Role of Scrib and Dlg in anterior-posterior patterning of the follicular epithelium during Drosophila oogenesis
© Li et al; licensee BioMed Central Ltd. 2009
Received: 9 June 2009
Accepted: 1 December 2009
Published: 1 December 2009
Proper patterning of the follicle cell epithelium over the egg chamber is essential for the Drosophila egg development. Differentiation of the epithelium into several distinct cell types along the anterior-posterior axis requires coordinated activities of multiple signaling pathways. Previously, we reported that lethal(2)giant larvae (lgl), a Drosophila tumor suppressor gene, is required in the follicle cells for the posterior follicle cell (PFC) fate induction at mid-oogenesis. Here we explore the role of another two tumor suppressor genes, scribble (scrib) and discs large (dlg), in the epithelial patterning.
We found that removal of scrib or dlg function from the follicle cells at posterior terminal of the egg chamber causes a complete loss of the PFC fate. Aberrant specification and differentiation of the PFCs in the mosaic clones can be ascribed to defects in coordinated activation of the EGFR, JAK and Notch signaling pathways in the multilayered cells. Meanwhile, the clonal analysis revealed that loss-of-function mutations in scrib/dlg at the anterior domains result in a partially penetrant phenotype of defective induction of the stretched and centripetal cell fate, whereas specification of the border cell fate can still occur in the most anterior region of the mutant clones. Further, we showed that scrib genetically interacts with dlg in regulating posterior patterning of the epithelium.
In this study we provide evidence that scrib and dlg function differentially in anterior and posterior patterning of the follicular epithelium at oogenesis. Further genetic analysis indicates that scrib and dlg act in a common pathway to regulate PFC fate induction. This study may open another window for elucidating role of scrib/dlg in controlling epithelial polarity and cell proliferation during development.
The follicle cell (FC) epithelium over the egg chamber in Drosophila ovary plays a pivotal role in the egg development. At oogenesis, follicular epithelial cells along the anterior-posterior (AP) axis are specified and differentiated into several distinct cell types that will subsequently either undergo a series of morphogenetic changes, or extend the germline-soma interactions [1–3]. While most of the epithelial FC subpopulations contribute to construction of the eggshell along with its specialized structures such as dorsal appendages through complex morphogenesis, the specified PFCs initiate establishment of the oocyte polarity, and determine the AP and dorsal-ventral (DV) axes of the resulting embryos. Thus, patterning of the follicular epithelium is an essential step for the proper development of the egg.
Drosophila oogenesis begins with formation of the 16-cell germline cyst in the germarium of the ovaries that is composed of 15 nurse cells and one oocyte. Each germline cyst is then encapsulated by a monolayer of the somatic stem cell-derived FCs [1, 4]. After the encapsulated cysts leave the germarium, the surrounding somatic FCs develop into a sheet of cuboidal epithelial cells and a pair of polar cells at each pole of the nascent egg chamber. As oogenesis proceeds, the follicular epithelium in the egg chamber becomes progressively polarized along the AP axis . Early on, two terminal domains are differentiated from the mainbody region in the epithelial FC layer. Further, cells in each terminal domain can adopt three different terminal fates, depending on their locations away from the poles of the chamber [1, 6, 7]. In this way, the mirror image prepattern of the terminal domains in the epithelium is generated. This symmetry was broken at mid-oogenesis when Gurken signal, produced in the oocyte, activates the EGFR signaling pathway in the surrounding FCs at the posterior of the egg chambers, defining a posterior fate in those cells [5, 8–11]. At this time, AP patterning of the epithelium is established. Starting from stage 7, the epithelial FCs cease proliferation and enter an endocycle [12, 13]. By stage 8, all specified FC types in the epithelium along the AP axis are differentiated into five distinct subpopulations, known as border, stretched, centripetal, main body and posterior cells.
