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First published online 30 November 2005
doi: 10.1242/dev.02179

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The centrosome-nucleus complex and microtubule organization in the Drosophila oocyte

Jens Januschke^1,*, Louis Gervais^1,*, Laurent Gillet¹, Guy Keryer², Michel Bornens² and Antoine Guichet^1,

¹ Institut Jacques Monod, Unité Mixte Recherche 7592, CNRS, Université Paris 6 et Paris 7, 2 place Jussieu, 75251 Paris Cedex 05, France.
² Institut Curie, Section Recherche, Unité Mixte Recherche 144, CNRS, 75248 Paris, France.

View larger version (117K): [in a new window]   Fig. 1. MTOC material and centrioles localize to the oocyte nucleus during mid-oogenesis. In stage 6 (A) and stage 8 (B), Tub37C, (Rb1011, red) is detected around the oocyte nucleus and as a dot close to it (arrowheads in A,B). (A-C) DNA green. (C) Tub23C (R77, red) is detected around the oocyte nucleus at stage 8. A similar distribution is observed at stage 6 (data not shown). (D-G) Egg chambers expressing GFP-cter-D-PLP. (D) 3D reconstruction of the oocyte nucleus. D-PLP is detected as a dot (light blue, arrowhead) on the surface of the nucleus (inset, original data). (E-G) D-PLP (single view in E, green) and Tub37C (single view in F, red) co-localize (G) during stage 8 to the same dot close to the nucleus (arrowheads). (H) Polyglutamylated MTs (ID5 antibody) are detected as a dot in the vicinity of the nucleus during stage 9 (arrowhead, inset magnification view). (I,J) Electron micrographs of the anterior dorsal corner of the oocyte; anterior is to the top, dorsal to the right. (I) Low power magnification of the area around the oocyte nucleus. The centrioles are found in the boxed area. (J) At high magnification, two centrioles (white arrowheads) are visible, most probably there are two more (black arrowheads). Golgi cisternae are in the vicinity of the centrioles (arrow). (K) Egg chamber expressing D-PLP. The centrosome is found close to the nucleus (arrow; oocyte outline indicated with dashed line). (L-N) Flies fed with colchicine. (L) The nucleus is misplaced and anti-Khc staining revealed MTs only in the follicle cells. The centrioles (arrows) are separated from the oocyte nucleus in a stage 8 (M) or stage 9 (N) oocyte expressing D-PLP. Asterisk, oocyte nucleus. Scale bars: 20 µm in A-H,K-N; 1 µm in I,J.

View larger version (103K): [in a new window]   Fig. 2. Comparative analysis of MTs in living and fixed egg chambers. MTs in fixed stage-8 egg chambers detected with anti--Tubulin-FITC (A) and anti-Khc (B) (inset in B specificity of the anti-Khc antibody, a Khc7.288 mutant germline clone shows no anti-Khc staining in the oocyte). (C-E) Stage 7 egg chambers; (F-H) stage 9 egg chambers. (C,F) Living egg chambers expressing GFP--Tubulin. (D,G) Fixed egg chambers stained with anti--Tubulin-FITC. (E,H) Fixed egg chambers stained with anti-Khc. (I,J) Stage 9 egg chambers, Khc (red) and aromatic c-ter amino acid of Tub (green). The arrow in J points to the posterior Khc pool.

View larger version (139K): [in a new window]   Fig. 3. Extraction procedure is not affecting egg chamber polarity. (A) Surface view of a fixed stage-10 egg chamber stained for anti--Tubulin-FITC. (B,C) MTs in stretched follicle cells revealed by anti--Tubulin-FITC in fixed egg chamber (B) and by GFP--Tubulin in living egg chamber (C). MTs in main body follicle cells revealed by anti--Tubulin-FITC in fixed egg chamber (D) and by GFP--Tubulin in living egg chamber (E). (F,G) Atypical protein kinase C. Posterior follicle cells fixed after a standard procedure using 4% paraformaldehyde (F) or after the extraction protocol (G). (H,I) Fixed oocyte expressing Kin::LacZ, stained for anti-Khc (red) and anti-ß-Gal (green) (arrow, posterior fraction of Khc). (J,K) Fixed oocyte expressing Nod::LacZ, stained for anti-Khc (red) and anti-ß-Gal (green) (Arrows, anterior distribution of Nod). Grk (L) and Osk (M) protein after the extraction protocol. Scale bar: 20 µm.

