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Keywords:

  • VM32E;
  • dpp;
  • eggshell;
  • oogenesis;
  • Drosophila

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

Among the members of the Drosophila melanogaster vitelline membrane protein gene family, VM32E has the unique feature of being a component of both the vitelline and the endochorion layers. The VM32E gene is expressed at stage 10 of egg chamber development in the main body follicle cells, and it is repressed in the anterior and posterior follicle cells. Here, we show that this spatial restriction of VM32E gene expression is conserved 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 gene and the activity of specific VM32E regulatory regions, in genetic backgrounds altering the Dpp pathway, we show that VM32E gene is negatively regulated by the Dpp signaling. Therefore, it appears that the Dpp signaling pathway executes its control on eggshell morphogenesis also by controlling the expression of eggshell structural genes. Developmental Dynamics 235:768–775, 2006. © 2005 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

In Drosophila melanogaster, the event of oogenesis takes place in the egg chamber, which consists of the oocyte and 15 nurse cells, surrounded by a monolayer of follicle cells. During oogenesis, the follicle cells interact with the germline cells, and this is critical in the establishment of polarities in the egg and the developing embryo (Gonzalez-Reyes et al.,1995; Roth et al.,1995; Ray and Schüpbach,1996). Cell–cell interactions cause the follicle cells to be divided into several subgroups that have specialized roles during oogenesis, including border cells, stretch cells, and centripetal cells at the anterior of the follicle, posterior terminal cells at the posterior of the follicle, and the main body follicle cells in the middle. The follicle cells are responsible for producing the eggshell, which is composed of a vitelline membrane that is the innermost layer, a wax layer, a crystalline innermost chorionic layer, an endochorion, and an exochorion, which is the outer layer of the eggshell (Margaritis et al.,1980). The eggshell of a mature egg is characterized by specialized anterior structures such as the micropile that allows sperm entry, the operculum that forms the exit hatch for the developed embryo, and two dorsal respiratory appendages (reviewed by Spradling,1993). The complex architecture of the eggshell suggests the underlying differentiation of the follicular epithelium in distinct spatial cell domains, each endowed with specific functions. The epidermal growth factor receptor (Egfr) signaling induces dorsal follicle cells fates (Ray and Schüpbach,1996; Perrimon and Perkins,1997; Dobens and Raftery,2000), leading to the definition of two separate populations of dorsal follicle cells (Wasserman and Freeman,1998; Peri et al.,1999) that will guide the production of the two dorsal appendages. The patterning of the eggshell along the anteroposterior (AP) axis also requires transforming growth factor-beta (Tgf-β) family member, Decapentaplegic (Dpp; Padgett et al.,1987; Twombly et al.,1996; Deng and Bownes,1997; Peri and Roth,2000). At stage 10 of egg chamber development, it is expressed in both the nurse cell associated follicle cells and in centripetal cells, and is required for the formation of the anterior eggshell. The centripetal cells consist of 30 to 40 follicle cells that migrate during stage 10B of oogenesis in between the nurse cells and the anterior of the oocyte. The size and placement of the operculum and dorsal appendages are quite sensitive to altered levels of Dpp signal (Twombly et al.,1996).

Although much is known about the induction and refinement of the signaling pathways involved in the formation of the anterior eggshell structures, little is known about the regulation and the function of the genes encoding eggshell structural proteins. The eggshell proteins are synthesized and secreted by the follicle cells surrounding the oocyte in a well-defined temporal order from stage 8 to stage 14 of egg chamber development (Petri et al.,1976; Waring and Mahowald,1979; Fargnoli and Waring,1982; staging of oogenesis is based on Spradling,1993). The formation of these extracellular structures is a complex process that requires time-coordinated synthesis, cleavage, and transport of various proteins and, finally, cross-linking at specific functional domains (Andrenacci et al.,2001; Manogaran and Waring,2004). Many aspects of eggshell biogenesis are still to be elucidated. Interestingly, vitelline membrane assembly requires the activity of fs(1)Nasrat and fs(1)polehole genes, which are needed for local activation of the Torso receptor tyrosine kinase that leads to the proper patterning of the termini of Drosophila (Cernilogar et al.,2001; Jimenez et al.,2002). In addition, the finding that the Torsolike protein, which is involved in Torso activation, is a component of the vitelline membrane (Stevens et al.,2003) indicates that the eggshell is an integral part of maternal signaling and opens up very interesting issues of a linkage between eggshell assembly and embryonic patterning.

