4B and C). This result suggests thatNotch signalling influences the activity of the vgQE in twoways: it represses the activity of the vgQE autonomously butit is also required for its activity in a non-autonomous way.

Wingless acts synergistically with Vestigial topromote the activity of the Quadrant Enhancer(vgQE)It is clear that the activity of the vgBE is required for theactivation of the vgQE (Kim et al., 1996; T. K. and M. A. M.unpublished data), but little is known about how this interactiontakes place. The observation that vgQE is activated in Sermutant discs in which UAS-vgis expressed ectopicallysuggests that the activation of the vgQE is mediated by Notchsignalling through the activity of Vestigial on the vgQE (Fig.4E,F). However, in this experiment, expression of wg is alsorestored (Fig. 4F) and this raises the possibility that activationof the vgQE is mediated through the presumed ‘organizing’activity of Wingless (Zecca et al., 1996; Neumann and Cohen,1996b). This is probably not the case since we find that ectopicexpression of UAS-wgalone does not lead to the activation ofthe vgQE in Sermutants (Fig. 4D). The inability of Winglessto activate the vgQE in this experiment is not due to a generalinsensitivity of the cells to Wingless signalling, since UAS-wgis always capable of inducing hinge fate ectopically (Klein andMartinez-Arias, 1998a).

These results clearly demonstrate a requirement for vg in theactivation of the vgQE. However, clonal analysis has shownthat vgacts cell autonomously (Kim et al., 1996) and therefore,in the wild type, the non-autonomous effects of Notchon thevgQE must be mediated by another, diffusible molecule(s)which is under control of Notch signalling. A number of

studies suggest that Wingless has an influence on theexpression of vgin the wing pouch and that its expression atthe wing margin is under control of Notchsignalling (Zecca etal., 1996; Neumann and Cohen, 1996b). Therefore, it ispossible that Wingless is mediating the non-autonomous effectof Notchon the vgQE. It might be that Wingless acts by actingon the vgQE to elevate and maintain the levels of vg expressionthat had been induced by Vestigial through the vgBE. Inagreement with this proposal, we find that the activity of thevgQE is elevated in response to the ectopic expression of UAS-wgwith dppGal4 (Fig. 5C,H) and that expression of a dominant

Fig. 4.Notchactivity is required for the activation of the vgQE.(A) In a Ser94C/SerRX106mutant wing disc, the vgQE is neveractivated. (B) The activation of the Notch/Su(H)pathway in theSer94C/SerRX106 wing disc by the expression of UASDl with dppGal4rescues the loss of wing tissue and leads to the activation the vgQE inthe posterior region of the recovered pouch (arrow). (C) Detection ofNotchactivity in Ser94C/SerRX106wing disc in which UASDlisexpressed by dppGAL4 by monitoring the expression of the targetgene wg. Arrow indicates the region corresponding to the sameregion in B. The comparison of the expression of wg and that of thevgQE suggests that the vgQE is induced non-autonomously by theectopic expression of Dl. The results also demonstrate therequirement of Notchactivity for the activation of the vgQE.(D) Expression of UAS-wgby dppGal4 does not activate the vgQE.(E) However, expression of UAS-vgin the same genetic backgrounddoes activate the vgQE. A clear stripe of suppression of the activityof the vgQE is observed in the region of the DV boundary (arrow)suggesting that Nsignalling occurs in this region. This is furthersupported by the fact that a small stripe of wgexpression isdetectable until mid/late third instar (arrow). The expression of wgalong the DV boundary is normally absent in this Ser mutantcombination and suggests that the provided activity of vg leads to theenhancement of the residual Nactivity detectable in Ser mutant wingdiscs during early wing development (Klein and Martinez Arias,1998b). Indeed, it has been previously shown that vg is collaboratingwith Notchsignalling to activate wgexpression along the DVboundary during normal wing development (Klein and MartinezArias, 1998a). For further information see text.

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negative Wingless molecule (see Materials and Methods)suppresses the activity of the vgQE and reduces the size of thewing pouch (Fig. 5J).

Altogether our results suggest that the upregulation of vgexpression in response to wg is mediated by the vgQE. Ourconclusion is supported by the existence of several putativeTCF-1 binding sites in the vgQE (data not shown). However,the effects of Wingless are always restricted to the normaldomain of vg expression (compare Fig. 3F,K), in agreementwith the results presented above that Wingless alone is notsufficient to initiate ectopic expression of vg through the vgQE.These effects are likely to be mediated by Vestigial itself and,as indicated above, the role of Wingless on this regulation isto maintain and modulate the levels of activity of the vgQE. Totest this possibility, we have expressed vg under the control ofdppGAL4 and observed that this leads to elevated activity ofthe vgQE and, in addition to weak, but reproducible amountsof ectopic activity of the vgQE in the notal region (arrows inFig. 5F). No ectopic expression of wg is observed in the dorsalside of the wing disc in these experiments and therefore theectopic activation of the vgQE in the dorsal is probablyindependent of Wingless signalling (Fig. 5L). Although thisdoes not rule out the existence of regulatory intermediaries, itdoes suggest that Vestigial has an effect on the activity of thisenhancer.

