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doi: 10.1242/10.1242/dev.00115

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Eyes Absent, a key repressor of polar cell fate during Drosophila oogenesis

Department of Biological Chemistry, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD, USA

View larger version (72K): [in a new window]   Fig. 1. Egg chamber development in wild-type and mutant ovaries. (A) A Drosophila ovariole showing the germarium and egg chambers from stages 3-10 (anterior towards the left). Each egg chamber contains 16 germ cells, including 15 nurse cells (nc) and one oocyte (o). Polar cells are shown in red. Stalk cells separate adjacent egg chambers. Border cells are shown in blue. (B-D) Normarski optics images of egg chambers from enhancer trap line PZ6356, stained for ß-galactosidase, which is expressed at highest levels in the border cells (arrows) and the oocyte nucleus (arrowheads). (B) A wild-type, stage 10 egg chamber with one cluster of border cells (BCs) at the nurse cell/oocyte border (ON, oocyte nucleus). (C) A stage 10 mosaic egg chamber containing 54C2 mutant cells and three clusters of border cells. (D) A stage 10 mosaic egg chamber containing cos2 mutant cells and two clusters of border cells. (E-G) Fluorescence micrographs of a border cell cluster double stained with anti-FAS3 (green) and rhodamine phalloidin (red). In wild-type egg (E), one pair of anterior polar cells stains. The polar cells (pc) are surrounded by border cells (bc). In both 54C2 (F) and cos 2 (G) mosaic egg chambers, extra border cells surround extra polar cells. (H-J) Normarski optics images of egg chambers stained for ß-galactosidase from enhancer trap line A101 (neu-lacZ), which is a marker for mature polar cells. (H) In wild-type, polar cells are located at the anterior and posterior poles. In egg chambers containing 54C2 (I) or ptc (J) mosaic chambers, polar cells are found at many locations. (K-P) Fluorescence micrographs of egg chambers stained with anti-FAS3 (K,L,N,O) or DAPI (M,P). (K) A series of wild-type egg chambers up to stage 5. (L) A wild-type, stage 9 egg chamber. In the germarium and stage 1 egg chamber, all follicle cells are FAS3 positive. Polar cells are indicated by white arrowheads. (M) A wild-type, stage 9 egg chamber stained with DAPI. (N-P) Egg chambers from eya3cs/eyaE(P)10 females. Egg chambers are either arrested early in oogenesis (N) or are covered with FAS3-positive cells (O). In the egg chamber in P, which is the same one as in O, the germ cells are degenerating. Scale bars: in B, 50 µm for B-D; in E, 10 µm for E-G; in H, 50 µm for H-J; in K and N, 25 µm for K-M and N-P, respectively.

View larger version (90K): [in a new window]   Fig. 2. EYA protein expression during Drosophila oogenesis. Fluorescence confocal micrographs of wild-type egg chambers stained for GFP (ub-Cadherin-GFP) (green) and EYA (red) (A,B), or for EYA (green) and ß-galactosidase (red) (C-F). In C,E,F, expression of ß-galactosidase (red) from enhancer trap line A101 labels polar cells. In D, ß-galactosidase expression from enhancer trap line 93F is shown, which labels stalk cells. EYA protein is expressed in the germarium (A) and in all follicle cells except polar cells (A-C) and stalk cells (D) up to stage 8. (E) A stage 9 egg chamber in which EYA is excluded from the polar cells (arrows), but is expressed in migrating border cells. (F) EYA expression in a stage 10 egg chamber. Scale bars: in A, 10 µm for A; in B, 10 µm for B; in C, 10 µm for C-E; in F, 50 µm for F. Arrows in A,B indicate regions lacking EYA expression; arrows in C,E,F indicate polar cells.

View larger version (140K): [in a new window]   Fig. 3. Loss of eya leads to ectopic polar cells in a cell autonomous fashion. Fluorescence confocal micrographs of epithelial follicle cells stained for GFP (green) (A-C) and for EYA (red in B), FAS3 (red in E) or ß-galactosidase from A101 (red in H,I). Co-localization appears yellow. GFP-positive cells are wild type, whereas the cells lacking GFP are eya mutant cells and are outlined in A-C. Scale bars: in A, 10 µm for A-C; in D, 10 µm for D-I.

View larger version (86K): [in a new window]   Fig. 4. The relationship between EYA and HH pathway genes. (A-C) Fluorescence confocal micrographs of a Pka mosaic egg chamber. GFP expression is shown in green. Both EYA (nuclear) and FAS3 (membrane) are shown in red. The Pka mutant cells lack GFP and are outlined by broken lines in A-C. (B) A composite image of a z-series of adjacent confocal sections showing the expression of EYA and FAS3 to demonstrate that some FAS3-positive cells have EYA expression, whereas others do not. The YZ plane corresponding to a slice through the Z series of the clone shown in B is shown in (B'). Of the FAS3-positive cells which have red membrane staining in (B), two cells (*) are EYA positive and four cells (arrows) are EYA negative and lack nuclear staining. (C) Merged picture of GFP, eya and FAS3 in a single section from A. (D-F) Fluorescence micrographs of a Pka mosaic egg chamber stained for EYA (green) and ß-galactosidase (red) from the enhancer trap line A101. Scale bars: in A, 5 µm for B' and 10 µm for A-C; in D, 10 µm for D-F.

View larger version (136K): [in a new window]   Fig. 5. The relationship between ci and eya. (A) Fluorescence micrographs of a ci mosaic egg chamber showing GFP expression in green and EYA expression in red. Cells that lack GFP are ci mutant cells. Co-localization of GFP and EYA appears yellow. (B,C) Fluorescence images of egg chambers double stained for FAS3 (green) and ptc-lacZ (red). (B) A wild-type egg chamber in which anterior polar cells (arrow) stain. (C) An eya mosaic egg chamber with two pairs of anterior polar cells (arrows), both of which have ptc-lacZ expression. (D) Fluorescence images of an egg chamber expressing the constitutively active form of CI (CIAC) (see Materials and Methods). Cells with ß-galactosidase expression (green) express CIAC. Expression of EYA is shown in red. (E,F) Fluorescence images of a Pka mosaic egg chamber double stained for CI (gray in E, red in F) and GFP in green. (G) Fluorescence micrographs of an eya mosaic egg chamber double stained for EYA (red) and CIN (green), which recognizes both CIAC and CIR (eya mutant clones are outlined) (H,I) Fluorescence images of an eya mosaic egg chamber double stained for CI (gray in H, red in I) and GFP in green. Scale bars: in A, 10 µm for A,D,G; in B, 25 µm for B,C; in E, 10 µm for E,F,H,I.

View larger version (87K): [in a new window]   Fig. 6. The phenotype of overexpression of eya in the ovary. Egg chambers stained with DAPI (A,C,C',E) to label nuclei or with anti-FAS3 (B,D,F,G) to label the polar cells. (A) A compound egg chamber produced by overexpression of eya in the ovary. (B) The same egg chamber as in A, which has four pairs of polar cells (arrows). (C) Compound egg chambers that never leave the germarium. (C') A wild-type germarium and the first two egg chambers for comparison. (D) A stage 8 egg chamber in which posterior polar cells express FAS3. Anterior polar cells appear to be missing. (E) A fused egg chamber. (F,G) Two different focal planes of the same egg chamber as in E. There are three pairs of polar cells (arrows) in this compound egg chamber. (H) A stage 10 egg chamber in which border cells express GFP and overexpress EYA. Migration is delayed. Arrowheads indicate the centripetal follicle cells and the normal extent of border cell migration at this stage. Scale bars: in A, 50 µm for A,B; in C, 25 µm for C,D; in C', 25 µm for C'; in E, 50 µm for E-G; in H, 50 µm for H.

View larger version (19K): [in a new window]   Fig. 7. The effect of activated N on EYA expression and polar cell fate. GFP (green) is expressed in cells in which activated N is expressed. A101 staining (red) labels polar cells, among which three are GFP-positive cells. EYA expression is shown in blue. Overlap of green and red appears yellow. Overlap of blue and yellow would appear white.

View larger version (67K): [in a new window]   Fig. 8. The so mutant phenotype in the ovary. (A) Nomarski optics images of a wild-type ovariole stained for ß-galactosidase activity to reveal the expression pattern of so5-lacZ in stalk cells (arrows) after stage 3. (B-D) Fluorescence images of two so mutant egg chambers (e1 and e2). (B) In the e1 egg chamber most follicle cells are mutant for so (lack GFP). In the e2 egg chamber, about half of the follicle cells are mutant for so and lack GFP (e2). (C) DAPI staining of the same two egg chambers. (D) Polar cells (arrows), revealed by anti FAS3 staining, develop normally. Scale bar: 50 µm.