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First published online February 18, 2004
doi: 10.1242/10.1242/dev.01001

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missing oocyte encodes a highly conserved nuclear protein required for the maintenance of the meiotic cycle and oocyte identity in Drosophila

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

View larger version (68K): [in a new window]   Fig. 1. mio is required for oocyte development. (A) The cell types and stages of Drosophila oogenesis. In region 1 of the germarium (R1), a germline stem cell divides to give rise to a cyst of 16 interconnected cystocytes. In region 2a (R2a), the two pro-oocytes (dark grey), as well as the two adjacent cells with three ring canals (light grey), construct SC. The meiotic gradient is restricted to the single oocyte by region 3 (stage 1). In region 3, the nurse cells enter the endocycle. (B) Microtubule-based directional transport to the oocyte (black). (C) A wild-type stage 5 egg chamber. Note that the oocyte DNA has condensed into a compact karyosome (arrow). (D) A mio2 egg chamber that contains 16-polyploid nurse cells. (E,F) Ovarioles from wild-type (E) and mio2 homozygous female (F). Ovarioles were stained with Hoechst to visualize nuclei. O, oocyte.

View larger version (39K): [in a new window]   Fig. 2. Orb staining in wild-type and mio ovaries. Wild-type (A,C) and mio mutant (B,D) germaria stained with Orb (green) and Hoechst (blue). (A) In wild type, weak Orb accumulation is first observed in a single cell in region 2a (arrow), and becomes strong by region 2b. (B) In mio mutants, Orb accumulation in a single cell is not visible until late region 2b (arrow) or sometime until region 3. (C) In wild type, Orb continues to accumulate at the posterior cortex of the oocyte during post-germarial stages (arrowheads). (D) In mio mutant egg chambers, some Orb proteins do not move to the posterior in stage 2 (see B), remain detectable in the anterior of the oocyte, and gradually disappear when the oocyte enters the endocycle.

View larger version (45K): [in a new window]   Fig. 3. mio affects meiotic progression. Wild-type (A) and mio mutant germaria (B) stained with C(3)G (red) and Orb (green). Magnified C(3)G staining of adjacent cysts are shown in small boxes. As is observed in wild type, in mio mutants fully developed SCs are formed in two to four cells in region-2a cyst. However, in mio cysts more than four cells enter the meiotic cycle and construct SC (see region-2a cyst traced with white line). (C,D) Anterior regions of wild type (C) and mio mutant germaria (D). Boxes indicate magnified regions of adjacent cysts stained with Hoechst (blue) and C(3)G (red). (C) Upper two cysts traced with white line are in early region 2a, determined by the absence of Orb expression. In wild type, early region-2a cysts sometimes show an early zygotene-like SC, with two to ten dots per nucleus observed in one or two cystocyte within a cyst. (D) In mio mutants, more extensive SCs are observed in early region 2a, which include numerous dots and short thread-like SCs that are often present in more than two cells per cyst.

View larger version (48K): [in a new window]   Fig. 4. egl, mio double mutants arrest prior to pachytene. mio is required at an early stage of meiosis. (A-C) Germaria double labeled with C(3)G (red) and Orb (green). Magnified C(3)G staining is shown in upper right panel. (A) mio mutant: two pro-oocytes have thick thread-like C(3)G pattern, indicating that, like wild type, the mio pro-oocytes progress to pachytene. (B) egl null mutant (egl1): all 16 cells form the less extended SC that is representative of late zygotene or early pachytene. (C) mio2, egl1 double mutants: all 16 cells show the dot pattern of C(3)G that is normally observed in early zygotene.

View larger version (69K): [in a new window]   Fig. 5. Identification of the mio gene (A) The physical map of the 23C-D cytogenetic interval. Black boxes indicate the regions that are deleted in each deficiency. Light hatched boxes are the regions of uncertainty. The mio (CG7074) ORF is shown as black box with arrow indicating the direction of transcription. The other 10 ORFs in the defined 50 kb interval are depicted as grey arrows. The mio gene is composed of seven exons. (B) The mio gene encodes a single transcript. Northern blot of total RNA from wild-type ovaries, probed with a mio cDNA. (C) The Mio protein contains an N-terminal domain with four WD40 repeats (grey box) and a RING/PHD finger-like domain near the C terminus (black box). An alignment of the four WD40 repeats and the RING/PHD finger-like domain of Mio, human FLJ20323, mouse BC20002, zebrafish BC047198, fission yeast SPAC630.02 and budding yeast YBL104c. The locations of the mio1 and mio2 truncations are indicated (arrowhead). Both yeast homologs lack the last WD40 repeat, while the mammalian homologs contain six WD40 repeats, two more than are found in Drosophila. All homologs except YBL104c contain a RING/PHD finger-like domain. (YBL104c shares only part of the conserved domain.) Black bar above the structure shows the region used as antigen. Stars indicate amino acid conservation between mio homologs and the indicated motifs. The consensus for the RING finger and PHD finger motifs are shown, respectively, above and below the mio alignments.

View larger version (54K): [in a new window]   Fig. 6. Mio localizes to oocyte nuclei. (A) Western blot analysis of ovarian protein extracts. The Mio antibody recognizes a single band that coincides with the predicted molecular size of Mio, 98.6 kDa in extract from wild type (WT) and the Mio overexpression line (UASp-mio). Additionally, the antibody recognizes a slightly smaller protein (asterisk) in mio2 ovarian extracts. mio2 is predicted to encode a truncated Mio protein of 92.6 kDa. The last lane is a higher contrast picture of the mio2 extract. (B) A wild-type ovariole stained with Mio. (C) Faint Mio signal is detected in two cells of a cyst in region 2a. The pattern is often not symmetric, with one nucleus (large arrow) frequently having a brighter signal. In region 2b and region 3, the staining is restricted to a single cell and dramatically increases in intensity (arrows). (D) C(3)G localization in the same germarium as depicted in C. Small box denotes magnified region-2b nucleus stained with Mio (red) and C(3)G (green). Arrows and arrowheads indicate Mio-positive cells in B and C(3)G-positive cells in D. E is a diagram showing the stages and localization of Mio (red) and C(3)G (green) in the germarium depicted in C and D. The Mio protein colocalizes to the nuclei that stain most intensely with C(3)G in region 2a (large arrowheads and arrows). However, the overlay of a region 2b nucleus demonstrates that the Mio protein has a different distribution than the SC component C(3)G (small box).

View larger version (60K): [in a new window]   Fig. 7. Mutations in mei-W68 suppress the mio mutant phenotype. Ovarioles from mio2 (A) and a mio2, mei-W681 double mutant (B), stained with Hoechst (blue) and Orb (red). Unlike the single mutant, oocytes from double mutants form normal karyosomes (arrows) and accumulate Orb protein beginning in region 2a (arrowheads). Although most mio2, mei-W681 egg chambers develop normally, some abnormalities are observed, such as the mispositioning of the oocyte (see third egg chamber in B where the oocyte is located in the center, rather than the posterior, of the egg chamber).

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