The eggshell proteins appear to be not functionally redundant, suggesting that each structural component may have a specific role in the eggshell assembly (Andrenacci et al.,2001). The complex expression pattern of the chorion genes (Parks and Spradling,1987; Tolias et al.,1993; Mariani et al.,1996) indicates that the synthesis of the different chorion proteins is spatially and temporally regulated. Although the vitelline membrane protein genes VM26A.1, VM34C, VM26A.2 are expressed in all follicle cells surrounding the oocyte, from stage 8 to stage 10 of oogenesis, the VM32E gene is active only at stage 10 (Burke et al.,1987; Popodi et al.,1988; Gigliotti et al.,1989), and it is differently expressed in the distinct domains of the follicular epithelium (Gargiulo et al.,1991). Our previous work on VM32E protein expression has identified several unique features of this protein compared with the other members of the same gene family. At the time of its synthesis (stage 10), the VM32E protein is not detectable in anterior and posterior follicle cells. However, it is able to spread in the extracellular space around the oocyte, and by stage 11 it is uniformly distributed in the vitelline membrane. During the terminal stages of oogenesis the VM32E protein is partially released from the vitelline membrane and becomes localized in the endochorion layer also.

An interesting issue on VM32E gene expression pattern concerns its repression in the polar domains of follicular epithelium. In the present study, we show that the Dpp signaling pathway negatively controls VM32E gene expression in the centripetal follicle cells. Because Dpp signaling is involved in defining the anterior eggshell patterning, the spatially restricted expression of VM32E gene may be part of the proper assembly process of the anterior region of the eggshell.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

Spatial Control of VM32E Gene Expression

The VM32E gene is transcribed only at stage 10 of egg chamber development (Gargiulo et al.,1991; Cavaliere et al.,1997; Andrenacci et al.,2000). The gene is transcribed in all main-body follicle cells but is absent from the most anterior and posterior domains (Fig. 1A, arrow and arrowhead, respectively). This finding is in contrast with the VM26A.2 gene (Fig. 1B), which encodes one of the most abundant vitelline membrane structural proteins (Pascucci et al.,1996) and is expressed from stage 8 to 10 of egg chamber development in all follicle cells surrounding the oocyte (Popodi et al.,1988). The RNA expression patterns of VM32E and VM26A.2 are reflected in their respective protein distribution patterns. We have raised polyclonal antibodies against these two proteins (anti-CVM32E and anti-VMP; Andrenacci et al.,2001; Cernilogar et al.,2001). As shown in Figure 1D, VM26A.2 is expressed by all oocyte-associated follicle cells. By contrast, VM32E is expressed by the main-body follicle cells but is missing in the anterior and posterior groups of the follicle cells (Fig. 1C, arrow and arrowhead, respectively).

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Figure 1. VM32E and VM26A.2 genes expression and proteins distribution in stage 10B egg chambers. A,B: Whole-mount in situ hybridization showing the spatial distribution of the Drosophila melanogasterVM32E and VM26A.2 transcripts, respectively. A: All main body columnar follicle cells express the 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-mount egg chambers stained with anti-CVM32E (C) and anti-VMP antibodies (D). Whereas the VM32E protein (C) is not synthesized in the anterior and posterior follicle cells, the VM26A.2 appears evenly distributed in all follicle cells (D). E,F: Distribution of VM32E and VM26A.2 orthologous proteins in D. pseudoobscura using the D. melanogaster anti-CVM32E (E) and anti-VMP antibodies (F). To better visualize the follicle cells in the anterior and posterior domains, after immunostaining, the D. pseudoobscura egg chambers nuclei were DAPI (4′-6-diamidino-2-phenylindole) stained (E,F). In A,C,E, the arrow and the arrowhead, respectively, indicate the most anterior and posterior follicle cells. In all the panels, the egg chambers are oriented with the anterior end to the left. In A and C, the dorsal side is at the top.

