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

The specification of organ primordia by intercellular signaling has been well described, but it is not yet clear how such tissue patterning events are translated into organ morphogenesis through cytoskeletal reorganization(Braga and Yap, 2005; Jamora and Fuchs, 2002). The actin cytoskeleton controls cell morphology and polarity, endocytosis,intracellular trafficking, contractility and cell division. Filamentous (F)actin is assembled from monomeric (G) actin subunits. Polymerization occurs predominantly by extension of the fast-growing barbed ends of the filaments,while filaments are disassembled by loss of monomers from the slow-growing pointed ends. Actin filament growth, stability and disassembly are controlled by a plethora of actin-binding proteins. Profilin, formins and Enabled/VASP(Ena/VASP) family proteins promote actin polymerization, while Cofilin,Cyclase-associated protein (CAP) and capping proteins (CPs) restrict actin polymerization by different mechanisms(dos Remedios et al., 2003; Wear and Cooper, 2004; Winder and Ayscough, 2005). Cofilin severs filaments and enhances dissociation of actin monomers from the pointed end (Bamburg, 1999);CAP sequesters actin monomers, preventing their incorporation into filaments(Amberg et al., 1995; Gottwald et al., 1996); and CPs restrict accessibility of the barbed end, inhibiting addition or loss of actin monomers (Schafer et al.,1995).

Functional CPs are a highly conserved αβ heterodimer that bind the barbed ends of actin filaments through the C-terminal regions of both subunits (Amatruda et al.,1992; Schafer et al.,1992; Wear and Cooper,2004). CPs and the Arp2/3 complex, which promotes filament branching, favor formation of the short highly branched actin filaments required to generate protrusive force at the leading edge of migrating cells. Ena/VASP proteins have the opposite activity, promoting formation of long unbranched parallel bundles of actin filaments(Bear et al., 2002; Pantaloni et al., 2000; Pollard and Borisy, 2003). In mouse or Dictyostelium cells, depletion of CPs can cause extensive formation of filopodia and increase the length and bundling of actin filaments, reducing cell motility (Hug et al., 1995; Mejillano et al.,2004). Another function of CPs is to cap a short filament of the actin-related protein Arp1 in the Dynactin complex, which is required for Dynein-mediated transport along microtubules(Schafer et al., 1994).

Drosophila imaginal discs are bilayered epithelial tissues consisting of a columnar monolayer epithelium covered by a squamous peripodial epithelium. The columnar epithelial cells are polarized along the apicobasal axis. Adherens junctions (AJ) composed of E-cadherin and α- andβ-catenin link the actin cytoskeleton of neighboring cells, forming an adhesive belt. In Drosophila, two complexes important for polarity are present in the subapical region just apical to the AJ: the Bazooka/Par-6/aPKC complex and the Crumbs/Stardust/Pals-associated tight junction (Patj) complex. Finally, the septate junction, basal to the AJ and formed by the Discs-large (Dlg)/Scribble/Lethal giant larvae complex,establishes a barrier preventing the diffusion of solutes across the epithelium (Gibson and Perrimon,2003). Cells that lack these apical complexes are defined as mesenchymal (Fristrom,1988).

The wing disc has a concentrically organized proximodistal (PD) axis; the primordium of the wing blade is in the center, surrounded by the wing hinge primordium, with the notum and pleura at the periphery. During the second larval instar, an antagonistic relationship between epidermal growth factor(EGF) and Wingless (Wg) signaling divides the disc into a dorsal region that gives rise to the notum, and a ventral region that forms the wing. The wing is further subdivided by expression of the selector genes vestigial(vg) and scalloped (sd) in the wing blade and homothorax (hth) in the wing hinge(Klein, 2001). Vg and Sd encode subunits of a heterodimeric transcription factor that controls wing identity (de Celis, 1999); vg is required for wing formation(Williams et al., 1991) and its misexpression can induce ectopic wing tissue(Kim et al., 1996).

