PATTERNS & PHENOTYPES

Dpp Signaling Down-Regulates the Expressionof VM32E Eggshell Gene During DrosophilaOogenesisFabio Bernardi, Valeria Cavaliere, Davide Andrenacci,† and Giuseppe Gargiulo*

Among the members of the Drosophila melanogaster vitelline membrane protein gene family, VM32E hasthe unique feature of being a component of both the vitelline and the endochorion layers. The VM32E geneis expressed at stage 10 of egg chamber development in the main body follicle cells, and it is repressed in theanterior and posterior follicle cells. Here, we show that this spatial restriction of VM32E gene expression isconserved in the D. pseudoobscura orthologous gene, suggestive of a conserved function of VM32E protein.The VM32E gene is not expressed in the centripetal migrating follicle cells, where the Decapentaplegic(Dpp) pathway is active in patterning the anterior eggshell structures. By analyzing the native VM32E geneand the activity of specific VM32E regulatory regions, in genetic backgrounds altering the Dpp pathway, weshow that VM32E gene is negatively regulated by the Dpp signaling. Therefore, it appears that the Dppsignaling pathway executes its control on eggshell morphogenesis also by controlling the expression ofeggshell structural genes. Developmental Dynamics 235:768–775, 2006. © 2005 Wiley-Liss, Inc.

Key words: VM32E; dpp; eggshell; oogenesis; Drosophila

Accepted 7 November 2005

INTRODUCTION

In Drosophila melanogaster, the eventof oogenesis takes place in the eggchamber, which consists of the oocyteand 15 nurse cells, surrounded by amonolayer of follicle cells. During oo-genesis, the follicle cells interact withthe germline cells, and this is criticalin the establishment of polarities inthe egg and the developing embryo(Gonzalez-Reyes et al., 1995; Roth etal., 1995; Ray and Schupbach, 1996).Cell–cell interactions cause the folli-cle cells to be divided into several sub-groups that have specialized rolesduring oogenesis, including bordercells, stretch cells, and centripetal

cells at the anterior of the follicle, pos-terior terminal cells at the posterior ofthe follicle, and the main body folliclecells in the middle. The follicle cellsare responsible for producing the egg-shell, which is composed of a vitellinemembrane that is the innermostlayer, a wax layer, a crystalline inner-most chorionic layer, an endochorion,and an exochorion, which is the outerlayer of the eggshell (Margaritis et al.,1980). The eggshell of a mature egg ischaracterized by specialized anteriorstructures such as the micropile thatallows sperm entry, the operculumthat forms the exit hatch for the de-veloped embryo, and two dorsal respi-

ratory appendages (reviewed by Spra-dling, 1993). The complex architectureof the eggshell suggests the underly-ing differentiation of the follicular ep-ithelium in distinct spatial cell do-mains, each endowed with specificfunctions. The epidermal growth fac-tor receptor (Egfr) signaling inducesdorsal follicle cells fates (Ray andSchupbach, 1996; Perrimon and Per-kins, 1997; Dobens and Raftery,2000), leading to the definition oftwo separate populations of dorsalfollicle cells (Wasserman and Free-man, 1998; Peri et al., 1999) that willguide the production of the two dor-sal appendages. The patterning of

Dipartimento di Biologia Evoluzionistica Sperimentale, Bologna, ItalyGrant sponsor: University of Bologna.†Dr. Andrenacci’s present address is Istituto di Genetica e Biofisica-C.N.R., Via Pietro Castellino 111, 80131 Napoli, Italy.*Correspondence to: Giuseppe Gargiulo Dipartimento di Biologia Evoluzionistica Sperimentale, Via Selmi 3, 40126 Bologna,Italy. E-mail: giuseppe.gargiulo@unibo.it

DOI 10.1002/dvdy.20660Published online 21 December 2005 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 235:768–775, 2006

© 2005 Wiley-Liss, Inc.

