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First published online December 1, 2003
doi: 10.1242/10.1242/dev.00853

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Germ line stem cell differentiation in Drosophila requires gap junctions and proceeds via an intermediate state

Lilach Gilboa¹, Alexandria Forbes^*, Salli I. Tazuke^2,3,, Margaret T. Fuller^2,4 and Ruth Lehmann^1,

¹ Developmental Genetics Program, Skirball Institute at NYU School of Medicine and Howard Hughes Medical Institute, 540 First Avenue, New York, NY 10016, USA
² Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305-5329, USA
³ Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, CA 94305-5329, USA
⁴ Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305-5329, USA

View larger version (68K): [in a new window]   Fig. 1. Germ cells that lack zpg fail to differentiate. (A) The Drosophila germarium. In all figures, anterior is to the left and posterior to the right. Germ line stem cells (GSCs) are located at the anterior of the ovariole. Progressively more developed germ cells are located posteriorly: a putative intermediate state (or pre-cystoblasts, Pre-Cb), cystoblasts (Cb), dividing cysts and egg chambers (EC). The somatic niche is composed of terminal filament cells (TF), cap cells (CpC) and inner-sheath cells (IS). (B) zpg ovaries were stained with anti-Vasa to mark the germ line (green) and with an antibody that recognizes the spectrosome, fusome and the cell cortex (mAb 1B1; red). Only a few germ cells at the anterior tip of the ovarioles could be observed. Some of those germ cells are attached to cap cells (arrowheads). (C,D) Wild-type (C) or zpg (D) ovaries stained with anti-Vasa (green) and anti-BamC (red) to mark cystoblasts and dividing cysts. In wild type, a full complement of germ line cells, from GSCs to dividing (yellow; arrow) and budding cysts could be observed. In zpg ovaries only a few germ cells at the tip of the ovariole could be observed, mostly lacking BamC staining (panel D depicts many such ovarioles, each containing a few cells). (E) Wild-type and zpg ovaries were co-labeled with anti-Zpg (green) and with mAb 1B1 (red). Zpg is expressed on the membrane of germ cells in the germarium. In zpg ovaries, no staining with the anti-Zpg antibody was observed, attesting to the specificity of the antibody and to the fact that the alleles used in this study may be protein nulls (E, inset). Scale bars: in B, 20 µm for B,C,E; in D, 50 µm.

View larger version (124K): [in a new window]   Fig. 2. Differentiation results in death of zpg cells. (A,B) Ovaries stained with anti-Vasa to mark the germ line (green) and anti-phospho-Histone H3 to mark mitotic cells (red). Mitotic cells at the GSC position can be observed in both wild-type (A) and zpg (B) ovaries. (C,D) Flies carrying a hs-bam transgene were heat-shocked, and their ovaries dissected and stained with anti-Vasa to mark the germ line (green), and with the mAb 1B1 antibody to mark the cell cortex, fusomes and spectrosomes (red). hs-bam, zpg heterozygous mutant ovaries showed a series of egg chambers attached to empty germaria (C). hs-bam, zpg homozygous mutant ovaries lacked germ line (D). Control ovaries of heat-shocked zpg mutants showed the zpg phenotype (D, inset). Scale bar: in A, 20 µm for A,B; in C, 50 µm for C,D.

View larger version (149K): [in a new window]   Fig. 3. pum mutant germ cells require zpg for differentiation. Ovaries from flies of the indicated genotypes were stained with anti-Vasa (green) and the mAb 1B1 antibody (red). pum mutant ovarioles were either devoid of germ line (B), or had a few defective, but differentiated, egg chambers and an empty germarium (A). zpg, pum double-mutant ovaries resembled zpg ovaries, with only a few germ cells at the tip of the ovariole, except that a few more germ cells were present. More cysts and egg chambers were also observed (D, arrow). Scale bar: in A, 20 µm; in B, 50 µm for B-D.

View larger version (155K): [in a new window]   Fig. 4. zpg is required for survival of bam `stem-cell tumors'. Ovaries from flies of the indicated genotypes were stained with anti-Vasa (green) and the mAb 1B1 antibody (red). zpg bam /+ bam ovaries showed the bam phenotype with many `stem cell-like' germ cells and no egg chambers (A). zpg bam / zpg+ flies had the zpg phenotype with few germ cells at the tip of the ovariole (B). zpg and bam or bgcn double mutant ovaries (C,D) had a similar phenotype to that of zpg, with only a few germ cells located at the tip of the ovary. However, the number of germ cells at the tip was higher than in zpg single mutants (compare panel B with panels C,D). Scale bar in D: 50 µm for A-D.

View larger version (84K): [in a new window]   Fig. 5. zpg is required for Dpp-induced over-proliferation of early germ cells. Flies of the indicated genotypes were either dissected (C,E), or subjected to heat shock prior to dissection (A,B,D,F), and then stained with anti-Vasa (green) and mAb 1B1 antibody (red; A,B) or anti-pMad (red; C-F). After heat shock, hs-dpp; zpg heterozygous ovaries were filled with many single germ cells (A). hs-dpp, zpg homozygous mutant ovaries did not show any proliferation of single germ cells (B). Staining of wild-type (OR) germaria revealed p-Mad staining in early germ cells (C), whereas all germ cells have nuclear p-Mad when Dpp is overexpressed (D). zpg cells also have nuclear p-Mad, whether Dpp is overexpressed (F) or not (E). Scale bar: in A, 20 µm for A,C,D; in B, 50 µm.

View larger version (47K): [in a new window]   Fig. 6. An average of 3.9 single cells do not stain with BamC antibody. (A) Individual germaria were scored for the number of cells that contain a spherical spectrosome and do not stain with BamC antibodies. The graph illustrates the number of germaria (y axis) that contained between 1 and 6 of such cells (x axis). (B) Wild-type ovaries were stained with anti-Vasa (blue), with the mAb 1B1 antibody (green) and with anti-BamC (red). Four cells that possess a single spectrosome but do not stain for BamC can be observed at the anterior of the ovariole (arrowheads). A cyst, which does stain with BamC antibody, can be seen just posteriorly (arrow). Scale bar: 20 µm. (C) An illustration of the possible outcomes of mitotic recombination in a pre-cystoblast or a GSC (see arrows). Cells carrying 0 copies of the lacZ transgene are depicted in white, whereas those carrying 1 or 2 copies of the transgene are light or dark green, respectively. (D) A germarium representing a GSC mitotic recombination event. Arrowheads mark the GSC and its progeny lacking lacZ (only one daughter cell is in plane). An arrow marks the `twin spot', which strongly expresses lacZ.

View larger version (15K): [in a new window]   Fig. 7. A model for GSC differentiation. Somatic cells of the niche (TF, terminal filament), cap cells (CpC) and inner sheath cells (IS) are depicted in red. Germ line cells are depicted in shades of green (GSC, germ line stem cell; Pre-Cb, pre-cystoblast; Cb, cystoblast). Stem cell maintenance is controlled by dpp, pum, and other unidentified factors (dashed lines). Zpg is needed for the differentiation of the stem cell. Bam and Bgcn are needed for the differentiation of the cystoblast and cysts.