Consistent with our conclusion that Wingless enhances theeffects of Vestigial, we find that coexpression of Wingless andVestigial with dppGAL4, which leads to the ectopic inductionof pouch and hinge fate in the notal regions (Klein andMartinez-Arias, 1998a), triggers a widespread and stableexpression of the vgQE throughout the wing disc (Fig. 5G).This synergistic effect is more clear in the case of the leg discswhere the vgQE enhancer is not active during normaldevelopment. Expression of wgalone in the leg disc does notactivate the vgQE and, similar to the wing disc, vgalone leadsto some weak activation of this enhancer (data not shown).However, their combined expression leads to very robust andwidespread expression of this enhancer (Fig. 5M).

These results suggest that the activity of wg is required toenhance the activity of the vgQE which is initiated by the Vgprotein itself. They further suggest that the role of Notch in theregulation on the activity of the vgQE is twofold. First, itactivates the expression of Vestigial (via the vgBE) and of wgalong the DV boundary. This sets up the conditions for theactivation of the vgQE through Wingless and Vestigial as thewing pouch begins to grow. Second, at the DV boundary, Notchsignalling leads to a suppression of the activity of the vgQEbut maintains the expression of wgand of the vgBE.

To confirm this conclusion, we have monitored the influenceof coactivation of Wingless and Notch signalling on the activityof the vgQE enhancer. We have shown previously that thedefinition of the size and the fate of the wing pouch requiresthe combination of Wingless and Notch signalling on the vgBE(Klein and Martinez-Arias, 1998a and this work). We now findthat ectopic expression of Dl or wg alone are not sufficient toactivate the vgQE (Fig. 5C,D; see above) but that thecoexpression of both results in the extension of the vgQE intothe notum as it has been previously shown for vg expressionby antibody staining (Fig. 5E; Klein and Martinez-Arias,1998a).

The interactions between Dpp and Wingless duringwing development take place at the level of thevgQE and require the vg gene productMultiple experiments have shown that dppprovides animportant input into the growth of the wing (see e.g. Zecca etal., 1995) but appears not to play a role in the initiation of wingdevelopment since wings derived from dppmutants that affectits function during wing development, have a normal margin(see for example Zecca et al., 1995). However, the vgQEcontains functional Dpp response sites (Kim et al., 1997a) andresponds to Dpp (Kim et al., 1996). These observations,together with those of other inputs on the expression of vg,have led to the suggestion that, as the wing grows, it integratesinputs from both wg and dpp(Kim et al., 1997a).

To test this integration directly, we have created a completeoverlap of the activities of wg, dppand vgby activating UAS-wgand UAS-dpp with vgBEGAL4 (Staehling-Hampton andHoffmann, 1994; Neumann and Cohen 1996a and Fig. 6A).Ectopic expression of wgalone in this manner leads to ectopicexpression of the vgQE in the posterior region of the domain ofvgBE expression (Fig. 6B), over a region in which dpp isnormally expressed (compare Fig. 6B with A and G).Interestingly, elevation of wgexpression achieved in the area ofthe DV boundary does not enlarge the wing pouch (compare Fig.1G and 6B). This suggests that, although wg is required for thecorrect growth of the pouch, it is not a limiting factor. Expressionof dppunder the same conditions leads to an enlargement of thewing blade along the AP axis, with the vgBE as a reference butwithout extending it into the notum (Fig. 6C).

Coexpression of wgand dppleads to a pronounced extensionof the wing blade towards the notum (Fig. 6D). In someinstances, we can observe the development of up to eight wingblades, all with a common origin over a point ventral to thenotum (Fig. 6E,F). All these wings express the vgQE and eachof them has a defined margin (Fig. 6F). It is possible that thesemultiplets are produced by splitting of an initial primordium.It is the case that the expression driven by vgBEGAL4 isdiscontinuous (see Fig. 6A) and therefore leads to thegeneration of several foci of overlap of the activities of wg anddpp, which appear to act as foci for growth and patterning.

In the wild type, the activity of the vgQE is initiated at theintersection of wgand dppexpression and radiates from thisfocus (see Fig. 1 and Kim et al., 1996). The results presentedhere indicate that this overlap determines important parametersof the morphogenesis of the wing. For example, it is possiblethat the distance from this focus – perhaps defined by the rangeof diffusion of the two molecules – defines a threshold thatcontributes significantly to the determination of the size andshape of the wing blade. By tampering with these overlaps, onecan alter the shape of the wing (Fig. 6) or, by generating aseries of them, trigger the development of multiple wings fromone primordium.

