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doi: 10.1242/10.1242/dev.00352

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The p27^cip/kip ortholog dacapo maintains the Drosophila oocyte in prophase of meiosis I

Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA

View larger version (24K): [in a new window]   Fig. 1. Drosophila ovarian cyst and germarium. (A) The 16 cells of an ovarian cyst are connected in an invariant pattern via actin-rich ring canals (rc, ring canal). The single oocyte (black) always develops from one of the two cells with four ring canals. The other 15 cells in the cyst, including the other cell with four ring canals (asterisk), enter the endocycle and develop as highly polyploid nurse cells. (B) A germarium. The four mitotic cyst divisions take place in region 1 of the germarium. In region 2a, all 16 cells enter premeiotic S phase. In region 2b, a meiotic gradient forms with the two cells with four canals progressing to pachytene. By the time cysts enter region 3 of the germarium (stage 1) only the cell that is destined to become the oocyte (black) remains in the meiotic cycle. By contrast, the 15 mitotic sisters of the oocyte enter the S phase of the first endocycle on their way to becoming highly polyploid nurse cells. NC, nurse cell.

View larger version (115K): [in a new window]   Fig. 2. Dap is differentially regulated in oocytes and nurse cells. (A) A germarium stained with Dap antibody. Regions of the germarium are noted below. (B,C) Confocal sections of (B) an early stage 1 egg chamber and (C) a late stage 1 egg chamber stained for Dap (green) and Orb (red) to highlight the oocyte. Note that Dap is at high levels in all cyst nuclei in B, but has begun to cycle in C, as evidenced by the absence of Dap in some nuclei (asterisks). (D,E) Egg chambers stained with (D) Dap antibody and (E) DAPI to highlight nuclei. Consistent with the cycling of the Dap protein, nurse cells with high, medium and low levels of Dap are observed. Asterisks mark nuclei that are negative for Dap protein. (F) A stage-10B egg chamber stained for Dap (green) and Orb (red). Note that the nurse cells, which have exited the endocycle, contain uniformly high levels of Dap protein. (G) Double labeling for Dap (green) and BrdU (red) indicates that S phase takes place when Dap levels are low. (H) Corresponding DAPI staining. o, oocyte.

View larger version (65K): [in a new window]   Fig. 3. dap oocytes enter the endocycle. (A) Wild-type egg chamber stained with DAPI to highlight nuclei, note the large size of the nurse cell DNA mass relative to the compact oocyte karyosome (arrowhead). (B-D) In dap germline clones, oocytes achieve varying degrees of polyploidy. Egg chambers containing dap germline clones in which (B) there are 16-polyploid nurse cell nuclei and no oocyte, (C) the oocyte is partially polyploid (arrowhead) and (D) the oocyte DNA has abnormal morphology but is not obviously polyploid. The broken circle outlines a magnification of the oocyte defect.

View larger version (104K): [in a new window]   Fig. 4. dap oocytes accumulate Orb protein and progress to pachytene. (A) A dap cyst (arrowhead) in region 2b of the germarium accumulates Orb protein (green) in a manner indistinguishable from an adjacent wild-type cyst (arrow). In both (B) wild type and (C) dap clones, in region 2b of the germarium the two pro-oocytes contain thread-like C(3)G staining (green) indicative of cells in pachytene. In both panels, the other 14 cells in the cyst that are negative for C(3)G are not shown. In B,C the cell with the stronger C(3)G staining is the true oocyte (arrow). In A,C dap clones were identified by the absence of ß-gal staining (red). Scale bar: 5 µm.

View larger version (114K): [in a new window]   Fig. 5. dap is required to maintain oocyte fate. (A,B) Wild-type and (C-F) dap4 clones double labeled with DAPI (A,C,E) to highlight nuclei and BicD antibody (B,D,F). Note that in D, where the dap4 oocyte has become highly polyploid, there is little preferential accumulation of the BicD protein. By contrast, in F, where the dap4 oocyte has undergone little to no DNA replication, BicD accumulates to levels similar to those observed in wild-type oocytes.

View larger version (82K): [in a new window]   Fig. 6. Entry into the endocycle disrupts the asymmetric distribution of -tubulin in dap cysts. (A-D) dap4 cysts double labeled with DAPI (A,C) and -tubulin antibody (B,D). In C, where the oocyte has become polyploid (arrowhead), there is no concentration of -tubulin staining (D) in the oocyte (compare with B).

View larger version (70K): [in a new window]   Fig. 7. A dap null allele dominantly suppresses the two oocyte phenotype of cycE01672 egg chambers. (A) Wild-type and (B) cycE01672 stage 10 egg chambers stained with Dap antibody. Note that Dap accumulates to high levels in two cells, the oocyte and an adjacent cyst cell, in the cycE01672 egg chamber. (C) In wild-type egg chambers, the posterior nurse cells have the highest DNA contents in the cyst. By contrast, in cycE01672 egg chambers(D,E), a cell that is normally specified to become a posterior nurse cell frequently undergoes a reduced number of endocycles and has an inappropriately low DNA content (arrow). This phenotype is variable and can range from egg chambers in which the second four-ring canal cell undergoes little to no DNA replication (D) to a relatively small reduction in DNA content (E). This reduction in DNA content is suppressed by 2.5 fold when a single copy of the null allele dap4 is placed in the cycE01672 background. (F) An egg chamber from a cycE01672,dap4/+ female in which the posterior nurse cells have wild-type ploidy values (compare C with F). (C-F) Egg chambers stained with DAPI to highlight nuclei.

View larger version (27K): [in a new window]   Fig. 8. A model for the maintenance of the two independent cell cycles of ovarian germline cysts. (A) High levels of Dap help maintain the oocyte in prophase of meiosis I (red) by inhibiting inappropriate DNA replication. In the nurse cells the cycling of Dap allows the periodic initiation of DNA replication during the endocycle (blue). (B) The balance of cyclinE-Cdk2 and its inhibitor Dap determine if a cyst cell enters the endocycle or arrests in meiosis (see text for details). Scissors indicate Dap proteolysis.

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