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Figure 2. The decapentaplegic (dpp) ectopic expression represses VM32E gene expression in the follicle cells. A: Confocal image showing the surface view of the anterior domain of follicle cells of a stage 10B egg chamber. We used a strain expressing the fused His2avDGFP protein (Clarkson and Saint,1999) that marks all the nuclei (green). VM32E protein (red) is not detected in a small group of dorsal anterior follicle cells (arrowhead) and in the 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, the dorsal 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 all oocyte-associated follicle cells of stage 8 and 10 egg chambers. D,E: In situ hybridization showing VM32E (D) and VM26A.2 (E) mRNA distribution in a dpp misexpression background. When dpp is ectopically expressed in all the follicle cells using the CY2 Gal4 driver, the VM32E expression is repressed (D), whereas the expression of VM26A.2 gene is unaffected (E). F: Immunostaining analysis with anti-CVM32E antibody showing that the VM32E 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 anterior end on the left. G: Low-magnification image of neutral red–permeable eggs from CY2 Gal4/UAS-dpp females. The uptake of neutral red generates uniformly stained eggs and eggs showing patched staining in the anterior region. H: Low-magnification image of wild-type eggs that are not permeable to neutral red. In G and H, all the eggs are oriented with the anterior end to the left. Scale bar = 20 μm in A.

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The precise temporal and spatial control of VM32E gene activity may be relevant for VM32E protein function, and we expected it would be conserved in other Drosophila species. The D. pseudoobscura (Dp) orthologous gene (GenBank accession no. EAL33865) encodes a protein that shares high sequence homology with the D. melanogaster (Dm) VM32E (67.2% identity, 74.6% similarity, data not shown). Indeed, using the same anti-CVM32E antibody, we show that the distribution of the DpVM32E protein appears very similar to the one shown for the DmVM32E (compare Fig. 1C and 1E) in which the protein is not synthesized in the anterior and posterior follicle cells (Fig. 1E, arrow and arrowhead, respectively). The evolutionary conservation of VM32E gene expression pattern between D. melanogaster and D. pseudoobscura, which are separated by 25–30 million years (Powell,1996), points out the interesting issue on the regulatory signals controlling the expression of this eggshell gene. We also analyzed the DmVM26A.2 orthologue, DpVM26A.2 protein (GenBank accession no. EAL34278; 79.7% identity, 87% similarity, data not shown) detected by anti-VMP antibody. As shown in Figure 1F, because the DpVM26A.2 signal appears weaker and more diffuse than that of DmVM26A.2, it is difficult to assess whether the DpVM26A.2 protein is expressed in all the anterior follicle cells as it occurs for the DmVM26A.2 or may have an expression pattern resembling the one shown for the Dm-DpVM32E proteins.

dpp Ectopic Expression Represses VM32E Gene Activity

To better define the D. melanogaster VM32E expression pattern in the anterior follicle cells, we took a close-up look, by confocal microscopy, of the VM32E protein localization in the anterior follicular domain. As shown in Figure 2A, the VM32E is absent in a small group of the dorsal–anterior follicle cells (arrowhead) and in the centripetally migrating follicle cells (arrows). The centripetal cells express the decapentaplegic (dpp) gene (Twombly et al.,1996; Mantrova et al.,1999), which encodes a transforming growth factor β-related signaling molecule (Padgett et al.,1987). The Dpp pathway plays a critical role in defining the position and morphology of anterior eggshell structures such as the operculum and the two dorsal appendages (Twombly et al.,1996). The follicular domains in which the Dpp signaling is active can be identified by visualizing the activated form of Mothers against dpp (Mad), the downstream effector of Dpp signaling, using an antibody against the phosphorylated form of the protein (pMad; Tanimoto et al.,2000). Because the anti-CVM32E and the anti-pMad antibodies were both raised in rabbits, to visualize these proteins in the same egg chamber, we used a transgenic fly strain expressing a VM32E protein fused with a MYC tag under the wild-type VM32E minimal promoter (Andrenacci et al.,2001). The VM32E-MYC protein was detected by using a monoclonal antibody against the MYC epitope. As shown in Figure 2B, pMad is detected in the centripetal follicle cells (Guichet et al.,2001), which are devoid of VM32E gene activity, suggesting a negative role of Dpp signaling on VM32E expression. To test whether Dpp signaling inhibits VM32E transcription, we examined the VM32E expression pattern in egg chambers ectopically expressing dpp by using the GAL4/UAS binary expression system (Brand and Perrimon,1993). UAS-dpp flies were crossed with flies from the CY2 Gal4 line (Queenan et al.,1997), which expresses the Gal4 transgene in all the follicle cells covering the oocyte from stage 8 (Fig. 2C). In this genetic background, VM32E expression is completely abolished (Fig. 2D,F), whereas VM26A.2 transcription is not affected (Fig. 2E).