Once becoming subdivided into various cell types along the AP axis, the epithelial FCs undergo dramatic cell shape change and directed migration at middle and late oogenesis [1, 2]. These morphogenetic processes include: (1) During stage 9, a group of 6-10 border cells delaminate from the anterior tip of the epithelium and migrate between the nurse cells to the anterior end of the oocyte; (2) At the same time, the majority of the FCs, including the centripetal, mainbody and PFC cells, move posteriorly to form a columnar epithelium covering the growing oocyte, while the stretched cells adjacent to the border cells flatten to be in association with the nurse cells as a squamous epithelium; (3) At stage 10b, the centripetal cells migrate between the nurse cells and the oocyte towards the center of the egg chamber to cover the anterior region of the oocyte. The unique morphogenesis above conferred by the patterned follicular epithelium is destined to bring the vast majority of the FCs into contact with the oocyte for ultimate formation of the eggshell with its specialized structures.
Apart from being involved in the deposition of a functional eggshell, the specified PFCs function in the axial patterning of the oocyte and the resultant embryo. During stage 6-7, the PFCs signal back to the oocyte, causing the reorganization of its microtubule (MT) cytoskeleton [14, 15]. This directs the MT-dependent localization of bicoid and oskar mRNA to the anterior and posterior pole of the oocyte respectively, thus defining the AP polarity of the oocyte, and the resulting embryo's AP axis [15–17]. The repolarization of the MT cytoskeleton also triggers the migration of the nucleus from the posterior to the dorsal anterior corner in the oocyte . At this time, the localized Grk around the nucleus signals for a second time to induce the overlying main body cells to adopt a dorsal, rather than a ventral fate, specifying the pattern of the DV axis [10, 11, 19–21]. Thus, the PFCs execute dual functions for the egg development.
Each cell in a given tissue or organ can interpret its positional information provided by signaling molecules from a local source to adopt a specific fate. Several lines of evidence have indicated that the JAK/STAT and EGFR pathways, elicited by the signaling ligands Unpaired from the polar cells and Gurken from the oocyte respectively, function in the AP patterning of the entire follicular epithelium. Prior to stage 6 of oogenesis, the graded JAK/STAT pathway activities induce the division of the FC layer into the two terminal domains and the main body domain, and three default anterior fates within each terminal [6, 7, 22]. Later on, activation of EGFR signaling pathway in the posterior terminal instructs those terminal cells to adopt a posterior, rather than an anterior fate. Meanwhile, the Notch signaling activity is necessary for differentiation of those specified FC types by controlling a switch from mitosis to endocycle in the epithelium at early mid-oogenesis [12, 13]. Although essential role of the JAK signaling in patterning the AP axis of the follicular epithelium is established, current data also suggest that other signals present at the terminal are required for specification of the distinct anterior fates, particularly induction of the stretched and centripetal cell subpopulations[1, 6, 7].
To date, several genes have been showed to regulate the above three well-characterized signalling pathways at oogenesis, thereby being implicated in the follicular patterning [23–28]. For example, the Hippo (Hpo) tumor suppressor pathway components are involved in the PFC fate induction through modulating the Notch activity [24–26, 29]. Recently, we reported that lgl is required in the FCs for specification and differentiation of the PFCs at mid-oogenesis . In this study we tested another two Drosophila tumor suppressor genes scrib and dlg for their role in PFC fate induction. Removal of scrib or dlg function from the epithelial FCs at the posterior resulted in a loss of the PFC fate, subsequently causing oocyte polarity defects. Further, we showed that the aberrant coordinated activities of EGFR, JAK/STAT and Notch pathways elicited by mutations in scrib/dlg are causally linked to defective specification of the PFC fate in the mosaic clones. We also investigated whether scrib/dlg are implicated in anterior patterning of the follicular epithelium. Inactivation of scrib or dlg in the FCs at the anterior disrupted specification of the stretched and centripetal cell fate, whereas differentiation of the border cells can still occur in the most anterior regions of the mutant clones. Finally, we provided genetic evidence that scrib and dlg function in posterior patterning of the epithelium in a common pathway.
scrib/dlgare required for specification and differentiation of the PFCs
Disruption of the signaling pathways may underlie the defective PFC fate induction in scrib/dlgmutants
To better understand how the EGFR signaling is disrupted in the mutant FCs at the posterior, we further examined whether mutations in scrib/dlg block activation of ERK, the key signal transducer of EGFR pathway [39, 40]. Our analysis revealed that ERK activation can still occur in the posterior scrib or dlg mutant FCs at stage 6-8 of oogenesis, as indicated in the presence of di-phosphorylated form of ERK (scrib2, n = 23; dlgm52, n = 28) (Fig 2I, J). These data suggest that disruption of the EGFR signaling pathway at the posterior domains induced by loss of scrib or dlg may take place downstream of ERK activation.