the eggshell along the anteroposte-rior (AP) axis also requires trans-forming growth factor-beta (Tgf-�)family member, Decapentaplegic(Dpp; Padgett et al., 1987; Twombly etal., 1996; Deng and Bownes, 1997; Periand Roth, 2000). At stage 10 of eggchamber development, it is expressed inboth the nurse cell associated folliclecells and in centripetal cells, and is re-quired for the formation of the anterioreggshell. The centripetal cells consist of30 to 40 follicle cells that migrate dur-ing stage 10B of oogenesis in betweenthe nurse cells and the anterior of theoocyte. The size and placement of theoperculum and dorsal appendages arequite sensitive to altered levels of Dppsignal (Twombly et al., 1996).

Although much is known about theinduction and refinement of the sig-naling pathways involved in the for-mation of the anterior eggshellstructures, little is known about theregulation and the function of thegenes encoding eggshell structuralproteins. The eggshell proteins aresynthesized and secreted by the fol-licle cells surrounding the oocyte in awell-defined temporal order fromstage 8 to stage 14 of egg chamberdevelopment (Petri et al., 1976; War-ing and Mahowald, 1979; Fargnoliand Waring, 1982; staging of oogen-esis is based on Spradling, 1993).The formation of these extracellularstructures is a complex process thatrequires time-coordinated synthesis,cleavage, and transport of variousproteins and, finally, cross-linking atspecific functional domains (An-drenacci et al., 2001; Manogaran andWaring, 2004). Many aspects of egg-shell biogenesis are still to be eluci-dated. Interestingly, vitelline mem-brane assembly requires the activityof fs(1)Nasrat and fs(1)poleholegenes, which are needed for local ac-tivation of the Torso receptor ty-rosine kinase that leads to theproper patterning of the termini ofDrosophila (Cernilogar et al., 2001;Jimenez et al., 2002). In addition,the finding that the Torsolikeprotein, which is involved in Torsoactivation, is a component of thevitelline membrane (Stevens et al.,2003) indicates that the eggshell isan integral part of maternal signal-ing and opens up very interesting

issues of a linkage between eggshellassembly and embryonic patterning.

The eggshell proteins appear to benot functionally redundant, suggest-ing that each structural componentmay have a specific role in the egg-shell assembly (Andrenacci et al.,2001). The complex expression pat-tern of the chorion genes (Parks andSpradling, 1987; Tolias et al., 1993;Mariani et al., 1996) indicates that thesynthesis of the different chorionproteins is spatially and temporallyregulated. Although the vitellinemembrane protein genes VM26A.1,VM34C, VM26A.2 are expressed in allfollicle cells surrounding the oocyte,from stage 8 to stage 10 of oogenesis,the VM32E gene is active only at stage10 (Burke et al., 1987; Popodi et al.,1988; Gigliotti et al., 1989), and it isdifferently expressed in the distinctdomains of the follicular epithelium(Gargiulo et al., 1991). Our previouswork on VM32E protein expressionhas identified several unique featuresof this protein compared with theother members of the same gene fam-ily. At the time of its synthesis (stage10), the VM32E protein is not detect-able in anterior and posterior folliclecells. However, it is able to spread inthe extracellular space around the oo-cyte, and by stage 11 it is uniformlydistributed in the vitelline membrane.During the terminal stages of oogene-sis the VM32E protein is partially re-leased from the vitelline membraneand becomes localized in the endocho-rion layer also.

An interesting issue on VM32E geneexpression pattern concerns its repres-sion in the polar domains of follicularepithelium. In the present study, weshow that the Dpp signaling pathwaynegatively controls VM32E gene ex-pression in the centripetal follicle cells.Because Dpp signaling is involved indefining the anterior eggshell pattern-ing, the spatially restricted expressionof VM32E gene may be part of theproper assembly process of the anteriorregion of the eggshell.