Vestigial contributes to the differential patterns ofgene expression in the wing bladeA variety of experiments have suggested that the expression ofwg at the wing margin is a source of Wingless which, in aconcentration-dependent manner, determines patterns of geneexpression (Zecca et al., 1996; Neumann and Cohen, 1996b).One of these targets is the vg gene and here we have shown



Fig. 5. Activity of the vgBE (A,B), the vgQE (C-G,I,J,M) and wglacZ(K,L) as a response to various inputs during wing development. In allcases, except J, ectopic expression was elicited with dppGAL4, which directs gene expression along the AP boundary, with higher levels on thedorsal than on the ventral sides (see Fig. 6G). In J, the expression of UAS-wg−C was expressed with sdGal4, which activates expression in thearea of the wing throughout wing development (A). Expression of vghas no effect on the activity of the vgBE and this is not altered even bycoexpression of vgwith wg (shown in B). (C) Expression of wg leads to an upregulation of the activity of the vgQE within its normal realm ofexpression (compare with anti-Vestigial staining in I and Fig. 1F). (D) Activation of Notch/Su(H)signalling with UAS-Nintra leads to asuppression of the activity of the vgQE within the anterior region of the disc, which is where Notchsignalling is being ectopically activated. Theeffects are restricted to the normal domain of the wing pouch, as indicated by the arrow. The effect is weaker over the ventral region of the disc,where the expression of dppGAL4 is weakest (see Fig. 6G). (E) Coexpression of Dl and wghas two different effects on the activity of the vgQE.Over the anterior region, it has an effect similar to Nintra and suppresses its activity (compare with D); however, over the posterior region, there isa large region of ectopic expression that reaches into the notum (arrow) and into the above lying peripodial membrane (arrowhead). SincedppGAL4 is not expressed over this region, this effect is non-autonomous and must be due to the activity of wg. Furthermore, because on theirown expression of Dl or wgnever produces this effect, this observation highlights the synergism of their combined activities (see also Klein andMartinez-Arias, 1998a for further details concerning the expansion of the wing pouch into the notal region in response to the coexpression of thetwo genes). (F) Expression of vg leads to ectopic expression of vgQE not only over the hinge region, but also extending into the notal region(small arrows). The ectopic expression of vgdoes not lead to ectopic activation of wg (see L) and therefore the effects of vgon the vgQE areprobably not mediated by wg. (G) Coexpression of vgand wg leads to a strong enhancement of the ectopic activity of the vgQE showing that wgcan enhance the induction of the vgQE by vg. Notice that ectopic expression of wgalone is not able to elicit ectopic induction of the activity ofthe vgQE (see C). (H) Expression of Vestigial protein after expression of wgunder the control of dppGAL4. Notice the elevated levels ofexpression along the AP boundary (arrowhead). (I) Wild-type expression of Vestigial revealed by antibody staining, for comparison with H.(J) Expression of a dominant negative form of wg, UAS-wg-C, results in the reduction of the wing pouch and the reduction of the activity of thevgQE. (K) Expression of wg (wglacZ) and vgQE, after expression of vgunder the control of dppGAL4. The expression of wg in the hinge regionindicate the limits of the wing area and indicates that high levels of activity of the vgQE are restricted to the wing region. The extension of theactivity of the vgQE into hinge domains indicates that vg transforms hinge into wing blade. (L) Expression of wg (wglacZ) after expression of vg.Notice that there is almost no effect on wgexpression other than the disruption of the normal pattern of expression around the hinge. This effectis likely to be due to the vg-mediated transformation of hinge into blade and shows that the effects of vgon the expression of the vgQE, shown inF,K, are independent of wg. (M) Coexpression of wgand vg leads to high levels of activity of the vgQE in the leg discs. On its own, theexpression of vg leads only to weak ectopic activation of the vgQE in the leg discs (data not shown) and we have never found any ectopic activityof the vgQE if wg is expressed in the leg disc. The vgQE is not active in the leg discs during normal development. This suggests that, similar tothe situation in the wing disc, wgcan enhance the induction of vgexpression by Vestigial protein but not initiate vg (see text).

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that, in the wing blade, these effects are likely to be targetedthrough the vgQE. However, here we have shown that Vestigialcan alter the spatial activity of the vgQE without altering theendogenous pattern of wg expression. Since vgis itselfexpressed in a gradient similar to that generated by thediffusion of Wingless (Williams et al., 1991; Zecca et al., 1996;Neumann and Cohen et al., 1996), and displays synergisticinteractions with Wingless (Figs 2, 3), this raises the possibilitythat Vestigial operates together with, rather than downstreamof, Wingless in the regulation of gene expression within thewing pouch. To test this, we have studied the effects ofVestigial on another target of Wingless signalling: Distalless(Dll).