Expression of the AFC markers in scrib/dlg mutant clone cells at the posterior terminal
Presence of The AFC Fate in PFC Clones
0% (n = 66)
0%(n = 54)
0% (n = 37)
0%(n = 12)
10%(n = 20)
Anterior Follicle cell
8.3%(n = 36)
Anterior Follicle cell
9.1%(n = 22)
In sum, loss-of-function mutations in scrib/dlg cause perturbation of coordinated activities of the three signaling pathways, which could well explain why the mutant FCs at the posterior lose the PFC fate and do not adopt an AFC fate either.
Mutations in scrib/dlgcause aberrant anterior patterning of the follicular epithelium
Induction of distinct AFC fate in scrib/dlg mutant clone cells at the anterior terminal
Absence of The AFC Fate in AFC Clones
4.5% (n = 67)
13.7% (n = 51)
26.5% (n = 34)
27.6% (n = 29)
77.8% (n = 27)
We next examined effects of scrib/dlg mutations in specification of the more central anterior fates, stretched and centripetal cells populations using enhancer trap line MA33 and BB127 respectively. As depicted in Fig 5E, F and Table 2, in a certain percentage of stage 10 scrib or dlg mutant egg chambers, MA33 expression was absent in clones at the stretched cell territory (scrib2, 26.5%, n = 34; dlgm52, 27.6%, n = 29), implying a defect in stretched cell differentiation. Starting from stage 9 specified stretched cells undergo a morphogenetic change to become a squamous epithelium covering nurse cells at the anterior . It is conceivable that defective patterning of the stretched cell population will subsequently block its spreading, which happens during the morphogenesis. As predicted, in vast majority of scrib/dlg mutant clones with defective differentiation of the stretched cells, the mutant cells failed to spread out and adopt the squamous morphology indicative of aberrant morphogenesis (scrib2, 88.8%, n = 9; dlgm52, 87.5%, n = 8) (Fig 5E). Likewise, loss of scrib function in FCs at the anterior resulted in a failure to express BB127 in a high percentage of stage 10b mosaic chambers harboring both stretched and centripetal cell clones (77.8%, n = 27) (Fig 5G and Table 2), indicating a loss of centripetal cell fate. Further study revealed that the defective "centripetal cells" can not migrate centripetally (90.4%, n = 21) (Fig 5G). Thus, we conclude that mutations in scrib/dlg perturb specification of the more central anterior fates. Given that the graded activities of JAK/STAT pathway are necessary for determining specific fates within the anterior terminal domain, we sought to analyze JAK signaling activity in clones harboring the stretched and centripetal cell populations by examining the nuclear accumulation of STAT92E protein. In the wild type, nuclear accumulation of STAT92E indicative of the JAK signaling activities is still present in the specified squamous FCs covering the nurse cells at stage 10 (Fig 6H), but absent in specified centripetal cells (date not shown). We, therefore, chose to determine whether mutations in scrib/dlg perturb JAK signaling during patterning and early morphogenesis of the stretched cell population. Strikingly, in all defective "stretched cells" of scrib/dlg mutant clones (scrib2, n = 25; dlgm52, n = 21), STAT92E protein accumulates in nuclei to a level comparable with that in wild type stretched cells (Fig 6I, J), indicating that activation of JAK/STAT pathway can still occur in the mutant cells. Thus, these data suggest that defective patterning of the stretched cell population induced by loss of scrib or dlg does not require disruption of the JAK/STAT pathway.