RESULTS AND DISCUSSION

Spatial Control of VM32EGene Expression

The VM32E gene is transcribed onlyat stage 10 of egg chamber develop-

ment (Gargiulo et al., 1991; Cavaliereet al., 1997; Andrenacci et al., 2000).The gene is transcribed in all main-body follicle cells but is absent fromthe most anterior and posterior do-mains (Fig. 1A, arrow and arrowhead,respectively). This finding is in con-trast with the VM26A.2 gene (Fig.1B), which encodes one of the mostabundant vitelline membrane struc-tural proteins (Pascucci et al., 1996)and is expressed from stage 8 to 10 ofegg chamber development in all folli-cle cells surrounding the oocyte(Popodi et al., 1988). The RNA expres-sion patterns of VM32E and VM26A.2are reflected in their respective pro-tein distribution patterns. We haveraised polyclonal antibodies againstthese two proteins (anti-CVM32E andanti-VMP; Andrenacci et al., 2001;Cernilogar et al., 2001). As shown inFigure 1D, VM26A.2 is expressed byall oocyte-associated follicle cells. Bycontrast, VM32E is expressed by themain-body follicle cells but is missingin the anterior and posterior groups ofthe follicle cells (Fig. 1C, arrow andarrowhead, respectively).

The precise temporal and spatialcontrol of VM32E gene activity may berelevant for VM32E protein function,and we expected it would be conservedin other Drosophila species. The D.pseudoobscura (Dp) orthologous gene(GenBank accession no. EAL33865)encodes a protein that shares high se-quence homology with the D. melano-gaster (Dm) VM32E (67.2% identity,74.6% similarity, data not shown). In-deed, using the same anti-CVM32Eantibody, we show that the distribu-tion of the DpVM32E protein appearsvery similar to the one shown for theDmVM32E (compare Fig. 1C and 1E)in which the protein is not synthesizedin the anterior and posterior folliclecells (Fig. 1E, arrow and arrowhead,respectively). The evolutionary con-servation of VM32E gene expressionpattern between D. melanogaster andD. pseudoobscura, which are sepa-rated by 25–30 million years (Powell,1996), points out the interesting issueon the regulatory signals controllingthe expression of this eggshell gene.We also analyzed the DmVM26A.2 or-thologue, DpVM26A.2 protein (Gen-Bank accession no. EAL34278; 79.7%identity, 87% similarity, data notshown) detected by anti-VMP anti-

VM32E GENE EXPRESSION 769

Fig. 1.

Fig. 2.

770 BERNARDI ET AL.

body. As shown in Figure 1F, becausethe DpVM26A.2 signal appearsweaker and more diffuse than that ofDmVM26A.2, it is difficult to assesswhether the DpVM26A.2 protein isexpressed in all the anterior folliclecells as it occurs for the DmVM26A.2or may have an expression pattern re-sembling the one shown for the Dm-DpVM32E proteins.

dpp Ectopic ExpressionRepresses VM32E GeneActivity

To better define the D. melanogasterVM32E expression pattern in the an-terior follicle cells, we took a close-uplook, by confocal microscopy, of theVM32E protein localization in the an-terior follicular domain. As shown inFigure 2A, the VM32E is absent in asmall group of the dorsal–anteriorfollicle cells (arrowhead) and in thecentripetally migrating follicle cells(arrows). The centripetal cells expressthe decapentaplegic (dpp) gene(Twombly et al., 1996; Mantrova etal., 1999), which encodes a transform-ing growth factor �-related signalingmolecule (Padgett et al., 1987). TheDpp pathway plays a critical role indefining the position and morphologyof anterior eggshell structures such asthe operculum and the two dorsal ap-pendages (Twombly et al., 1996). Thefollicular domains in which the Dppsignaling is active can be identified byvisualizing the activated form of