In the wild-type wing, Dllis expressed in the wing pouch ina circle of cells that lies within that defined by the expressionof vestigial (Zecca et al., 1996; Neumann and Cohen, 1996band Fig. 7A). We find that expression of vg with dppGAL4elevates the levels of Dll expression within the developing wingblade and triggers ectopic expression of Dll outside this area(Fig. 7B). This differs from the effects of ectopic expressionof wg, which are always restricted to the developing wing bladeand never induce ectopic expression of Dll(Zecca et al., 1996;Neumann and Cohen, 1996b and Fig. 7C). Coexpression ofboth, wg and vg, results in synergistic effects similar to thosedescribed before for other targets and an increase in the areaof ectopic expression (Fig. 7D).

The effects of vgare independent of wg (Fig. 7E), since theyare achieved even in ap mutant where wg is never expressedalong the DV boundary (Fig. 7F-H and see also Ng et al.,1996). In these mutants, there is no Notch/Su(H)signalling, butectopic expression of vgcan rescue the loss of the wing pouchcaused by the loss of apfunction (Klein and Martinez Arias,1998a). Although vg is required for wgexpression, there is notmuch wgexpression in the rescued apmutants. However, thereis a clear stripe of Dllexpression that correlates with theexpression of vg.

These results, together with our previous observation thatVestigial can contribute to the expression of wg at the wingmargin, indicate that Vestigial can indeed regulate geneexpression in the wing blade. In addition, the correlationbetween levels suggests that different concentrations ofVestigial elicit different effects and that, in some instances,these effects seem to be independent of the levels of Wingless.Thus Vestigial might act in concert with Wingless, and notsimply as an effector of wg, in the regulation of geneexpression.

In our experiments, we notice that very high levels ofexpression of Vestigial, or long-term exposure to vestigialexpression, results in the cessation of proliferation, loss of geneexpression and, eventually, in cell death (T. K. and A. M. A.,unpublished data). This might account for the small of wingsthat are often visible when vgis overexpressed in thedeveloping wing and would correlate with the zone of non-


Fig. 6. Effects of the ectopic expression of wg (B), dpp(C) or both(D-F) under the control of vgBEGAL4 on wing development.(A) Pattern of expression of the vgBEGAL4 line used in theseexperiments revealed with UAS-lacZ. The pattern is very similar tothat of the vgBE (compare with Fig. 1C). (B) Activity of the vgQEafter expression of wgin the pattern of the vgBE. Notice that there isan extension of the activity of this enhancer over the posterior dorsalregion of the disc. This pattern of ectopic activity follows the line ofendogenous expression of dpp(see G), which overlaps the pattern ofexpression of the vgBE in this region. Arrowhead points to a regionwhere the expression of vgBEGAL4 is not overlapping with that ofthe endogenous dppexpression (compare A with arrowhead in G). Inthis area, the vgQE is not activated. If dppis coexpressed with wg,the vgQE is activated in this region as well (see arrowhead in D).(C) Activity of the vgQE after expression of dpp in the pattern of thevgBE. Notice that there is an extension of the normal pattern alongthe AP axis, but no change along the DV axis and no extension of thewing into the notum as in the case of ectopic wgexpression(arrowhead indicates the same position as in B). (D-F) Activity of thevgQE in wing discs resulting from the coexpression of wgand dppwith vgBEGAL4. The outcome ranges from the generation of acontinuous large wing blade indicated by the continuous expressionof the vgQE (E,F), to discs where several wing pouches are defined(up to 8) as indicated by the activity of the vgQE (E) and wg-lacZexpression in F. This ‘twinning’ effect might reflect the establishmentof several primordia early in development and is probably the resultof the patchy expression observed in the notal area (see A and text).Each winglet has a margin outlined by the band of no activity of thevgQE (in E). These wings indicate that dppcan enhance the effectsof wgand suggests that the overlap of dpp, wgand vgexpression isrequired for the activation of the vgQE and the generation of acomplete wing during normal development. (G) The expressiondomain dppGAL4 revealed by the activation of UAS-GFP. GFP isexpressed in a stripe along the AP boundary in a AP gradient withthe highest levels at the posterior. Arrowhead points to the regionwhere its expression domain does not overlap with that ofvgBEGAL4.


proliferation that appears at the wing margin, where the levelsof vg expression are elevated.