In a mosaic genetic screen for genes required during early eye differentiation (Janody et al.,2004), we identified loss-of-function mutations in the genes encoding Capping protein α (Cpa), the CAP homolog Capulet (Capt) and the Cofilin homolog Twinstar (Tsr). Here, we show that Cpa, as well as the previously identified Capping protein β (Cpb), prevent extrusion and death of cells in the wing blade epithelium, but are not required for this function in other regions of the wing disc. Although cpa, capt and tsr mutations all increase actin filament polymerization, only cpa is required to maintain vg-expressing cells within the epithelium. Furthermore, Vg enhances transcription of cpa in the wing blade region. These results provide a link between pattern formation controlled by Vg and morphogenesis of the wing blade through cytoskeletal regulation mediated by actin capping proteins.

The mosaic screen for mutations affecting early eye development has been described in detail (Janody et al.,2004). Five lethal alleles of cpa(cpa^36P, cpa^43D, cpa^66A,cpa^69E and cpa^107E) and four lethal alleles of tsr (tsr^76E, tsr^99E,tsr^100A and tsr^110M) were recovered from a screen of 45,000 flies on the right arm of the second chromosome. Recombination mapping relative to P(w⁺) elements localized the cpa gene between 57A8-9 and 57D11-12, 1.5 cM proximal to P element l(2)K02206 and 0.9 cM distal to P element domK08108 (FlyBase). Our alleles failed to complement the lethal P element CG10540^KG02261(FlyBase), which is inserted in the cpa gene. Recombination mapping relative to P(w⁺) elements localized the tsralleles to 0.42 cM distal to P element elF6(K13214) (FlyBase) and identified a lethal P element in the tsr gene, l(2)k05633^k05633(FlyBase), which failed to complement our tsr alleles. Other fly stocks used were cpb^M143, khc^k13314, bs² (FlyBase), capt^E636,capt^E593 (Benlali et al.,2000), da-Gal4(Wodarz et al., 1995), tub-Gal4 (Lee and Luo,1999), puc-lacZ(Martin-Blanco et al., 1998),UAS-th (Ryoo et al.,2002), UAS-vg23 (Kim et al., 1996), UAS-N^intra(Doherty et al., 1996),UAS-bsk^DN(Adachi-Yamada et al., 1999b),UAS-Mal-D-ΔN and UAS-dia^CA(Somogyi and Rorth, 2004). To generate clones marked by the absence of GFP in the wing disc, y, w;FRT42D, cpa or tsr/CyO P(y⁺) or y, w;FRT40, capt/SM6-TM6B males were crossed to y, w, hsFLP122;FRT42D or 40, ubi-GFP females. To generate clones positively labeled by GFP, y, w; FRT42D, cpa or tsr/CyO P(y⁺) or y, w; FRT40, capt or cpb/CyO P(y⁺) males were crossed to y, w,hsFLP122, UAS-GFP; FRT42D or FRT40, tub-GAL80; tub-GAL4/TM6B. puc-LacZ, UAS-th, UAS-vg or UAS-N^intra transgenes were combined with the FRT42D or FRT42D, cpa chromosomes. The offspring were heat-shocked for 1 hour at 37°C at both 24 and 48 hours after a 24 hour egg collection,corresponding to the first and second larval instar. To visualize cpaor tsr mutant clones at different times after induction, larvae were heat-shocked for 1 hour at 37°C at 60 hours after a 24 hour egg collection. Wing discs were dissected from crawling late third instar larvae either 36 or 60 hours after the heat shock; these had clones induced at early third instar or at second instar, respectively. All experiments were performed at 25°C. To rescue survival of cpa mutants, da-GAL4 or tub-GAL4; cpa/SM6-TM6B flies were crossed to UAS-HA-cpa,cpa/SM6-TM6B. To rescue cpa mutant clones in the wing imaginal disc, y, w, hsFLP122, UAS-GFP; FRT42D, tub-GAL80; tub-GAL4/TM6B flies were crossed to FRT42D, cpa;UAS-HA-cpa/SM6-TM6B.