Mothers against dpp (Mad), the down-stream effector of Dpp signaling, us-ing an antibody against the phosphor-ylated form of the protein (pMad;Tanimoto et al., 2000). Because theanti-CVM32E and the anti-pMad an-tibodies were both raised in rabbits, tovisualize these proteins in the sameegg chamber, we used a transgenic flystrain expressing a VM32E proteinfused with a MYC tag under the wild-type VM32E minimal promoter (An-drenacci et al., 2001). The VM32E-MYC protein was detected by using amonoclonal antibody against the MYCepitope. As shown in Figure 2B, pMadis detected in the centripetal folliclecells (Guichet et al., 2001), which aredevoid of VM32E gene activity, sug-gesting a negative role of Dpp signal-ing on VM32E expression. To testwhether Dpp signaling inhibitsVM32E transcription, we examinedthe VM32E expression pattern in eggchambers ectopically expressing dppby using the GAL4/UAS binary ex-pression system (Brand and Perri-mon, 1993). UAS-dpp flies werecrossed with flies from the CY2 Gal4line (Queenan et al., 1997), which ex-presses the Gal4 transgene in all thefollicle cells covering the oocyte fromstage 8 (Fig. 2C). In this genetic back-ground, VM32E expression is com-pletely abolished (Fig. 2D,F), whereasVM26A.2 transcription is not affected(Fig. 2E).

Ectopic dpp expression in the wholeegg chamber resulted in eggs with ex-

panded opercula and reduced dorsalappendages (data not shown; Dobenset al., 2000). In addition, we founddefects on vitelline membrane integ-rity. We incubated dechorionated eggsfrom CY2 Gal4/UAS-dpp females withneutral red, a dye that is not normallyable to cross the vitelline membraneand, therefore, used to detect defectsin vitelline membrane assembly(LeMosy and Hashimoto, 2000; Cer-nilogar et al., 2001). In contrast towild-type eggs (Fig. 2H), those derivedfrom CY2 Gal4/UAS-dpp femalesshowed significant uptake of neutralred. A total of 245 of 588 eggs fromCY2 Gal4/UAS-dpp females (41.7%)were permeable to neutral red dye,whereas only 20 stained eggs were de-tected over 542 wild-type eggs (3.7%).The range of staining in the mutanteggs permeable to dye varied fromuniform staining to patches of intensestaining in the anterior region of theegg (Fig. 2G). After the brief bleachtreatment and dye incubation, mor-phological changes were often seenwith the most permeable eggs show-ing shrinkage. Therefore, it appearsthat dpp ectopic expression affectsvitelline membrane integrity.

VM32E Expression IsNegatively Regulated byDpp Signaling Pathway inthe Centripetal Follicle Cells

