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First published online 22 August 2007
doi: 10.1242/dev.008466

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Rab11 maintains connections between germline stem cells and niche cells in the Drosophila ovary

University of Kansas, Department of Molecular Biosciences, 1200 Sunnyside Avenue, Lawrence, KS 66045, USA.

View larger version (79K): [in this window] [in a new window]   Fig. 1. Rab11 is enriched on the fusome of GSCs and germline cysts. (A) Diagram of the Drosophila germarium. Niche terminal filament (TF), cap (CC), and escort stem (ESC) cells are shown in gray. The fusome (red) lies at the anterior cortex of germline stem cells (GSCs, black) and extends throughout the cytoplasm of germline cysts (white). Oocyte (green); somatic follicle cells (FC); ring canals (blue crescents). (B-E) Region 1 of a wild-type germarium immunostained for E-cadherin (green), Rab11 (red), and the fusome marker -Spectrin (blue). Merged image (E). Anterior is to the left in this and all subsequent figures. A single GSC is outlined in B, with the break in the tracing (dashed line) revealing strong E-cadherin staining at the GSC-cap cell interface. The smaller dot of E-cadherin staining (arrow) superimposes with Rab11 on the fusome (arrows in C and D). (F-H) Wild-type germarial regions 1 and 2 immunostained for Rab11 (red) and -Spectrin (blue). Merged image (G). Strong accumulation of Rab11 is apparent on the GSC (arrowhead) and fusome of the germline cyst (arrow). (I,J) Mosaic germarium immunostained for nuclear GFP (nGFP, green), Rab11 (red) and -Spectrin (blue). Note the absence of Rab11 protein in the rab11-null cells, which are marked by the absence of nGFP in this and all subsequent images. (J) Rab11 channel only. (K,L) Germarial region 1 of rab11::GFP transgenic germarium immunostained for GFP (red) and -Spectrin (blue). Arrowheads indicate strong Rab11::GFP expression on the fusome. Scale bars: 10 µm.

View larger version (74K): [in this window] [in a new window]   Fig. 2. Rab11 is required to maintain E-cadherin at the cap cell-GSC junction and to anchor the fusome to the anterior cortex of the GSC. (A,B) Region 1 of a mosaic Drosophila germarium immunostained 8 days after clone induction (ACI) for E-cadherin (red), -Spectrin (blue) and nGFP (green). (A) A 2-cell rab11-null germline cyst is outlined (dashed line). A dividing wild-type GSC (boxed) is shown to the left and magnified in B, where the dashed line outlines the cap cells. Note that the plane of division (evident by the stretched-out fusome) is such that both daughter cells remain in the niche, with one filling the vacancy created by a lost GSC. (C-E) Wild-type (C) and mosaic (D,E) germaria immunostained for nGFP (green), E-cadherin (red) and -Spectrin (blue) 2 days ACI. Wild-type and rab11-null GSC-cap cell junctions are indicated with arrowheads and arrows, respectively. E-cadherin staining is reduced at the mutant junctions at the expense of increased fusome staining, especially in E, where several strong dots of staining are evident. (F) Dividing rab11 (arrow) and wild-type (arrowhead) GSCs immunostained for -Spectrin (blue), Vasa (cytoplasmic green, germline only) and nGFP (green) and stained with DAPI (red). The DAPI-stained nuclei at the left correspond to cap cells. The rab11 GSC fusome is splayed and displaced from the anterior cortex. (G) Mosaic germarium immunostained for nGFP (green), -Spectrin (red) and Bam (blue) 2.2 days ACI. The rab11-null GSC (outlined in yellow) exhibits only background levels of Bam expression. A wild-type GSC and a 2-cell germline cyst (outlined in white) are shown for comparison. Scale bars: 10 µm.

View larger version (116K): [in this window] [in a new window]   Fig. 3. rab11-null germline cysts arrest early and display defects in fusome segregation and bulk membrane trafficking. (A,B) Mosaic Drosophila germarium immunostained for (A) -Spectrin (red), Vasa (cytoplasmic green) and nGFP (green) or (B) E-cadherin (red), -Spectrin (blue) and nGFP (green). Severely affected rab11-null germline cysts with no detectable fusomes and reduced numbers of germline cells are outlined in yellow. A less affected rab11-null germline cyst with a normal fusome is outlined in white in A and indicated with an arrow in B. (C) Wild-type germarium immunostained for E-cadherin (red) and -Spectrin (blue). The arrowheads point to the posterior of the oocyte, where enriched accumulation of E-cadherin is evident, especially in the region 2B oocyte (left arrowhead). (D) Mosaic germarium immunostained for E-cadherin (red), -Spectrin (blue) and nGFP (green). The arrow points to a region 2B rab11-null oocyte with greatly reduced E-cadherin accumulation (compare with left arrowhead in C). The arrowhead points to a wild-type region 3 oocyte, where enriched E-cadherin expression is still apparent. A severely affected, rab11-null germline cyst, similar to those seen in B, is outlined. (E,E') Mosaic germarium immunostained for Orb (red, oocyte) and nGFP (green). The bracketed area in E is shown at a different focal plane in E'. Note that the oocyte is positioned at the posterior end of the wild-type germline cysts (arrowheads), but at the center of the rab11-null germline cyst (arrow). (F,G) Mosaic germarium immunostained for nGFP (green) and HtsRC (red) to label ring canals. (F) Left arrow points to a rab11-null germline cyst, with clumped ring canals. The right arrow points to a mosaic germline cyst, where the ring canals are only clumped in the rab11-null (GFP-negative) portion. (G) Germarium with a completely rab11-null germline. All ring canals are clumped in the center of the cysts. Scale bars: 10 µm.