Third instar larval imaginal discs were stained with antibodies as described (Lee and Treisman,2001). Antibodies used were guinea pig anti-Dlg (1:300; gift from P. J. Bryant), mouse anti-Arm [N2 7A1, 1:10; Developmental Studies Hybridoma Bank (DSHB)], rabbit anti-Caspase 3 (1:500; BD Bioscience), mouse anti-β-galactosidase (1:200; Promega), rabbit anti-Vg (1:20);(Williams et al., 1993), mouse anti-HA (1/5000; Covance). Secondary antibodies were from Jackson Immunoresearch, used at 1:200 or Molecular Probes, used at 1:500, conjugated to FITC, Texas Red or Cy5. Rhodamine-conjugated phalloidin (Sigma) was used at a concentration of 0.3 μM. Fluorescence images were obtained on a Leica TCS NT confocal microscope or on a LSM 510 Zeiss confocal microscope. For in situ hybridization, antisense or sense RNA probes, labeled with digoxigenin-UTP(Roche), and encompassing the entire cpa cDNA were used. In situ hybridization was performed as described(Maurel-Zaffran and Treisman,2000), except that a biotinylated anti-digoxigenin antibody (1:500 Jackson Immunoresearch) was followed by TSA enhancement (TSA-indirect Perkin Elmer Life Science) coupled to Streptavidin Texas Red or Fluorescein (1:100,Perkin Elmer Life Science). Imaginal discs were mounted in Vectashield medium(Vector Laboratories).

The cpa cDNA clone (GH10050) was obtained from the Berkeley Drosophila genome project, and was amplified by PCR and cloned into HA-pUAST as an EcoRI fragment to generate UAS-HA-cpa. Transgenic flies were generated by standard methods. For each of the five cpa or four tsr alleles recovered in our screen, the coding region was amplified by PCR from DNA obtained from homozygous mutant first instar larvae and from the isogenic FRT42D line. cpa^107E,cpa^36P and cpa^69E contain nonsense mutations at amino acids 162, 174 and 180, respectively, that would truncate the Cpa protein before the actin-binding domain. cpa^43Dcontains a nucleotide substitution that changes Asn190 to Gly. The four tsr alleles contain missense mutations: tsr^76E changes Gly79 to Asp, tsr^99E changes Asp146 to Asn, tsr^100A changes Leu89 to Gln, and tsr^110M changes Gly67 to Glu.

In a mosaic genetic screen for genes required during early eye differentiation (Janody et al.,2004), we identified lethal alleles of cpa, tsr and capt. Capping protein, Cofilin and CAP all restrict actin filament polymerization (Bamburg, 1999; Gottwald et al., 1996; Schafer et al., 1995), and loss of any of these genes results in a severe degenerative phenotype in the adult eye (Delalle et al.,2005) (and data not shown).

Surprisingly, we found a region-specific requirement for cpa in the wing disc: cpa mutant clones induced at the first or second larval instar could not be recovered in the wing blade epithelium, although mutant clones could develop in the remainder of the wing disc(Fig. 1A). By contrast, clones mutant for tsr or capt survived equally well in all regions of the disc (Fig. 1B,C). Optical cross-sections through the wing disc showed that in the wing blade region, positively labeled cpa mutant cells were found on the basal surface of the disc rather than within the epithelium(Fig. 1D). However, tsr and capt mutant clones were maintained within the epithelium in all regions of the wing disc(Fig. 1F,G). Cpa functions as a heterodimer with its partner Capping protein β (Cpb)(Amatruda et al., 1992; Schafer et al., 1992), and the stability of each subunit depends on its association with the other(Casella and Torres, 1994; Mejillano et al., 2004). As expected, we found that cpb mutant cells were also extruded on the basal surface in the wing blade region, but not in the remainder of the wing disc (Fig. 1E). Thus, cpa and cpb are both required to maintain epithelial integrity specifically in the wing blade primordium, suggesting that this subregion of the epithelium has a distinct cellular organization.