To confirm the negative role played byDpp pathway on the VM32E expres-

Fig. 1. VM32E and VM26A.2 genes expression and proteins distribution in stage 10B egg chambers. A,B: Whole-mount in situ hybridization showingthe spatial distribution of the Drosophila melanogaster VM32E and VM26A.2 transcripts, respectively. A: All main body columnar follicle cells expressthe VM32E gene but the most anterior and posterior follicle cells are silent. B: The VM26A.2 gene is expressed in all follicle cells. C,D: Whole-mountegg chambers stained with anti-CVM32E (C) and anti-VMP antibodies (D). Whereas the VM32E protein (C) is not synthesized in the anterior andposterior follicle cells, the VM26A.2 appears evenly distributed in all follicle cells (D). E,F: Distribution of VM32E and VM26A.2 orthologous proteins inD. pseudoobscura using the D. melanogaster anti-CVM32E (E) and anti-VMP antibodies (F). To better visualize the follicle cells in the anterior andposterior domains, after immunostaining, the D. pseudoobscura egg chambers nuclei were DAPI (4�-6-diamidino-2-phenylindole) stained (E,F). InA,C,E, the arrow and the arrowhead, respectively, indicate the most anterior and posterior follicle cells. In all the panels, the egg chambers are orientedwith the anterior end to the left. In A and C, the dorsal side is at the top.Fig. 2. The decapentaplegic (dpp) ectopic expression represses VM32E gene expression in the follicle cells. A: Confocal image showing the surfaceview of the anterior domain of follicle cells of a stage 10B egg chamber. We used a strain expressing the fused His2avDGFP protein (Clarkson andSaint, 1999) that marks all the nuclei (green). VM32E protein (red) is not detected in a small group of dorsal anterior follicle cells (arrowhead) and inthe centripetal follicle cells (arrows). B: Confocal image of a sagittal section of a stage 10B egg chamber showing the expression of phosphorylated-Mad (pMad, red) and the VM32E-MYC protein (green). The arrow indicates the centripetal cells that express pMad but not VM32E. In A and B, thedorsal side is at the top. C: View of the UAS-GFP reporter expression driven by the CY2 Gal4 enhancer trap line. The expression is detected in alloocyte-associated follicle cells of stage 8 and 10 egg chambers. D,E: In situ hybridization showing VM32E (D) and VM26A.2 (E) mRNA distribution ina dpp misexpression background. When dpp is ectopically expressed in all the follicle cells using the CY2 Gal4 driver, the VM32E expression isrepressed (D), whereas the expression of VM26A.2 gene is unaffected (E). F: Immunostaining analysis with anti-CVM32E antibody showing that theVM32E protein is not detected when dpp is ectopically expressed by the CY2 Gal4 driver. In A to F, the egg chambers are oriented with the anteriorend on the left. G: Low-magnification image of neutral red–permeable eggs from CY2 Gal4/UAS-dpp females. The uptake of neutral red generatesuniformly stained eggs and eggs showing patched staining in the anterior region. H: Low-magnification image of wild-type eggs that are not permeableto neutral red. In G and H, all the eggs are oriented with the anterior end to the left. Scale bar � 20 �m in A.

VM32E GENE EXPRESSION 771

Fig. 3.

Fig. 4.

sion, we investigated the effect of aconstitutively active form (TkvQ253D;Nellen et al., 1996) of the Dpp type Ireceptor Thick veins (Tkv). As shownin Figure 3A, the activity of the UAS-tkvQ253D transgene driven by CY2 Gal4completely suppresses the VM32E ex-pression. Mad and Medea (Med) arecentral components of the Dpp signaltransduction pathway and they actdownstream of Tkv in responding cells(reviewed by Raftery and Sutherland,1999). Expression of UAS-Med or UAS-Mad transgenes driven by CY2 Gal4line disrupts VM32E expression. Theectopic activity of UAS-Med transgenesignificantly reduces the VM32E tran-scription in the whole follicular epi-thelium (Fig. 3B), whereas in theUAS-Mad egg chambers, the VM32Eexpression is completely wiped out(Fig. 3C). The stronger repressionshown by Mad could be due to theexpression of two copies of UAS-Madtransgene driven by CY2 Gal4.

Our results on the ectopic expres-sion of different components of theDpp signaling clearly point out thenegative role of this pathway onVM32E gene transcription. To furtherconfirm this repression activity, we in-duced mutant clones of follicle cellshomozygous for the null allele Mad12.As shown in Figure 3D–J, the VM32Eexpression is turned on in centripetal

follicle cells lacking Mad function,whereas the VM32E gene activity isunaffected in clones within the mainbody follicle cells. This finding demon-strates that the Dpp pathway activityin the centripetal cells is responsiblefor the repression of VM32E expres-sion in this cellular domain.

Our previous work on VM32E gene de-fined within the minimal promoter(�348/�39) the cis-acting regulatory re-gions dictating the VM32E gene expres-sion in the different follicle domains(Cavaliere et al., 1997; Andrenacci et al.,2000). Interestingly, the 3�-most segmentof the minimal promoter, �112/�39, byitself dictates specific lacZ expression inthe centripetal cells (arrows in Fig. 4A;Cavaliere et al., 1997). However, whenthe upstream region �253/�113 wasadded to the �112/�39 fragment (�253/�39), only the ventral expression appearsand the centripetal expression is re-pressed (arrows in Fig. 4B; Cavaliere etal., 1997). Therefore, the �253/�113 frag-ment, in addition to containing the posi-tive element(s) that specify VM32E geneexpression in the ventral domain, mustalso contain the cis-acting element(s) re-pressing the gene activity in the centrip-etal follicle cells. We analyzed the effect ofdpp misexpression on the �253/�39-lacZ construct. In CY2 Gal4/UAS-dppbackground the expression of the lacZgene was completely abolished (Fig. 4C),