Ectopic dpp expression in the whole egg chamber resulted in eggs with expanded opercula and reduced dorsal appendages (data not shown; Dobens et al.,2000). In addition, we found defects on vitelline membrane integrity. We incubated dechorionated eggs from CY2 Gal4/UAS-dpp females with neutral red, a dye that is not normally able to cross the vitelline membrane and, therefore, used to detect defects in vitelline membrane assembly (LeMosy and Hashimoto,2000; Cernilogar et al.,2001). In contrast to wild-type eggs (Fig. 2H), those derived from CY2 Gal4/UAS-dpp females showed significant uptake of neutral red. A total of 245 of 588 eggs from CY2 Gal4/UAS-dpp females (41.7%) were permeable to neutral red dye, whereas only 20 stained eggs were detected over 542 wild-type eggs (3.7%). The range of staining in the mutant eggs permeable to dye varied from uniform staining to patches of intense staining in the anterior region of the egg (Fig. 2G). After the brief bleach treatment and dye incubation, morphological changes were often seen with the most permeable eggs showing shrinkage. Therefore, it appears that dpp ectopic expression affects vitelline membrane integrity.

VM32E Expression Is Negatively Regulated by Dpp Signaling Pathway in the Centripetal Follicle Cells

To confirm the negative role played by Dpp pathway on the VM32E expression, we investigated the effect of a constitutively active form (TkvQ253D; Nellen et al.,1996) of the Dpp type I receptor Thick veins (Tkv). As shown in Figure 3A, the activity of the UAS-tkvQ253D transgene driven by CY2 Gal4 completely suppresses the VM32E expression. Mad and Medea (Med) are central components of the Dpp signal transduction pathway and they act downstream of Tkv in responding cells (reviewed by Raftery and Sutherland,1999). Expression of UAS-Med or UAS-Mad transgenes driven by CY2 Gal4 line disrupts VM32E expression. The ectopic activity of UAS-Med transgene significantly reduces the VM32E transcription in the whole follicular epithelium (Fig. 3B), whereas in the UAS-Mad egg chambers, the VM32E expression is completely wiped out (Fig. 3C). The stronger repression shown by Mad could be due to the expression of two copies of UAS-Mad transgene driven by CY2 Gal4.

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Figure 3. Decapentaplegic (Dpp) signaling pathway represses VM32E gene expression in the centripetal follicle cells. A–C: In situ hybridization showing VM32E expression. A: When the constitutively active form (TkvQ253D) of the type I receptor Tkv is ectopically expressed in all the follicle cells using the CY2 Gal4 driver, the VM32E expression is completely abolished. B: The VM32E expression is drastically reduced when the CY2 Gal4 ectopically expresses UAS-Med transgene. C:VM32E gene expression is completely repressed when two copies of UAS-Mad are expressed in all follicle cells by the CY2 Gal4 line. D–J: VM32E expression (red) in stage 10B Mad12 mosaic egg chambers. Absence of GFP expression (green) marks the cells that are homozygous for Mad12 (dotted area). The dotted line marks the limit of the anterior columnar follicle cells that normally express the VM32E gene in wild-type egg chambers. D–F: Confocal surface section of a stage 10B egg chamber showing three independent Mad12 clones, two of which are in the main body follicle cells and one in the centripetal cells (arrow). F: The merged picture clearly shows that the VM32E gene is derepressed and switched on in the Mad12 clone located in the centripetal cells, while its level of expression is unaffected in Mad12 clones located in the main body follicle 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: Confocal images 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 population and 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. Scale bars = 20 μm in the inserts.

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Our results on the ectopic expression of different components of the Dpp signaling clearly point out the negative role of this pathway on VM32E gene transcription. To further confirm this repression activity, we induced mutant clones of follicle cells homozygous for the null allele Mad12. As shown in Figure 3D–J, the VM32E expression is turned on in centripetal follicle cells lacking Mad function, whereas the VM32E gene activity is unaffected in clones within the main body follicle cells. This finding demonstrates that the Dpp pathway activity in the centripetal cells is responsible for the repression of VM32E expression in this cellular domain.

