Figure 2. Rab11 is required for the survival of follicle epithelial cells.

(A–E) Confocal images of immunostained germaria and/or egg chambers 10–12 days ACI, with rab11-null cells marked by the absence of nGFP. All egg chambers are derived from mosaic germaria that contained one rab11-null FSC and one wildtype FSC as evidenced by direct examination of the adjacent germarium and/or the presence of rab11-null cells in the egg chambers themselves. The stages of the compound and fused egg chambers are estimates based on the size of the nurse cell nuclei, the distance of the fused/compound egg chamber from the germarium, and/or its position relative to other more easily staged egg chambers within the same ovariole. (A) An s5 egg chamber fused to a compound egg chamber containing two ∼s6/7 germline cysts encased in a single continuous epithelium and immunostained for nGFP (green) and E-cad (red). rab11-null cells, which are marked with asterisks, are more abundant in the s5 egg chamber than in the adjacent older ones. The positions of the rab11-null cells in the compound egg chamber are consistent with a stalk or stalk-like identity (discussed more fully in Text and see Fig. 3). (B) An s5 egg chamber fused to an s8 or s9 egg chamber immunostained for nGFP (Green) and Fas3 (red). The arrowhead points to a cluster of 2–3 rab11-null cells that over express Fas-3. While such expression is indicative of a polar cell fate, we cannot rule out a stalk cell fate for these cells as stalk cells that fail to incorporate themselves into a functional stalk are also known to over-express Fas3 [43]. No rab11-null epithelial cells are seen in this plane of focus. Other focal planes (not shown) contained no or only a few rab11-null epithelial cells (not shown). The two strongly expressing Fas3 cells at the posterior end of the s8/9 egg chamber are wildtype polar cells, although the GFP signal is weak at the focal plane shown. (C) Germarium and s3 egg chamber immunostained for nGFP (Green) and E-cad (red). The arrow points to a putative wildtype FSC. A large rab11-null clone in the germarium is outlined in white. The dashed yellow line in the adjacent s3 egg chamber highlights two large gaps in the epithelium as evident by the absence of E-cad expression and also Nomarski imaging (not shown). (D) Germarium and s2 and s3 egg chambers immunostained for nGFP (green) and counterstained for activated caspase 3, a marker for PCD. Approximately half of the rab11-null cells (outlined with the dashed line) in the s3 egg chamber stain positively for activated caspase 3. The apparent weaker staining of some of the rab11-null cells is not evident at other focal planes (not shown). The activated caspase 3 staining activity at the junction between germarial regions 1 and 2a (arrowhead) is likely to correspond to escort cells, which are known to be targeted for PCD at this stage [44]. (E) Control rab11-null ; P[rab11+] clones in s4 and s7 egg chambers immunostained for nGFP (green), E-cad (red).