Overall, mutations in scrib/dlg cause an aberrant anterior patterning of the follicular epithelium, particularly a defect in specification of the stretched and centripetal cell fates. In this circumstance, defective AFC cell fate induction is closely correlated with the aberrant morphogenesis.
scrib genetically interacts with dlgin posterior patterning of the epithelium
We next investigated whether scrib interacts genetically with dlg in subdivision of the follicular epithelium into distinct cell types. GR1-Gal4-driven expression of dlg RNAi in the entire epithelium heterozygous for dlgm52 caused failure of the FCs at the posterior to differentiate properly in a certain percentage of the mutant chambers (Fig 7A). Strikingly, heterozygosity for scrib2 increases the penetrance of defective PFC fate induction in dlgm52/+; dlg RNAi mutant chambers (Fig 7A, Bb and 7Bb'), strongly suggesting that scrib and dlg act in a common pathway to function in posterior patterning of the epithelium. Likewise, genetic interactions of scrib with dlg were also observed for FC overaccumulation at the anterior (Fig 7A and 7Bc). In this case, however, development of the mutant chambers with multilayered FCs at the anterior was completely blocked before expression of the enhancer trap markers for the AFC fates appears at stage 10. This impeded a direct assay of the AFC fate in the mutant epithelium with cell overaccumulation at the anterior. For those mutant chambers with the FCs at the anterior remaining a monolayer, we detected expression of the AFC fate marker L53b in anterior mutant FCs (data not shown). Thus, study of genetic interactions between scrib and dlg in the anterior patterning is inconclusive.
In the present study we show that scrib/dlg function in both anterior and posterior patterning of the follicular epithelium. While removal of scrib or dlg function from the FCs at the posterior terminal completely blocked specification and differentiation of the PFCs, loss-of-function mutations in scrib/dlg at the anterior domain resulted in a partially penetrant phenotype of defective AFC cell fate induction as indicative of absence of the stretched and centripetal cell types at stage 10 of egg chambers. The differential regulation of the PFC and AFC cell differentiation by scrib/dlg could be attributable to the distinct signaling basis underlying the follicular patterning at the two terminals. In the case of posterior patterning, the combinatorial and sequential activities of JAK/STAT, EGFR and Notch signaling pathways play key roles in this process. The fully penetrant phenotype of aberrant PFC fate specification in posterior scrib/dlg mutant clones can be explained by the fact that inactivation of scrib or dlg completely perturb the EGFR signaling. Further investigation demonstrated that scrib/dlg mutation also causes defects in coordinated activation of JAK/STAT and Notch pathways in each multilayered clone cell, as evident in localized activity of JAK signaling and Notch signaling in the outer layer and inner layer respectively (Fig 4B, C, E, G, H, and 4J). Thus, the mutant cells at the posterior terminals generally do not adopt a terminal cell fate either. At the anterior, AFC fate induction requires JAK and Notch signaling activities. Current data, however, do not exclude a possibility that other unknown signals are involved in specifying the distinct AFC cell types, e.g. the stretched and centripetal cells[1, 6, 7]. We observed that while JAK signaling activity is present in the FCs surrounding the polar cells, preferentially in outer cells of the multilayered clones at the anterior terminals, activation of Notch pathway occurs in almost all mutant cells. The combined pattern of JAK and Notch signaling activity in the anterior mutant cells provides a good basis that the border cell, at least Slbo-expressing cell fate, can be induced in the most anterior region of the multilayered clones. However, our clonal analysis revealed specification of the stretched or centripetal cell types in corresponding mutant clones at the anterior is disrupted, albeit to a lesser extent. Considering the presence of JAK signaling activity in the mutant clone cells, we assume that loss-of-function mutation of scrib/dlg may perturb other unidentified signals implicated in patterning of the stretched and centripetal cell subpopulations.