DISCUSSION

The vestigialgene plays a central role in the development ofthe wing of Drosophila. Previous studies have shown that thespatial and temporal modulation of vg expression throughspecific enhancers is a driving force in the development andpatterning of the wing (Williams et al., 1994; Kim et al., 1996,1997; Neumann and Cohen, 1996a,b; Klein and MartinezArias, 1998a,b). This regulation is exerted by three majorsignalling pathways, Notch/Su(H), Wingless andDecapentaplegic, acting differentially on two collections ofenhancers: one early acting (vgBE) and one later acting(vgQE). Here we have shown that the vggene product itself isan important input in the regulation of vg gene expressionthrough the vgQE and that, in this process, Vestigial actssynergistically with the three signalling pathways that regulateits expression to drive the development of wing blade tissue.This result might provide an answer as to why the activity ofthe vgBE is required for the activation of the vgQE. The findingexplains also the non-autonomousrequirement of the Notch signallingcascade, since it is required for theactivation of the vgBE.

We have also shown that, during earlystages of wing development, theexpression of vg is controlled by thevgBE and that Notch signalling is notonly required for the maintenance of itsactivity but also for its initiation.Furthermore, we have provided someevidence to suggest that the activity ofWingless is required for the upregulationof this enhancer. These results addsupport to, but do not prove, thesuggestion that wing development isinitiated by the cooperation of Notch andWingless signalling on the vg gene(Couso et al., 1995; Klein and MartinezArias, 1998a).

The existence of an input of Winglesson the vgBE would be consistent with theexistence of two putative TCF-1 bindingsites in the first 80 bp of this enhancer.Deletion of this region of the vgBEsubstantially reduces its activity over thedomain of wg expression. Furtherevidence for a participation of wgin theactivity of the vgBE is provided by theobservation that a dominant negativeform of the Wingless receptor Dfrizzled2leads to a strong reduction of the activityof the vgBE (Zhang and Carthew, 1998).

These results contrast with the reportthat loss of winglessfunction does notaffect the activity of the vgBE(Neumann and Cohen, 1996a,b). Thisdifference might reflect different ways

of carrying out the experiments. The published results arebased on clonal analysis, which might not be suitable for thestudy of the vgBE: our results suggest that the loss of wgactivity reduces dramatically the activity of the enhancer butdoes not abolish it (Klein and Martinez Arias, 1998 and thiswork). This means that, taking into account the perduranceof lacZ and the limitations imposed by the need of the clonesto survive, loss of Wingless in clones might affect theendogenous enhancer but have no observable effect on thereporter. This caveat and our results would account for theobservation that large clones of winglessmutant cells do notaffect the expression of a vgBElacZbut display nicks withoutaffecting the size of the wing (see Neumann and Cohen,1996a): it is likely that in these clones the activity of theendogenous enhancer is affected in a manner that is notreflected by the lacZfusion.

Once vg expression is initiated in the wing through thevgBE, our results suggest that it is maintained in the growingpouch by a synergistic activity of Wingless and Vestigial onthe vgQE. This model predicts that loss of the vgBE will resultin the loss of the wing, and that loss of the vgQE should leadto small wings with a well-defined margin. The first predictionis fulfilled by the vg83b27 mutation, which removes the vgBE

Fig. 7.Effects of the ectopic expression of vgon the expression of Dll in the developing wing.All experiments make use of dppGAL4 to drive ectopic expression. (A) Pattern of Dllexpression in a wild-type third instar wing disc as revealed with anti-Dll antibody. (B) Ectopicexpression of vg leads to ectopic expression of Dllin a stripe that overlaps the expression ofvgand which extends into the notum region of the disc (arrowhead). (C) Ectopic expression ofwg leads to elevated levels of Dllexpression within its normal domain of expression, but notto ectopic expression. (D) Coexpression of vgand wgleads to widespread ectopic expressionof Dll over the domain of ectopic expression. (E) Ectopic expression of vgdoes not lead toectopic expression of wginstead suppresses the expression of wg within the hinge regionwhich is transformed to blade (see Fig. 5K,L). (F) Expression of vgin an apUG035mutant discrescues the loss of wing pouch tissue of this mutant (Klein and Martinez Arias, 1998a) andleads to ectopic expression of Dllover the domain of vgexpression (arrowhead).(G) Expression of wgin a disc as in F, showing that, although the wing is rescued (see Kleinand Martinez Arias, 1998a), there is no induction of wgexpression associated with the ectopicexpression of vg.

922

and abolishes the development of the wing (Williams et al.,1994).