indicating that the repression activity ofDpp signaling pathway on VM32E genetranscription acts within the �253/�113VM32E promoter region. To test this hy-pothesis, we analyzed the �253/�39-lacZ gene expression in Mad12 mosaicegg chambers, and we found that the�253/�39-lacZ is derepressed in Mad12

clones located in the centripetal cells (in-dicated by arrows in Figure 4D–I).Within the centripetal cells, this dere-pression is not influenced by the positionof the clone along the dorsoventral axis.Mad12 clones located in the dorsal andventral main body follicle cells (respec-tively indicated by asterisk and bracketin Fig. 4D–I) have no effect on the �253/�39-lacZ gene expression pattern.

The control of the expression ofstructural genes is crucial in morpho-genesis. This finding suggests differ-ential expression of transcription fac-tors, which in turn regulates thetissue-specific activity of structuralgenes. The elaborate patterning of thefollicular epithelium that defines theanterior structures of the eggshell iswell documented, whereas the molec-ular mechanisms controlling the ex-pression of the eggshell structuralgenes remain poorly understood.From our data, it appears that theDpp signaling pathway controls egg-shell morphogenesis also by regulat-ing the expression of eggshell struc-

Fig. 3. Decapentaplegic (Dpp) signaling pathway represses VM32E gene expression in the centripetal follicle cells. A–C: In situ hybridization showingVM32E expression. A: When the constitutively active form (TkvQ253D) of the type I receptor Tkv is ectopically expressed in all the follicle cells using theCY2 Gal4 driver, the VM32E expression is completely abolished. B: The VM32E expression is drastically reduced when the CY2 Gal4 ectopicallyexpresses UAS-Med transgene. C: VM32E gene expression is completely repressed when two copies of UAS-Mad are expressed in all follicle cellsby the CY2 Gal4 line. D–J: VM32E expression (red) in stage 10B Mad12 mosaic egg chambers. Absence of GFP expression (green) marks the cells thatare homozygous for Mad12 (dotted area). The dotted line marks the limit of the anterior columnar follicle cells that normally express the VM32E genein wild-type egg chambers. D–F: Confocal surface section of a stage 10B egg chamber showing three independent Mad12 clones, two of which arein the main body follicle cells and one in the centripetal cells (arrow). F: The merged picture clearly shows that the VM32E gene is derepressed andswitched on in the Mad12 clone located in the centripetal cells, while its level of expression is unaffected in Mad12 clones located in the main bodyfollicle cells. G–J: Stage 10B egg chamber showing a Mad12 clone comprising a group of anterior follicle cells and the neighboring centripetal cells.G: Nomarski view of a stage 10B egg chamber showing the ectopic expression of VM32E gene in the centripetal follicle cells (arrow). H–J: Confocalimages of the boxed area in G. H,I: Within the Mad12 clone (H), the cells residing to the left (anterior) of the line are within the centripetal populationand they now express VM32E (I). J: Merged image of H,I. In all the panels the egg chambers are oriented with the anterior region toward the left. Scalebars � 20 �m in the inserts.Fig. 4. The Decapentaplegic (Dpp) response element is within the �253/�113 VM32E promoter region. A: A stage 10B egg chamber showing thelacZ expression (green) in the centripetal follicle cells (indicated by arrows) driven by the �112/�39 VM32E promoter region. B: A stage 10B eggchamber showing the ventral lacZ expression pattern (green) driven by the �253/�39 VM32E promoter region and the absence of lacZ expression inthe centripetal follicle cells (indicated by arrows). C: The ectopic expression of decapentaplegic (dpp) in all follicle cells represses the �253/�39 VM32Epromoter activity. D–I: Confocal cross-sections of stage 10B Mad12 mosaic egg chambers showing the lacZ expression pattern (red) driven by the�253/�39 VM32E promoter region. D,G: Absence of green fluorescent protein (GFP) expression (green) marks the cells that are homozygous forMad12 in different follicle cell populations, centripetal cells (arrows) ventral follicle cells (bracket) and dorsal follicle cells (asterisk). E,H: The�253/�39-lacZ gene is derepressed and switched on in the Mad12 clones (arrows) located in the ventral (E) and dorsal (H) centripetal cells. Therefore,derepression in the centripetal cells is not influenced by the position of the clone along the dorsoventral axis. Mad12 clones located in the main bodyfollicle cells do not influence the �253/�39-lacZ gene expression; in the Mad12 clone of ventral follicle cells (bracket in E) the expression of the lacZgene is unaffected, while in Mad12 clone of dorsal follicle cells (asterisk in H) the gene is not turned on. F: Merged image of D and E. I: Merged imageof G and H. In all the panels, the egg chambers are oriented with the anterior region toward the left and the dorsal side is at the top. Scale bars � 20�m in the inserts.