We have identified in this report aberrant EGFR signaling pathway as the mechanism underlying defective PFC fate induction in scrib/dlg mutant FCs. In addition to EGFR pathway, loss of scrib or dlg function at the terminal domains can differentially affect JAK and Notch signaling activities in the multilayered clone cells. JAK signaling was absent in inner cells of the multilayered clones at the two terminals. Conversely, loss of Notch activity was localized to outer cells of the posterior multilayered clones. This discrepancy is likely to be linked to the spatial and temporal control of each signaling activity with respect to growth and patterning of the FC layer during oogenesis. Starting from stage 6 of oogenesis, Notch signaling is activated in all epithelial FCs for inducing the mitotic-to-endocycle transition [12, 13]. Given that at this time the multilayered cells have been formed in scrib/dlg mutant clones at the terminals, it can be imagined that activation of Notch pathway occurs only in the mutant cells directly contacting the Delta-producing germ cell, as shown in the inner cells of the posterior mutant clones (Fig 4E, G, H and 4J). This scenario was further justified by the recent report that loss of lgl causes the same aberrant Notch signaling pattern in the posterior multilayered FCs as scrib/dlg mutation does . Surprisingly, the multilayered scrib/dlg mutant clones at the anterior display a distinct Notch signaling pattern in which almost all FCs regardless of their spatial relation with the oocyte are positive for Notch activation (Fig 6E, F and 6G). Although at this point we do not understand the basis for this controversial pattern of Notch activity in the mutant clones at different terminals, this mechanism may partly underlie the observation in the present study that loss-of-function mutations in scrib/dlg differentially affect anterior and posterior patterning of the epithelium.
Likewise, the distinct pattern for JAK/STAT pathway activity between the inner and outer cells in scrib/dlg mutant clones is probably due to the spatial location of the ligand sources relative to the multilayered cells. Indeed, analysis of the polar cell positioning in the mutant clones revealed that the polar cells are in close proximity to the single layer of outer cells that retain JAK signaling activity (scrib2, 95.3%, n = 43; dlgm52, 88.9%, n = 27) (see Additional file 1) (Fig 4B, C). The positioning of the polar cells led us to argue that inner cells in the multilayered clones do not respond to the signaling ligand Unpaired secreted from polar cells, presumably due to their spatial relation with the ligand source. However, unlike the Notch pathway, JAK signaling is activated in FCs at the terminal domains of the egg chambers at early oogenesis after the polar/stalk cells are specified [6, 7, 22]. This temporal regulation might be alternatively responsible for the distinct JAK/STAT activity pattern in the multilayered clone cells. In this model, we assume that JAK/STAT pathway is activated in the mutant FCs at the terminal domains prior to occurrence of the cell overaccumulation. Thus, the presence of JAK signaling activity in single outer cell layer of the multilayered scrib/dlg mutant clones may indicate the initial activation of JAK/STAT pathway induced by the polar cells for specifying the terminal fate at early oogenesis. On the contrary, the inner cells deriving from the overaccumulation fail to respond to Unpaired ligand. Further studies in this direction will better define the underlying mechanisms for defective follicular patterning elicited by loss of scrib or dlg.
The phenotypic effects of scrib/dlg mutation in posterior patterning of the epithelium are similar to those of the Hpo pathway deficiency [24–26]. Further characterization of the patterning defects, however, reveals distinct underlying mechanisms for these two instances. In the case of the Hpo pathway, loss of the pathway component Hippo, Salvador, or Warts disrupts Notch signaling in all mutant FCs at the posterior via interfering with endocytosis of the Notch receptor, thereby resulting in aberrant PFC cell specification and differentiation at mid-oogenesis [24–26]. By contrast, activation of the Notch signaling is evident in inner cells of the multilayered scrib/dlg mutant clones at the posterior (Fig 4E, G, H and 4J) and almost all the multilayered clone cells at the anterior (Fig 6E, F and 6G). Furthermore, the fully disrupted EGFR pathway associated with the posterior patterning defects conferred by loss of scrib or dlg clearly distinguishes the Hpo pathway from scrib/dlg in the signaling basis for the mutant phenotype of defective PFC fate induction ([24–26] and this paper). In Drosophila, scrib/dlg are known to encode scaffolding proteins that are localized at the septate (basolateral) junctions of epithelial cells, and regulate the apico-basal cell polarity[31, 35, 50–52]. Previous studies have demonstrated that mutations in scrib/dlg disrupt the epithelial polarity in the FCs at the terminal domains of egg chambers, exhibiting mislocalized cell polarity proteins [31, 48, 53]. Based on this fact, one would surmise that the polarity defects observed in the posterior scrib/dlg mutant FCs perturb the apical accumulation of EGFR receptors, rendering these cells incompetent to respond to EGF signals due to failure of EGFR activation. However, further studies in this paper disapproved this simple scenario. Instead, we found that ERK is di-phosphorylated in the posterior FCs lacking scrib or dlg, suggesting that the EGFR in the mutant cells can still be activated in response to Grk signal. Thus, this finding points out that blocking in signal transduction from the activated ERK to the downstream targets elicited by loss of scrib or dlg may result in failure of the mutant FCs at the posterior to respond to EGFR signals.