There are no mutations that remove the vgQE withoutaffecting all functions of vg and which would allow us to testwhether or not the growth of the wing requires the expressionof vg through the vgQE. However, support for a requirementof the vgQE in the growth of the wing blade is provided by theclonal analysis, which demonstrates the requirement of vgactivity for the survival of the cells in the wing pouch and bya recent report on the effects of dominant mutations in the vggene (Kim et al., 1996; Simmonds et al., 1997). The dominantmutation vgW leads to a very small wing pouch, which stillcontains a wing margin (Simmonds et al., 1997). Indeeddetailed analysis of this mutation shows that, whereas vgW

mutant flies have vg function from the vgBE, the vgQE incompletely inactive (Simmonds et al., 1997). Our resultspredict that the loss of the activity of the vgQE should lead tothe loss of the wing pouch, but not of the margin, as is observedin the vgw mutant. Interestingly, the presence of the vgw

mutation causes extensive cell death at the early third instar(Simmonds et al., 1997), at the time of extensive cell growthin the wing pouch. This cell death is probably due to the lossof vg expression in descendants of cells that are determined atthe DV boundary and which come to lie outside the domain ofthe vgBE due to cell division. These cells would normallymaintain the expression of vg (and therefore their fate) throughthe activation of the vgQE, which is inactive in the presence ofvgw. Several other recently reported results are in agreementwith our conclusion: the clonal analysis suggests anautonomous requirement for vgby the cells of the wing pouchfor survival (Simpson et al., 1981; Kim et al., 1996) andreduction of wgfunction results in the reduction of the size orcomplete loss of the pouch with vgexpression reduced to theDV boundary (Couso et al., 1995; Zecca et al., 1996; Neumannand Cohen, 1996a,b).

Functional parameters in the development of thewing blade in DrosophilaOur results highlight some features of the process of wingdevelopment in Drosophila. In particular they highlightinteractions between the vgBE and the vgQE which can explainhow the wing grows and is patterned. During wingdevelopment, the vgBE has two well-defined roles. First, it isrequired to express vgin the primordium for the wing blade atthe end of the second larval instar. Second, vgBE-mediatedexpression of vgin the primordium is a prerequisite to initiatethe activity of the vgQE which will drive vgexpression in thedeveloping wing blade outside the domain of the vgBE. Thus,failures in the activity of the vgBE will result in failures in theactivation of the vgQE and thus in the early stages of thedevelopment of the wing blade. Later expression of vgfromthe vgQE is likely to be sufficient to maintain the expressionand function of vestigial(and identity) in the growing blade.

These interactions suggest a framework to consider how thewing blade develops and, in particular, about the interactionsobserved between the DV interface and the wing blade duringthe growth of the wing (Fig. 8). The primordium for the wingis defined, in the late second instar, precisely at the DVinterface through interactions between Notch and Winglesssignalling (Fig. 8; for further details see Klein and MartinezArias, 1998a and Fig. 8A-C). These local activities are

channelled through the vgBE and can account for the differentmutant phenotypes and the regulatory and genetic interactionsbetween these molecules.

In the early stages of the third instar, the primordiumexpands (Klein and Martinez Arias, 1998a,b), begins to growand cells that come to lie outside the DV interface maintain theexpression of vgthrough the activity of the vgQE (Fig. 8D).Clonal analysis and studies of cell proliferation (Milan et al.,1996) have not revealed any specific pattern to this phase ofdisc growth. However, it is clear that the DV interface and theAP boundary play some role in this process. Our resultsindicate that this role is likely to be the maintenance of theexpression of vg through the vgQE (Fig. 8D,E) by theconvergent effects of Vestigial, Wingless and Dpp. However,although vg as well as wgare required for the proper growthof the wing pouch, overexpression of these genes along the DVboundary does not lead to a larger pouch size (T. K. and A. M.A., unpublished data; see above), suggesting that the role ofthese genes during cell proliferation is more permissive ratherthan instructive.

At the beginning of the third instar, the wing primordium issubdivided into a proximal region, which will develop as hinge,and a distal one, which will develop as blade and upon whichthe margin will define the most distal point (Klein and MartinezArias, 1998a, Klein et al., 1998). These subdivisions seem tooccur in a sequence and as the wing blade grows they continueas revealed by the expression of Dll in a subset of cells withinthe wing blade. The expression of Dll requires Vestigial and islikely to define a domain within which high levels of Winglesssignalling will induce bristle formation. Consistent with this,loss of Dll function results in the loss of bristles in the wingmargin (Gorfinkiel et al., 1997). Dll delimits the distalmostregion of the leg disc and its expression within the wing blademight reflect the homology that has been suggested to existbetween the wing and the leg discs and highlights a clearproximodistal organization of the wing disc (Klein et al.,1998).

Analysis of the vg1 and Sermutant phenotypes suggests thatthe activity of vg in the initiation of wing development is onlyrequired for a limited period of time i.e. it is stabilized veryquickly (Klein and Martinez Arias, 1998a,b). In both mutantsituations although vg expression is initiated normally, it israpidly lost and the mutant flies develop a small winglet, whichis not observed in apmutant flies or flies mutant for vgnullalleles (T. K. and A. M. A., unpublished data). Therefore theshort-lived activity of vg in these mutants is sufficient for thestable commitment of cells to the wing pouch fate. It is likelythat wg plays a very important role in this commitment, whichis reflected in its synergistic effects with Notch.