VM32E GENE EXPRESSION 773

tural genes. The exquisite temporaland spatial regulation of the VM32Eeggshell structural gene, partly medi-ated by the critical morphogenic Dppsignaling system, suggests a crucialrole of VM32E protein in the complexprocess of eggshell assembly.

EXPERIMENTALPROCEDURES

Fly Strains

The following stocks were used: Drosoph-ila pseudoobscura wild-type stock (Tuc-son Drosophila Species Stock Center),CY2 Gal4 (Queenan et al., 1997), Mad12

FRT40A/SM6a, and ubiquitin-GFP FRT-40A; GR1-GAL4 UAS-FLP (Gupta andSchupbach, 2003), UAS-Mad, UAS-Med(Marquez et al., 2001), UAS-tkvQ253D

(Nellen et al., 1996), His2avDGFP (Clark-son and Saint, 1999), UAS-dpp (Staeh-ling-Hampton and Hoffman, 1994),�253/�39-lacZ, �112/�39-lacZ (Cava-liere et al., 1997), and VM32E-MYC (An-drenacci et al., 2001). Stocks were raisedon standard cornmeal/yeast/agar me-dium at 25°C, and crosses were made at18°C unless otherwise stated. yw67c23/yw67c23 was used as the wild-type stock inthis study.

In Situ Hybridization

Whole-mount in situ hybridizationwith digoxigenin-labeled (Roche)probes was performed as described byTautz and Pfeifle (Tautz and Pfeifle,1989). The 3� end of the VM32E cDNA(Gigliotti et al., 1989) and theVM26A.2 DNA region (�40/�230)were used as probes. The egg cham-bers were viewed with Nomarski op-tics on a Nikon Eclipse 90i microscope.

Clonal Analysis

Follicle cell clones were induced usingthe directed mosaic GAL4/UAS-FLPtechnique (Duffy et al., 1998). Folliclecell clones mutant for Mad were gener-ated using a GR1-GAL4 UAS-FLP line,which is expressed in all the folliclecells, including the follicle cell stem cells(Gupta and Schupbach, 2003). Mad12

FRT40A/SM6a females were crossedwith ubiquitin-GFP FRT40A; GR1-GAL4 UAS-FLP males. Females of thegenotype Mad12 FRT40A/ubiquitin-GFP FRT40A; GR1-GAL4 UAS-FLP/�were collected and maintained on

yeasted vials at 25°C for 3 days beforedissection. To analyze �253/�39-lacZgene expression in Mad12 follicle cellclones, females carrying the �253/�39-lacZ construct on the X chromosomeand the balancer TM3 Ser were crossedwith ubiquitin-GFP FRT40A; GR1-GAL4 UAS-FLP males. The TM3 Sermale offspring was crossed with Mad12

FRT40A/SM6a females. Females of thegenotype �253/�39-lacZ/�; Mad12

FRT40A/ubiquitin-GFP FRT40A; GR1-GAL4 UAS-FLP/� were collected andmaintained on yeasted vials at 25°C for3 days before dissection.