Once the specified FC cell types are induced, each cell population will undergo a unique morphogenetic change and execute respective functions . Remarkably, we observed a concurrent defect in morphogenesis of those anterior scrib/dlg mutant clone cells with aberrant patterning of the stretched or centripetal cell subpopulation (Fig 5E, G and Fig 6I, J). It would be interesting and important to determine whether scrib/dlg is implicated in morphogenesis of the patterned follicular epithelium as well. For this purpose, we need to identify a scrib or dlg mutant allele for certain genetic background in which the morphogenesis can be uncoupled from the patterning process. Under such circumstance could we generate scrib/dlg mutant clones with proper patterning of the AFC cell types, and then test how the subsequent morphogenesis occurs in the specified cell subpopulations, e.g. the stretched and centripetal cells. Likewise, a hypomorphic scrib or dlg allele with certain reduced activity could be of great value to understanding better how the specified PFCs function in polarization of the oocyte at mid-oogenesis. Interestingly, we found that RNAi-mediated knockdown of the endogenous dlg expression alone in the follicular epithelium can disrupt the oocyte polarity as indicative of mislocalization of Stau, but properly induce the PFC fate (our unpublished data). This unexpected observation led us to propose that expression of this dlg RNAi transgene may specifically perturb the process in which the specified PFCs control formation of the oocyte polarity. Thus, screening a Drosophila mutant library such as the transgenic RNAi library for gene(s) modifying the dlg RNAi phenotype would unveil the mechanisms responsible for involvements of dlg in regulation of the PFC function.
In this paper we present the first demonstration that the tumor suppressor genes scrib and dlg are required in the FCs for patterning of the follicular epithelium along the AP axis during Drosophila oogenesis. Genetic interaction of scrib with dlg in specification and differentiation of PFCs indicates a cooperative role between these two genes. While the data clearly show a differential role of scrib/dlg in anterior and posterior patterning of this epithelial layer, the underlying mechanisms await further investigations. Overall, study in this direction may provide alternatives for addressing scrib/dlg-mediated regulation of cell polarity and proliferation in epithelial tissues.
Fly stocks and genetics
All Drosophila stocks were maintained and crossed at 25°C according to standard procedures. Egg chamber stages are according to Spradling . The Canton S (CS) strain was used as wild type. scrib2 and scrib1 are null alleles of scrib [31, 52], and dlgm52 is a null allele of dlg . The transgenic RNAi line for dlg, UAS-dlg RNAi , was obtained from Vienna Drosophila RNAi Centre (VDRC, Transformant ID 41134). en-Gal4 (gift from A Bergmann)  and GR1-Gal4 (gift from T Schüpbach)  were used to drive its expression.
