Asymmetric Germ Line Divisions

In the adult ovary, the germ-line stem cells lie at the anterior of the tip of the germarium. Genetic (32) and laser ablation studies (33) showed that each ova-riole contains two to three self-renewing germ-line stem cells that undergo bursts of two to five successive divisions. Germ-line stem cells contain a spectrosome (the fusome precursor), which arises as the embryonic gonad is formed (34). The spectrosome associates with one pole of the metaphase spindle, and so is asymmetrically partitioned between the stem cell and cystoblast daughters during cytokinesis (35,36) (see Figs. 3, 4A,I).

Cystoblasts/cystocytes also divide asymmetrically, and in doing so, the spectrosome evolves into a branched fusome in the 16-cell cyst (5,37), accumulating proteins like Bam, ankyrin, a- and P-spectrins, the adducinlike product of the hu-li tai shao gene (HtsF), dynein (Dhc64C), and Cyclin A (5,35,36). Each polarization cycle begins in mitosis, with one pole of the spindle positioned near the fusome (see Fig. 4I), in a process mediated by cytoplasmic dynein and DLis-1 (38-40). As the polarization cycle progresses, the preexisting fusome and ring canal are inherited by only one of the two daughter cells. Following mitosis, a "fusome plug" forms in each newly formed ring canal, and then the fusome plug, together with the associated ring canal, moves toward, and finally fuses with the pre-existing fusome. As a result, a highly branched fusome forms within each cyst, with the older cystocytes containing larger networks of fusome.

Following the formation of the 16-cell cyst, the fusome breaks down and disappears (regions 2b-3 of germarium), whereas inside the cyst, a polarized transport is initiated and the nurse cells and oocyte differentiate. Studies on mutants that affect formation of the 16-cell cyst suggest that proper fusome organization is a precondition of oocyte specification. The fusome not only generates a branched pattern of interconnections between cystocytes, it also synchronizes and controls the number of cystocyte divisions.

Immunocytological analysis of stem cell and cystoblast/cystocyte divisions is a very demanding task, because it requires visualization of the spectrosome/ fusome together with the spindles, ring canals, nuclei/chromosomes, and cen-trosomes. Antibodies against these structures are listed in Appendix B. Because, as described earlier, the orientation of the spindle with respect to the spectrosome/fusome is determinative for proper development of the germ-line cyst, methods allowing the simultaneous visualization of these two structures are crucial (see Figs. 4A,I). Such a method is described in Subheading 3.1.

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