Gal4-Driven Expression inFollicle Cells

Females CY2 Gal4/UAS-GFP; CY2Gal4/UAS-dpp; CY2 Gal4/UAS-tkvQ253D; CY2 Gal4/UAS-Mad; CY2Gal4/UAS-Med and CY2 Gal4/UAS-dpp/�253/�39-lacZ were obtainedby crossing the parental strains. Thecrosses were performed at 18°C andthe progeny was transferred toyeasted vials at 29°C for 3 days be-fore dissection.

ImmunofluorescenceMicroscopy

Fixation and antibody staining ofhand dissected ovaries were carriedout as previously described (An-drenacci et al., 2001). Anti-CVM32E(1:100) or anti-VMP (1:50) antibodieswere used and detected with Cy3-con-jugated anti-rabbit secondary anti-body (1:100, Sigma). Rabbit anti-phos-phorylated SMAD (PS1) was used at1:500 dilution (Tanimoto et al., 2000)and detected with Cy3-conjugated an-ti-rabbit secondary antibody (1:100).Anti-MYC monoclonal antibody(Santa Cruz Biotechnology) was usedat 1:100 dilution and reacted with flu-orescein isothiocyanate (FITC) -conju-gated anti-mouse secondary antibody(1:250, Molecular Probes). Anti-�galmonoclonal antibody (DevelopmentalStudies Hybridoma Bank) was used at1:25 dilution and reacted with FITC(1:250) or Cy3-conjugated anti-mousesecondary antibody (1:100, Sigma).

DAPI (4�-6-diamidino-2-phenylin-dole) staining was carried out by incu-bating for 10 min the egg chamberswith DAPI at 1�g/ml in phosphatebuffered saline (PBS) and, after sev-

eral washes with PBS, the egg cham-bers were mounted.

Stained egg chambers mounted inFluoromount-G (Electron MicroscopySciences) were analyzed with conven-tional epifluorescence and with TCSSL Leica confocal system attached to aZeiss Axiophot microscope or viewedwith Nomarski optics on a NikonEclipse 90i microscope.

Neutral Red Assay

Neutral red staining of 0- to 12-hr eggswas carried out essentially as previ-ously described (Cernilogar et al., 2001).Dechorionated eggs were stained with 5mg/ml neutral red (Sigma) in PBS andincubated at room temperature for 5–10min. Eggs were washed six times withPBS, 0.05% Triton X-100 to removeneutral red, mounted in 50% glycerol inPBS, and viewed with Nomarski opticson a Nikon microscope.

ACKNOWLEDGMENTSWe thank Trudi Schupbach, StuartNewfeld, and Franco Graziani for flystrains. We also thank the Blooming-ton Stock Center and the Tucson Dro-sophila Species Stock Center for pro-viding us with fly stocks. We thankTetsuya Tabata and the Ludwig Insti-tute for Cancer Research for the rab-bit phospho-Smad1 antiserum and theDevelopmental Studies HybridomaBank for anti-�gal monoclonal anti-body. We thank Alessandra Donati forhelpful discussion. We also thank Sil-via Gigliotti and Franco Graziani forcritical reading of the manuscript. Avery special thanks goes to Tien Hsufor his advice on manuscript prepara-tion and helpful suggestions. We alsothank Marco Privitera for the graphicworks. This work was supported bythe University of Bologna in theframework of the Project “Meccanismie segnali molecolari della sopravvi-venza cellulare”; MIUR (Ex 40%,2004/2006) and University of Bolognain the framework of the Project“Nuove strategie di controllo degli in-setti con geni di antagonisti naturali”.

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