Mutant clones were generated by mitotic recombination using FLP/FRT Technique . Homozygous scrib2, scrib1 or dlgm52 clones were generated by crossing FRT82B scrib2/TM3 Sb (gift from D Bilder) or FRT82B scrib1/TM3 Ser (gift from HE Richardson) to yw hsFLP;FRT82B ubi-GFPnls, or crossing FRT101 dlgm52/FM7 (gift from S Goode) to FRT101 hGFP/FM7; hsFLP/CyO (gift from DA Harrison) or FRT101 tub-lacZ hsFLP/FM7 (gift from S Goode). To obtain follicle cell clones, the flies were heat-shocked as 3rd instar larvae and pupae at 37°C for 1 h on 4 consecutive days. Before dissection, all adults were put into fresh food vials for 2 days. The following enhancer trap markers were incorporated into the above fly strains for making scrib2 or dlgm52 clones: 998/12 (gift from D St Johnston) , Kinesin-lacZ (yw Kinesin-lacZ, gift from D St Johnston and KZ503, gift from YN Jan) , kek enhancer trap line BB142 (gift from T Schüpbach) [23, 37], m7-lacZ (gift from T Xie) [24, 46], MA33, BB127, L53b (gift from DA Harrison) [8, 41], dpp-lacZ (Bloomington Drosophila Stock Center) . 998/12 was recombined onto the FRT82B, scrib2 chromosome using meiotic recombination.
Antibodies and immunofluorescence
For antibody staining, ovaries were dissected into phosphate buffered saline (PBS) with 0.1% bovine serum albumin, fixed in 4% paraformaldehyde for 30 min and washed three times with PBST (0.3% Triton X-100 in PBS) except for anti-STAT92E and anti-dp-ERK staining. Then ovaries were permeabilized in PBS with 1% Triton X-100 for 1 h at room temperature (RT) followed by a 2 h incubation in PBST with 10% normal goat serum. Primary antibodies were incubated with ovaries at 4°C overnight. On the following day, ovaries were washed with PBST three times for 20 min and blocked for 1 h at RT. Then they were incubated with secondary antibodies or Phalloidin-TRITC (Sigma) at RT for 2 h, and stained with DAPI (Molecular Probes) for 10 min. Finally, ovaries were rinsed three times with PBST and mounted in VECTASHIELD Mounting Medium (Vector Laboratories). For anti-STAT92E staining ovaries were washed and incubated in PBS with 0.3% Tween-20. For anti-dp-ERK staining ovaries were fixed for 30 min in 8% formaldehyde, rinsed for an hour in PBS with 0.1% Tween-20, and stored overnight in methanol. After progressive rehydration and block, the ovaries were incubated with anti-dp-ERK antibodies.
The following primary antibodies were used in this work: rabbit anti-Stau (1:2000 gift from D St Johnston) , mouse anti-β-gal (1:10 DSHB 40-1a), rabbit anti-β-gal (1:50000 Cappel), rabbit anti-DG (1:3000 gift from WM Deng) , mouse anti-dp-ERK (1:200 Cell Signaling) [39, 40], rabbit anti-STAT92E (1:1000 gift from SX Hou) , mouse anti-Hnt (1:200 DSHB 1G9), mouse anti-Cut (1:100 DSHB 2B10), rat anti-Slbo (1:500 gift from P Rørth) , mouse anti-Dlg (1:1000 DSHB 4F3), mouse anti-Fas3 (1:200 DSHB 7G10). Secondary antibodies conjugated with Alexa Fluor 488, 546, 568 (Molecular Probes) were used at 1:1000 dilutions.
Confocal images were captured on Zeiss LSM 510 META laser scanning microscope and processed in Adobe Photoshop.
P-values were calculated by applying χ2-test.
We deeply thank David Bilder, Helena E. Richardson, Scott Goode, Douglas A. Harrison, Andreas Bergmann, Trudi Schüpbach, Daniel St Johnston, Yuh Nung Jan, Ting Xie, Wu-Min Deng, Steven X. Hou, Pernille Rørth, the Bloomington Drosophila Stock Center, the Vienna Drosophila RNAi Centre and the Developmental Studies Hybridoma Bank for providing fly strains and antibodies. We are also grateful to Bernard Mechler at the DKFZ, Heidelberg for helpful discussion and Shiyun Feng for her technical assistance. This work was supported by National Natural Science Foundation of China (30871409, 30800648), National Basic Research Program of China (2007CB947301), the Shanghai Pujiang Program (05PJ14075) and Shanghai Leading Academic Discipline Project (B205).
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