The cpa alleles exhibiting this phenotype included likely null alleles that would truncate the protein before the actin-binding domain. To confirm that the loss of cells from the wing pouch epithelium was due to mutations in the cpa gene, we generated transgenic fly lines expressing the full-length cpa transcript under the control of UAS sequences (Brand and Perrimon,1993). Expression of this transcript rescued the cell extrusion phenotype of cpa mutant clones in the wing blade epithelium(Fig. 1H). Driving expression of this transcript ubiquitously throughout development using daughterless (da)-GAL4 or tubulin(tub)-GAL4 was also sufficient to rescue the lethality of cpa mutants, although some of the rescued adults exhibited wing, eye or bristle defects (Table 1;data not shown).

Our failure to recover cpa mutant clones in the adult wing suggested that extruded mutant cells might be eliminated by apoptosis. Indeed,we found that most cpa mutant cells on the basal surface of the wing blade epithelium contained activated Caspase 3(Fig. 2A-B″). Extruded cpa mutant clones also expressed puckered-lacZ(puc-lacZ; Fig. 2C), a transcriptional target of the proapoptotic Jun N terminal Kinase (JNK) pathway(Adachi-Yamada et al., 1999a),indicating that JNK signaling is activated.

Vertebrate epithelial cells destined for death are surrounded by a contractile ring of actin and extruded basally(Rosenblatt et al., 2001). To determine whether extrusion of cpa mutant cells from the wing blade epithelium reflects a preliminary stage of apoptosis, we used the MARCM system to express the Drosophila Inhibitor of Apoptosis Protein(DIAP1; th - FlyBase) to prevent apoptosis of cpamutant cells. Expression of th in wild-type clones had no visible effect (Fig. 2E,H). Survival of cpa mutant cells was greatly enhanced when th was overexpressed (compare Fig. 2Dwith 2F); however, mutant cells still lost contact with the apical epithelial surface and were extruded basally (Fig. 2I). The same result was obtained using the caspase inhibitor P35 to block cell death (data not shown). This suggests that extrusion of cpa mutant cells is independent of programmed cell death.

In epithelial cells, the actin cytoskeleton is connected to adherens junctions (AJ) and septate junctions (SJ), which are necessary for stable cell-cell adhesion (Gibson and Perrimon,2003). Disruption of cortical actin assembly and apicobasal polarity can induce cell extrusion in the wing disc, as in moesinmutants (Speck et al., 2003). By contrast, tkv mutant cells are extruded as cysts that maintain apical junctions with one another (Gibson and Perrimon, 2005; Shen and Dahmann, 2005). Interestingly, we found that an HA-tagged form of Cpa, which could rescue extrusion of cpa mutant cells, accumulated at the apical cell membrane in all regions of the wing disc(Fig. 3A). In the wing blade primordium, HA-Cpa partly colocalized with components of epithelial junctions,including Armadillo (Arm) (Fig. 3B-B‴), Discs large (Dlg) and Crumbs (data not shown). We therefore examined the apicobasal polarity of cpa mutant cells. Cells that had extruded from the epithelium no longer expressed the AJ components Arm (Drosophila β-catenin) or DE-cadherin (DE-Cad), the SJ component Dlg, or the Crumbs complex component Pals-associated tight junction protein (Patj) (Fig. 3C-C″ and data not shown). When we examined discs 36 hours after clone induction, cpa mutant cells in the wing pouch were still present within the epithelium. In these cells, Arm(Fig. 3D-D″) and DE-Cad(data not shown) were mislocalized at basolateral positions, while Dlg localization was unaffected. However, Arm localization was normal in clones induced in the notum region (Fig. 3F-F″). This suggests that, in the wing blade epithelium,Cpa localized at cellular junctions maintains the localization of AJ components. However, Arm was also mislocalized at basolateral positions in tsr mutant clones (Fig. 3E-E″), indicating that mislocalization of AJ components is not sufficient to cause cell extrusion.

CPs tightly cap the fast-growing barbed ends of actin filaments, inhibiting the addition of actin monomers to the filament(Schafer et al., 1995). Decreased expression of cpa or cpb in the Drosophila eye imaginal disc causes actin filament accumulation(Delalle et al., 2005). To confirm that cpa also restricts growth of actin filaments in the wing disc epithelium, we stained wing imaginal discs with phalloidin 36 hours after clone induction, allowing visualization of mutant clones before cell extrusion. As expected, cpa mutant clones accumulated actin filaments in all regions of the wing disc(Fig. 4A-A′). Optical cross-sections through the notum showed that, like capt mutant cells(Fig. 4E-E′)(Baum and Perrimon, 2001), cpa mutant cells accumulated actin filaments near the apical cell membrane (Fig. 4C-C″). However, cpa mutant cells in the wing blade accumulated actin filaments around the entire cell cortex(Fig. 4B-B″), resembling tsr mutant cells (Fig. 4D-D′). As Cpa is concentrated at the apical cell membrane(Fig. 3A-B‴), the presence of excess actin filaments in basolateral regions may be due to the effect of cpa on apicobasal polarity. Indeed, some of these actin filaments colocalized with Arm displaced from apical adherens junctions(Fig. 4B-B″).

We observed extrusion of cpa mutant clones only in the wing pouch,indicating that either the position or identity of these cells makes them dependent on cpa. Signaling pathways activated by Wg, Decapentaplegic(Dpp) and Notch (N) can influence growth and/or survival in the wing blade primordium (Klein, 2001). Target genes of these pathways, including Vg(Klein and Arias, 1999), were still expressed in cpa mutant clones(Fig. 5A-A″ and data not shown), indicating that cpa is not required for the reception of these signals. Vg is a nuclear protein that confers a wing blade fate on cells in which it is expressed (Williams et al.,1991). We wondered whether transforming hinge or notum cells to wing blade identity by misexpressing Vg would induce extrusion of cpamutant cells. When we misexpressed vg in cpa mutant clones,we found that clones in all regions of the wing disc were extruded from the epithelium and contained activated Caspase 3 (compare Fig. 5B,D with Fig. 5E,G). Misexpressing vg in clones that were not mutant for cpa had neither of these effects (Fig. 5C,F). Thus, cpa is autonomously required for the survival and maintenance of vg-expressing cells in the wing disc epithelium, independently of the identity of the surrounding cells.

The requirement for cpa to maintain vg-expressing cells in the wing pouch epithelium suggests that capping actin filaments is crucial for Vg-induced wing blade morphogenesis. We wondered whether cpaexpression might itself be induced by Vg. Indeed, in situ hybridization showed that cpa mRNA was more strongly expressed in the wing pouch than in the hinge primordium or the notum regions, accumulating primarily at the basal surface of the epithelium (Fig. 6A,C; Fig. 6Bindicates the background level of the cpa sense probe). As vg is required for the survival of cells in the wing blade, we could only test its effect on cpa transcription in gain-of-function experiments. Interestingly, misexpressing vg in the notum increased cpa transcript levels (Fig. 6D-D′). The same effect was observed in clones expressing the constitutively active intracellular domain of N(N^intra) (Fig. 6E-E′), which induces misexpression of vg(Fig. 6F-F′). These results suggest that Vg alters the cytoskeletal structure of cells fated to form the wing blade in part by upregulating the expression of actin capping proteins.

Wing blade cells lacking either cpa or cpb are extruded from the epithelium and subsequently die. A number of possible mechanisms might account for this loss of CP mutant cells. As extrusion of cpamutant cells still occurs in the presence of the apoptotic inhibitors p35 or th, apoptosis is likely to be a secondary consequence of extrusion;extruded cells might undergo apoptosis because they are deprived of anti-apoptotic signals present in their normal niche. In addition, JNK activity is not essential for extrusion, as cpa mutant clones were not rescued by expression of a dominant-negative form of basket(bsk), which encodes JNK (see Fig. S1E in the supplementary material). However, we cannot exclude the possibility that the p35 or Th inhibitors block apoptosis too late to prevent release of an extrusion signal,as inhibition of caspases with z-VADfmk does not block extrusion of apoptotic MDCK cells (Rosenblatt et al.,2001).