Our previous work on VM32E gene defined within the minimal promoter (−348/−39) the cis-acting regulatory regions dictating the VM32E gene expression in the different follicle domains (Cavaliere et al.,1997; Andrenacci et al.,2000). Interestingly, the 3′-most segment of the minimal promoter, −112/−39, by itself dictates specific lacZ expression in the centripetal cells (arrows in Fig. 4A; Cavaliere et al.,1997). However, when the upstream region −253/−113 was added to the −112/−39 fragment (−253/−39), only the ventral expression appears and the centripetal expression is repressed (arrows in Fig. 4B; Cavaliere et al.,1997). Therefore, the −253/−113 fragment, in addition to containing the positive element(s) that specify VM32E gene expression in the ventral domain, must also contain the cis-acting element(s) repressing the gene activity in the centripetal follicle cells. We analyzed the effect of dpp misexpression on the −253/−39-lacZ construct. In CY2 Gal4/UAS-dpp background the expression of the lacZ gene was completely abolished (Fig. 4C), indicating that the repression activity of Dpp signaling pathway on VM32E gene transcription acts within the −253/−113 VM32E promoter region. To test this hypothesis, we analyzed the −253/−39-lacZ gene expression in Mad12 mosaic egg chambers, and we found that the −253/−39-lacZ is derepressed in Mad12 clones located in the centripetal cells (indicated by arrows in Figure 4D–I). Within the centripetal cells, this derepression is not influenced by the position of the clone along the dorsoventral axis. Mad12 clones located in the dorsal and ventral main body follicle cells (respectively indicated by asterisk and bracket in Fig. 4D–I) have no effect on the −253/−39-lacZ gene expression pattern.

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Figure 4. The Decapentaplegic (Dpp) response element is within the −253/−113 VM32E promoter region. A: A stage 10B egg chamber showing the lacZ expression (green) in the centripetal follicle cells (indicated by arrows) driven by the −112/−39 VM32E promoter region. B: A stage 10B egg chamber showing the ventral lacZ expression pattern (green) driven by the −253/−39 VM32E promoter region and the absence of lacZ expression in the centripetal follicle cells (indicated by arrows). C: The ectopic expression of decapentaplegic (dpp) in all follicle cells represses the −253/−39 VM32E promoter 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 for Mad12 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 body follicle 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 lacZ gene 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 image of 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 μm in the inserts.

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The control of the expression of structural genes is crucial in morphogenesis. This finding suggests differential expression of transcription factors, which in turn regulates the tissue-specific activity of structural genes. The elaborate patterning of the follicular epithelium that defines the anterior structures of the eggshell is well documented, whereas the molecular mechanisms controlling the expression of the eggshell structural genes remain poorly understood. From our data, it appears that the Dpp signaling pathway controls eggshell morphogenesis also by regulating the expression of eggshell structural genes. The exquisite temporal and spatial regulation of the VM32E eggshell structural gene, partly mediated by the critical morphogenic Dpp signaling system, suggests a crucial role of VM32E protein in the complex process of eggshell assembly.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

Fly Strains

The following stocks were used: Drosophila pseudoobscura wild-type stock (Tucson Drosophila Species Stock Center), CY2 Gal4 (Queenan et al.,1997), Mad12FRT40A/SM6a, and ubiquitin-GFP FRT40A; GR1-GAL4 UAS-FLP (Gupta and Schüpbach,2003), UAS-Mad, UAS-Med (Marquez et al.,2001), UAS-tkvQ253D (Nellen et al.,1996), His2avDGFP (Clarkson and Saint,1999), UAS-dpp (Staehling-Hampton and Hoffman,1994), −253/−39-lacZ, −112/−39-lacZ (Cavaliere et al.,1997), and VM32E-MYC (Andrenacci et al.,2001). Stocks were raised on standard cornmeal/yeast/agar medium at 25°C, and crosses were made at 18°C unless otherwise stated. yw67c23/yw67c23 was used as the wild-type stock in this study.

In Situ Hybridization

Whole-mount in situ hybridization with digoxigenin-labeled (Roche) probes was performed as described by Tautz and Pfeifle (Tautz and Pfeifle,1989). The 3′ end of the VM32E cDNA (Gigliotti et al.,1989) and the VM26A.2 DNA region (−40/+230) were used as probes. The egg chambers were viewed with Nomarski optics on a Nikon Eclipse 90i microscope.