The contributions of the DV and AP axes to the patterningof the wing are becoming clear. However, little is still knownabout what triggers the foci of proliferation that drive thegrowth of the wing blade or how the interactions that we havedescribed here control the final size of the wing. Our resultshere suggest a more indirect role of wg and vg in cellproliferation than previously suggested. Although the loss offunction of each of these genes has a significant influence onthe cell proliferation in the wing, the overexpression of bothwith the vgBEGal4 line do not lead to the increase of the wingpouch of the late third instar (see above). This suggests a morepermissive role of the two genes in cell proliferation, probably



to maintain the identity of the wing pouch. Studies that suggestthat the proliferation is induced by local clues and is a cellautonomous property (Milan et al., 1996) support thisconclusion.

The role of Vestigial during the growth of the wingbladevestigialencodes a nuclear protein with an unknown molecularfunction, but which can trigger the development of wing-tissue-specific properties upon developmentally unrelatedgroups of cells (Kim et al., 1996; Klein and Martinez Arias,1998a and this work). Although there is no evidence thatVestigial interacts directly with DNA, our results suggest thatit can influence patterns of gene expression and that this isprobably the way in which it exerts its function duringdevelopment.

It is possible that the role of Vestigial is to ‘select’ acombination of downstream genes that will lead to thedevelopment of wing tissue. In this situation, the interactionsthat we observe between Vestigial and the different signallingpathways might be essential in channelling the nuclear

effectors of these signalling pathways to the promoters of genesthat provide the identity of the wing. For example, wingless(Struhl and Basler, 1993; Wilder and Perrimon, 1995), dpp(Lecuit and Cohen, 1997) and Notch (Couso et al., 1994) arealso required for the development and patterning of legs;however, if vgis expressed in cells from the leg discs, theirdevelopment is altered and they develop wing tissue. Thissuggests that Vestigial provides an essential molecular contextfor wing development.

E

} wg-marginexpr.

vgDllAS-C

+vg+vg

+Dll

N+vg=

posterior

dorsal

ventral

anterior

vg+N*= wg-expr.(margin)

wg+vg= wing blade

N*+wg= vg (vgBE)A

B

C

N*= vg-margin expr. (BE);wg+vg+dpp = maintenanceof vg-expr. in the growingblade (QE)

D

Fig. 8. Model for the sequential interactions that lead to thedevelopment and patterning of the wing blade (see text for furtherdetails). (A) In a first step, Notch/Su(H)signalling induces theexpression of vgby activating the vgBE. The stabilization of this steprequires the activity of wgand therefore only takes place where theactivation of Notchintersects the activity of wg. This point alsodefines the future DV boundary and is highlighted by the square ofthe wing disc that is magnified in the cartoon. (B) Once vg isactivated, it establishes the wing blade fate in collaboration with wg(see Klein and Martinez Arias, 1998a). (C) In a third step,Notch/Su(H)signalling and Vestigial collaborate to induce along theDV interface the expression of genes that are required for theestablishment and patterning of the margin, among them wg. As aconsequence of the restriction of Notch/Su(H)signalling to the DVinterface, the initial primordium of the wing blade is, at this stage, astripe that straddles the DV interface. (D) After the establishment ofthe expression of wgand vgin the wing primordium, the wing bladebegins to grow. As cells proliferate, the expression of vg ismaintained in the cells of the blade that lie outside of the domain ofNotch/Su(H)signalling through the initiation and maintenance of theactivity of the vgQE by Vestigial, Dpp and Wingless. The activity ofthe vgBE and the expression of wgat the wing margin continues tobe controlled by Notch/Su(H)signalling and is therefore restricted tothe DV boundary. The expression of dppis restricted to a domainalong the AP boundary through interactions between hedgehogandpatched. Both Wingless and Dpp diffuse from their sources and,together with Vestigial, control vgexpression in the wing bladethrough the vgQE. During the time of early wing development theexpression of wg is changing rapidly and expands along the AP andthe DV axes (see Klein and Martinez Arias, 1998a). This expansionis dependent on the Notchactivity at the DV boundary (Klein andMartinez Arias, 1998b) and results in the recruitment of more cellsinto the wing primordium. A second consequence of this process isthat cells at the periphery that are solely exposed to the activity of wgand therefore adopt the hinge fate (see Klein and Martinez Arias,1998b for more details). (E) Diffusion from the DV boundarygenerates a gradient of Wingless that is translated in a gradient ofexpression of the Vestigial protein. Wingless and Vestigialcollaborate in the developing wing blade to activate the expression ofgenes required for its further subdivision. In a first step thecollaboration between Wingless and Vestigial results in theexpression of Distalless within the domain of expression of vg. Lossof Dll function results in the loss of bristles and hairs at the wingmargin (Gorfinkiel et al., 1997) in a manner similar to that caused bythe loss of wgfunction. This suggests that, in the wild type, Dll isrequired together with high concentration of Wingless to induce thedevelopment of the bristles characteristic for the anterior margin (bigcircles) and the hairs characteristic for the posterior margin (smallcircles). This suggests a proximodistal organization of the wing thatis generated in the course of development. Early on the wing anlagebecomes subdivided along the proximodistal axis into the primordiafor the proximal (hinge) and distal (blade). In the blade, the mostdistal point is defined as the wing margin (Couso et al., 1995; Kleinet al., 1998). Later on the blade becomes subdivided into furtherdomains reflected in the nested expression of Dll and members of theAchaete/Scute-Complex (AS-C).

924

Pattern formation is often described in terms of ‘positionalinformation’. In this paradigm, the informational input isthought to be universal, but its ‘interpretation’ is cell typespecific (Wolpert, 1969). The widespread and recurrent use ofa few signalling pathways in different developmental systemscould be viewed as the proof of the universality of positionalinformation, at least with regard to some of its elements. Ourresults suggest that Vestigial is an example of a key element inthe interpretation of this information: wherever it is expressed,it channels the output of these signalling pathways into wingtissue. In some sense, Vestigial is an example of a moleculedirectly involved in the interpretation of positional information.It is likely that in different organs and structures, there aremolecules that perform a similar role to that of Vestigial duringwing development in Drosophila. Furthermore, thearchitecture of regulatory networks allows some flexibility asto the position and molecular nature of these molecularinterpreters in the signalling networks.

Work during the last few years has begun to clarify the roleof Vestigial during wing development (Williams et al., 1993,1994; Kim et al., 1996, 1997a,b; Couso et al., 1995; Klein andMartinez Arias, 1998a). Analysis of epistatic relationshipsbetween genes involved in wing development has shown thatvestigial does not lie at the bottom of a linear hierarchy (seee.g. Neumann and Cohen, 1996b; Zecca et al., 1996), but thatit is a central element of a dynamic molecular network in whichWingless, Notch and Vestigial interact in a sequence ofinterdependent regulatory steps that set up the wingprimordium development (Klein and Martinez Arias, 1998a,band this work). The results presented here suggest that, duringthe growth of the wing, these interactions continue with theaddition of one more element to the network, Dpp, and withVestigial continuing to play a central role in the control of geneexpression.

During the third larval instar, the expression of vg, like thatof Dll , is regulated by Wingless (Kim et al., 1996; Zecca et al.,1996; Neumann and Cohen, 1996b). However, we have shownhere that Vestigial can itself regulate the expression of targetsof Wingless, even in the absence of wgexpression and thatVestigial, but not Wingless, can rescue the loss of wingdevelopment that results from the loss of Notchsignalling (seeabove and Klein and Martinez Arias, 1998a). Since Vestigialitself is distributed in a gradient across the developing wing(Williams et al., 1991; Zecca et al., 1996; Neumann and Cohen,1996b), these observations suggest that Vestigial might play amore instructive role in the development and patterning of thewing.

The results that we have presented here and elsewhere (seeKlein and Martinez Arias, 1998a), raise questions about theproposal that Wingless acts as some sort of organizer duringthe development of the wing blade (Diaz-Benjumea andCohen, 1995; Neumann and Cohen 1996b, Zecca et al., 1996)but they also provide some insights as to what might be theactual function of Wingless. In the instances that we havetested, it seems that Wingless cannot elicit de novo geneexpression alone and always acts synergistically with othersignalling systems that direct gene expression during wingdevelopment. In particular, the function of wg appears to be tostabilize and maintain patterns of gene expression rather thandirect patterns of gene expression. This function is very similarto the one that it plays in the embryo where Wingless does not

initiate gene expression but rather reveals and maintainspatterns of gene expression that have been established by otherregulatory events (see Martinez Arias, 1998 for a review).Interestingly, a similar role for other Wnt proteins can beinferred from experiments that test their potential during thespecification and patterning of the mesoderm in Xenopus(Cuiet al., 1996; Hoppler and Moon, 1998).

We want to thank S. Carroll for the gift of the vg antibody, severalvg enhancer-lacZconstructs and many useful discussions. We alsowant to thank S. Bray and S. Cohen for providing the anti-Dll antibodyand K, Brennan, J. Modolell and N. Lavery for comments on themanuscript. This work was supported by The Wellcome Trust andpartly by the Deutsche Forschungs-Gemeinschaft.

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