The function of CPs in organelle or vesicle transport is unlikely to explain the extrusion phenotype. CPs are thought to stabilize the barbed end of the Arp1 microfilament in the Dynactin complex, which is required for transport along microtubules (Schafer et al., 1994). cpa and cpb, like other Dynactin complex subunits, are required to maintain the position of nuclei in Drosophila photoreceptor neurons(Whited et al., 2004) (and data not shown). However, removal of kinesin heavy chain(khc), which counteracts Dynein/Dynactin-based transport, failed to rescue extrusion of cpa mutant cells in the wing disc (see Fig. S1C in the supplementary material).

We considered the possibility that the cpa phenotype was due to its effect on monomeric G-actin levels rather than on the filamentous actin cytoskeleton. G-actin has been shown to negatively regulate the nuclear localization and activity of Mal, a transcriptional cofactor for SRF(Miralles et al., 2003), and overexpression of Mal or of its activator diaphanous(Somogyi and Rorth, 2004) can cause extrusion and death of wing epithelial cells (see Fig. S1F,G in the supplementary material). However, overactivity of the MAL/dSRF pathway is unlikely to be responsible for extrusion of cpa mutant cells in the wing blade, as clones mutant for both cpa and blistered(bs), which encodes Drosophila SRF, were still extruded from the wing epithelium (see Fig. S1H in the supplementary material).

Extrusion of cpa or cpb mutant cells might be a direct result of defects in the actin cytoskeleton. Consistent with the requirement for CPs to inhibit addition of actin monomers to the fast-growing end of actin filaments (Schafer et al.,1995), we observed a strong accumulation of actin filaments in cpa mutant clones. However, tsr and capt mutations also induce excessive actin filament polymerization(Fig. 4)(Baum and Perrimon, 2001) but do not cause cell extrusion. The major function of Tsr (Cofilin) is to promote dissociation of ADP-actin from the pointed end of the filament, while Cpa prevents elongation of the barbed end of each branch and Capt sequesters actin monomers. The phenotypic differences between cpa, tsr and capt might therefore be due to different degrees of branching of the actin network formed in mutant cells (Fig. 7A). Possibly long unbranched filaments do not provide a framework of sufficient strength to withstand forces that place tension on the cell within the epithelium.

Extrusion is associated with dispersion of the adherens junction components Arm and DE-Cad along the lateral membranes. However, this defect is also observed in tsr mutant clones, and mislocalization of adherens junction components caused by overexpression of a dominant form of the polarity gene crumbs (Fan et al.,2003; Grzeschik and Knust,2005; Izaddoost et al.,2002) or a dominant-negative form of Dcdc42(Eaton et al., 1995) does not lead to cell extrusion. Therefore, mislocalization of AJ components is unlikely to be sufficient to cause extrusion of cpa mutant cells. By contrast, expression of dominant-negative Rac1, which prevents actin localization to adherens junctions, induces cell extrusion and death(Eaton et al., 1995). Thus,another possibility is that CPs may be crucial for linking actin filaments to the membrane, as has been previously proposed in other systems(Hutchings et al., 2003; Schafer et al., 1992; Schafer et al., 1998). Loss of cpb displaces actin bundles from the cell membrane in Drosophila bristles by increasing the concentration of non-bundle actin snarls (Hopmann et al.,1996; Frank et al.,2006) and CPs may specify actin filament position in the sarcomere(Schafer et al., 1995). In the Drosophila wing blade epithelium, loss of CPs might disrupt attachment of the actin cytoskeleton to the adherens junctions, breaking the connection between cells and inducing cell extrusion(Fig. 7C). The localization of HA-Cpa to apical junctions and the mislocalization of actin filaments throughout cpa mutant cells in the wing blade are consistent with this possibility. Such a role would be restricted to the wing blade, as cpa mutant cells within the notum epithelium accumulate actin filaments only at the apical cell membrane.