Clonal Analysis

Follicle cell clones were induced using the directed mosaic GAL4/UAS-FLP technique (Duffy et al.,1998). Follicle cell clones mutant for Mad were generated using a GR1-GAL4 UAS-FLP line, which is expressed in all the follicle cells, including the follicle cell stem cells (Gupta and Schüpbach,2003). Mad12FRT40A/SM6a females were crossed with ubiquitin-GFP FRT40A; GR1-GAL4 UAS-FLP males. Females of the genotype Mad12FRT40A/ubiquitin-GFP FRT40A; GR1-GAL4 UAS-FLP/+ were collected and maintained on yeasted vials at 25°C for 3 days before dissection. To analyze −253/−39-lacZ gene expression in Mad12 follicle cell clones, females carrying the −253/−39-lacZ construct on the X chromosome and the balancer TM3 Ser were crossed with ubiquitin-GFP FRT40A; GR1-GAL4 UAS-FLP males. The TM3 Ser male offspring was crossed with Mad12FRT40A/SM6a females. Females of the genotype −253/−39-lacZ/+; Mad12FRT40A/ubiquitin-GFP FRT40A; GR1-GAL4 UAS-FLP/+ were collected and maintained on yeasted vials at 25°C for 3 days before dissection.

Gal4-Driven Expression in Follicle Cells

Females CY2 Gal4/UAS-GFP; CY2 Gal4/UAS-dpp; CY2 Gal4/UAS-tkvQ253D; CY2 Gal4/UAS-Mad; CY2 Gal4/UAS-Med and CY2 Gal4/UAS-dpp/−253/−39-lacZ were obtained by crossing the parental strains. The crosses were performed at 18°C and the progeny was transferred to yeasted vials at 29°C for 3 days before dissection.

Immunofluorescence Microscopy

Fixation and antibody staining of hand dissected ovaries were carried out as previously described (Andrenacci et al.,2001). Anti-CVM32E (1:100) or anti-VMP (1:50) antibodies were used and detected with Cy3-conjugated anti-rabbit secondary antibody (1:100, Sigma). Rabbit anti-phosphorylated SMAD (PS1) was used at 1:500 dilution (Tanimoto et al.,2000) and detected with Cy3-conjugated anti-rabbit secondary antibody (1:100). Anti-MYC monoclonal antibody (Santa Cruz Biotechnology) was used at 1:100 dilution and reacted with fluorescein isothiocyanate (FITC) -conjugated anti-mouse secondary antibody (1:250, Molecular Probes). Anti-βgal monoclonal antibody (Developmental Studies Hybridoma Bank) was used at 1:25 dilution and reacted with FITC (1:250) or Cy3-conjugated anti-mouse secondary antibody (1:100, Sigma).

DAPI (4′-6-diamidino-2-phenylindole) staining was carried out by incubating for 10 min the egg chambers with DAPI at 1μg/ml in phosphate buffered saline (PBS) and, after several washes with PBS, the egg chambers were mounted.

Stained egg chambers mounted in Fluoromount-G (Electron Microscopy Sciences) were analyzed with conventional epifluorescence and with TCS SL Leica confocal system attached to a Zeiss Axiophot microscope or viewed with Nomarski optics on a Nikon Eclipse 90i microscope.

Neutral Red Assay

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

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

We thank Trudi Schüpbach, Stuart Newfeld, and Franco Graziani for fly strains. We also thank the Bloomington Stock Center and the Tucson Drosophila Species Stock Center for providing us with fly stocks. We thank Tetsuya Tabata and the Ludwig Institute for Cancer Research for the rabbit phospho-Smad1 antiserum and the Developmental Studies Hybridoma Bank for anti-βgal monoclonal antibody. We thank Alessandra Donati for helpful discussion. We also thank Silvia Gigliotti and Franco Graziani for critical reading of the manuscript. A very special thanks goes to Tien Hsu for his advice on manuscript preparation and helpful suggestions. We also thank Marco Privitera for the graphic works. This work was supported by the University of Bologna in the framework of the Project “Meccanismi e segnali molecolari della sopravvivenza cellulare”; MIUR (Ex 40%, 2004/2006) and University of Bologna in the framework of the Project “Nuove strategie di controllo degli insetti con geni di antagonisti naturali”.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES