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First published online 14 June 2006
doi: 10.1242/dev.02423

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Csk differentially regulates Src64 during distinct morphological events in Drosophila germ cells

¹ Department of Biological Sciences, Stanford University, 385 Serra Mall, Stanford, CA 94305, USA.
² Zoologisches Institut, Universität Zürich, Winterthurerstr. 190, CH-8057 Zürich, Switzerland.

View larger version (37K): [in a new window]   Fig. 1. Src64KO fertility defects. (A) Confocal projections of stage 10 egg chambers stained with phalloidin (green) to visualize filamentous actin and anti-Src64 (red) to visualize Src64 protein. Genotypes are abbreviated. (B) Immunoblot of ovary extracts using anti-Src64 antibody or anti-BicD (loading control). (C) Egg lay rates for wild-type (WT) females versus Src6417 or Src64KO expressed as the number of eggs laid/fly/hour. (D) Hatch rates for WT versus Src64 mutants. Scale bars: 10 µm.

View larger version (95K): [in a new window]   Fig. 2. Src64 mutants exhibit ring canal morphology defects. (A) Confocal projections of stage 10 WT, Src64 mutant (Src64KO, Src6417), or rescued Src64 mutant ([Src64WT]/+;Src64KO or [Src64WT]/+;Src64 17) egg chambers stained with anti-Hts-RC to visualize ring canals. (B) Average size ring canals labeled with anti-Hts-RC. (C) Partial confocal projections of stage 10 egg chambers labeled with phalloidin (green) to visualize filamentous actin and propidium iodide (red) to visualize DNA. Arrows and brackets indicate nurse cell fusion events. Scale bars: 10 µm.

View larger version (44K): [in a new window]   Fig. 3. Csk does not regulate ring canal growth. Confocal projections of stage 10 egg chambers stained with anti-Hts-RC to visualize ring canals are shown for (A) FRT82B, CskD589N, CskQ156Stop. (B) WT, overexpression of wild-type Src64 ([Src64WT]/+), expression of activated Src64 ([Src64Y547F]/+). For ring canals sizes, see Table 2. Scale bars: 10 µm.

View larger version (51K): [in a new window]   Fig. 4. Src64LOF egg chambers exhibit packaging defects. (A-E,G) Partial confocal projections of Src64LOF egg chambers labeled with phalloidin (green) to visualize filamentous actin and propidium iodide (red) to visualize DNA. (A) Src6417 egg chamber containing 30 nurse cells (NC) + two oocytes (OO) (32 germ cells (gc) total). (B) Src6417 egg chamber containing 60 NC + 4 OO (64 gc total). (C) Src64KO egg chamber containing 22NC+1OO (right) next to egg chamber containing 8NC+1OO (32 gc total). (D) Src64KO egg chamber containing 24NC+2OO (right) next to egg chamber containing 6NC (32 gc total). (E) Src64KO ovariole. (F) hsFlp/+;Src6417,FRT2A/GFP,FRT2A egg chamber with too many germ cells labeled with phalloidin (red) and anti-GFP (green). Src64 mutant cells lack GFP. (G) Src64KO egg chamber containing too many cells as well as catastrophic membrane collapse. (H) w,hsFlp/+;Src6417,FRT82B/Src6417,FRT82BGFP egg chamber labeled with anti-GFP (green) and phalloidin (red). Half the cells in the egg chamber are labeled with GFP (green) and half are unlabeled, indicating that the cells come from independently derived germline cysts that were packaged together. Scale bars: 10 µm.

View larger version (27K): [in a new window]   Fig. 5. Increasing Src64 activity leads to packaging defects. Partial confocal projections of egg chambers containing too few (*) or too many (**) cells. (A) [Src64Y547F]-expressing egg chambers labeled with phalloidin (green) to visualize filamentous actin and propidium iodide (red) to visualize DNA. (B) CskD589N egg chambers labeled with phalloidin. (C) CskE481K mutant tissue is marked by the absence of GFP (green). Nuclei are labeled with propidium iodide (red). The youngest egg chamber includes labeled and unlabeled cells; thus CskE481K germline mutation leads to packaging defects. Scale bars: 10 µm.

View larger version (101K): [in a new window]   Fig. 6. Germ cell differentiation and oocyte specification are normal in Src64 mutants. (A) Wild-type or Src6417 egg chambers stained with phalloidin (green) to visualize filamentous actin and anti-Vasa (purple). (B) WT or Src64KO egg chambers labeled with phalloidin (green) and anti-Orb (purple). Src64KO oocytes are normally labeled with anti-Orb (middle), but leaking of Orb from the oocyte (arrow) is seen when the cortical membrane breaks down (bottom). Scale bars: 10 µm.

View larger version (72K): [in a new window]   Fig. 7. Follicle cells are normal in Src64 mutants. Mispackaged egg chambers are indicated by asterisks in all panels. Wild type (WT), Src64LOF (Src64KO or Src6417), or [Src64Y547F] egg chambers labeled with (A) anti-FasIII, (B) anti-Cut (yellow) and phalloidin (blue), (C) WT (w; P[w+BL-93FlacZ]/+) or Src6417 (w; Src6417, P[w+BL-93FlacZ]/Src6417) egg chambers labeled with anti-ß-gal (yellow) to visualize stalk cells and phalloidin (blue). (D) anti-Baz (apical marker). (E) anti-Arm (lateral marker). (F-I) Germaria labeled with anti-Vasa (teal) and anti-FasIII (red). (F) WT, (G) Src6417, (H) Src64KO, (I) [Src64Y547F].

View larger version (48K): [in a new window]   Fig. 8. Src64 is activated on ring canals. (A) Wild-type ovariole stained with phalloidin to visualize filamentous actin (green) and anti-pY434 activation-specific Src64 (red). (B) Rescue of Src64 activation (right) by expressing wild-type ([Src64WT]/+;Src6417), but not kinase dead ([Src64KD]/+;Src6417) Src64 in Src6417 mutants. Filamentous actin is labeled with phalloidin (left). (C) Reducing Csk in Src64 hypomorphs can suppress the loss of Src64 activation at the ring canal. Src6417,Csk1T6/Src6417 egg chamber stained with phalloidin and anti-pY434. Boxes (green or pink) surround a ring canal that is weakly stained with anti-pY434. Below is a larger magnification view of the same ring canal.

View larger version (76K): [in a new window]   Fig. 9. Anti-pY434 labels contact points between germ cells and follicle cells in germaria. (A) Wild-type (WT) or Src6417 germaria stained with phalloidin to label filamentous actin or anti-pY434. (B) Src6417 mutant follicle cells (left) or germ cells (right) are marked by the absence of GFP (green). Anti-pY434 stains ring canals and follicle cell-germ cell contacts only when the germ cells express Src64. Scale bars: 10 µm.