Surprisingly, we found that cpa and cpb are required to prevent cell extrusion and death only in the region of the wing disc giving rise to the wing blade, but not in the primordia of the hinge or notum, or in the eye or leg discs (Delalle et al.,2005) (and data not shown). The requirement for cpadepends on the wing blade selector gene Vg, as expression of Vg in notum cells is sufficient to induce their extrusion in the absence of cpa. Vg also enhances the transcription of cpa in the wing blade primordium. Taken together, these results imply that patterning genes regulate cytoskeletal properties in order to achieve distinct morphological outcomes(Fig. 7B,C).

The molecular mechanism that makes Vg-expressing cells dependent on CPs for their maintenance in the epithelium is unknown, although our data support a cell-autonomous target of Vg. One possibility is that Vg promotes the expression or recruitment of an actin filament polymerizing factor. The role of CPs might be to restrict its activity at barbed ends, preventing the formation of a specific actin-based structure that induces loss of cell-cell contacts and extrusion. For example, Vg activates the expression of the type II transmembrane protein Four jointed (Fj), which regulates the activity of the cadherin Fat (Cho and Irvine,2004). Mammalian Fat1 can recruit Ena/VASP proteins, which promote actin polymerization at cell-cell contacts by antagonizing CPs(Moeller et al., 2004; Tanoue and Takeichi, 2004). However, misexpression of fj does not induce extrusion of either wild-type or cpa mutant cells in the notum(Cho and Irvine, 2004) (and data not shown). DE-cadherin levels are also higher in the wing pouch(Jaiswal et al., 2006), but increasing them in the hinge or notum by activating Wg signaling does not cause extrusion of cpa mutant cells (data not shown). Alternatively,Vg might control the expression of factors that promote the remodeling of cell junctions required for morphogenesis of the wing. Cpa could be required to maintain the connection between cells in the epithelium during these morphogenetic movements.

The non-uniform distribution of and requirement for cpa suggests that cytoskeletal organization varies in different regions of the wing disc. Gibson and Perrimon (Gibson and Perrimon,2005) observed that lateral wing disc cells had moderately reduced levels of basolateral cortical F-actin. In addition, filopodial extensions called cytonemes are oriented towards the AP and/or DV boundary within the wing pouch, while hinge cells do not extend cytonemes and notum cells radiate short cytonemes in all directions (Hsiung et al., 2005). Changes in cytoskeletal organization have been shown to establish cell affinity boundaries(Major and Irvine, 2005), to control the subcellular localization of transcription factors(Miralles et al., 2003) and to modulate the transport of signaling molecules(Benlali et al., 2000; Hsiung et al., 2005). Investigating the control of cpa by Vg may help us to understand how and why patterning genes regulate cell architecture. In addition, identifying additional target genes of Vg may illuminate how actin dynamics and changes in intercellular adhesion control the formation of the wing blade.

We thank S. Carroll, B. Mollereau, M. Affolter, P. J. Bryant, K. Hofmeyer,P. Rorth, the Bloomington Drosophila Stock Center, the Drosophila Genomics Resource Center and the Developmental Studies Hybridoma Bank for fly stocks and reagents. We thank Neal Jahren and William Hu for their contributions to the early stages of the project. Special thanks to Michel Sémériva and Michel Piovant, in whose laboratory many of these experiments were performed. The manuscript was improved by the critical comments of Inés Carrera, Reza Farajian, Kerstin Hofmeyer,Grant Miura, Jean-Yves Roignant, Michel Sémériva, Laurent Perrin and Josie Steinhauer. This work was supported by the National Institutes of Health (grant EY13777 to J.E.T.). F.J. was the recipient of a fellowship from the Association pour la Recherche sur le Cancer.