View larger version (123K): [in a new window]   Fig. 4. Two different MT subsets coexist inside the oocyte. Optical planes from confocal Z-sectioning of oocytes stained with anti-Khc according to the sectioning experimental setup presented in A. The numbers 1-5 represent the position of representative optical sections in a wild-type oocyte (1 represents the most proximal plane close to the cortex, and 5 the most distal plane). (B) Stage 6, (C) stage 7, (D) stage 8 and (E) stage 9 oocytes. Two MT subsets can be distinguished according to orientation and stage-dependent changes. The DV subset: throughout oogenesis, cortical MTs oriented along the DV axis could be observed. At the proximal and distal cortices, MTs running parallel to the DV axis can be seen from stage 7 on (C1, C5; D1, D5; E1, E5; white arrowheads) and to a lesser extent in stage 6 (B1, B5; white arrowheads). The AP subset: (B) Before oocyte repolarization at stage 6, MTs extend along the cortex and fold back slightly at the anterior (B2 and B3, red arrowheads). The nucleus is encapsulated by MTs and short fibers point from the nucleus toward the anterior (B3 and B4, arrows). (C) By stage 7, long MTs are rolled up in the shape of a `diaphragm' in the center of the oocyte (C2-C4). MT bundles emanating from the perinuclear region project toward the cortex (C2, arrow). The center of the cytoplasm contains only a few MT fibers. (D) In stage 8, the `diaphragm' has opened and MT bundles project from the anterior in the AP direction (D2, arrow). MTs bundle at the posterior and join the center (D3, arrow). Transverse sections of MTs appear as dots on the ventral side of the oocyte (D3, arrowhead). Free MT ends are found in the center of the oocyte (D4, arrowheads). (E) In stage 9, the central MTs originate from the anterior and the oocyte nucleus (E2 and E3, arrows). One or two thick bundles that resemble a `horseshoe'-like pattern orient in the AP direction (E4, arrows). (B6,C6,D6,E6) Schematic representations of the MT distribution at mid-oogenesis. (B6) MT distribution before repolarization is shown in blue. After repolarization, the invariant DV subset is depicted in black, the dynamic AP subset in red. (C6) Stage 7 (`diaphragm' state). (D6) Stage 8 (`open diaphragm' state). (E6) Stage 9 (`horseshoe' state). (F-H) Living egg chambers expressing Tau-GFP. (F) The cortical DV (arrowhead) subset can be detected in living stage 8 oocyte. (G) Stage 10A oocyte, in which two MT bundles in AP orientation can be seen in the center (arrows). (H) Stage 11: thin bundles of MTs can be detected in the DV orientation (arrowhead) close to the cortex. Red dotted lines mark the outline of the oocyte. Asterisk, location of oocyte nucleus. Scale bar: 20 µm.

View larger version (84K): [in a new window]   Fig. 5. The nucleus is an active center for MT polymerization. (A) Schematic representation of the experimental setup for cold-induced MT disassembly. Complete depolymerization: dissected ovaries were extracted (30 minutes), placed on ice (30 minutes), fixed and analyzed for MTs by anti-Khc. To allow MT regrowth, ovaries were incubated at 25°C for 3-30 minutes. (B) Stage 9 oocyte, complete MT depolymerization. Khc was only detectable at the posterior (arrowhead). (C) Cold-shock treatment did not alter Tub37C localization (arrowhead). (D,E) Centers of MT polymerization (arrowhead) were detected in the vicinity of the oocyte nucleus, never at the posterior or at cortical regions after short recovery times. (F) MTs appear along the anterior margin after 10 minutes regrowth. (G) MT regrowth along the cortex in the DV orientation during intermediate recovery times. (H) Optical section taken in the center of the same oocyte as in G. Clouds of Khc positive dots at the ventral and dorsal sides (arrowheads) represent transverse cuts of the cortical MTs. The AP subset in the center was not yet established. (I,J) Optical section of the same oocyte. Thirty minutes recovery at 25°C was sufficient to regrow MTs to the control situation. Cortical DV subset (I, arrowhead) and, in the center (J), MTs in the AP direction (arrow). Asterisk, oocyte nucleus. Scale bar: 20 µm.

View larger version (111K): [in a new window]   Fig. 6. grk mutant oocytes conserve the MT configuration of wild-type stage 6 oocytes. (A,B) grk mutant egg chambers stained for anti-Khc. In oocytes comparable to wild-type stage 8 (A) and stage 9 (B), the nucleus is misplaced to the posterior and surrounded by MTs. (C) Schematic representation of the MT network in grk oocytes. (D) grk stage 9 oocyte showing the accumulation of Khc (arrow) between the MT extremities of the long cortical MTs (red arrowheads) and the short central MTs (white arrowhead). (E,F) Wild-type stage 6 oocytes showing a similar MT distribution. MT bundles project from the nucleus at the posterior along the cortex to the anterior, where they bend back to the center (red arrowheads). Short bundles stretch out from the nucleus into the center of the oocyte (white arrowhead). To better visualize MTs, a projection of 10 optical sections (4 µm) is shown in F. (G) Egg chamber expressing Kinesin:ßGal (green) co-stained for Khc (red). (H) Tub37C (R46) localizes to the nucleus at the posterior in grk mutant oocytes and the centrosome close to it (arrow). (I) grk mutant oocyte, showing complete depolymerization of MTs after 30 minutes cold-shock. (J) grk mutant oocyte (projection of 5 µm) after a 15-minute recovery at 25°C. MTs regrow from the misplaced nucleus at the posterior. Asterisk, oocyte nucleus. Scale bar: 20 µm.

View larger version (59K): [in a new window]   Fig. 7. Correlation of the stage-dependent MT organization and grk as well as osk mRNA localization from stage 7 through stage 9. grk (green) and osk (blue) are visualized by fluorescence in-situ hybridization. At stage 7 (left), grk and osk are found mostly at the anterior cortex. grk is rapidly transported toward the oocyte nucleus in the transition from stage 7 to stage 8, exploiting the DV MT subset (black). The DV subset comprises long cortical MT fibers as well as the shorter cortical fibers. osk mRNA localization correlates with the successive progression of AP MT subset (red) formation. osk is transported from the anterior toward the center following the open diaphragm MTs in the center. By stage 9 (right), upon oocyte growth and complete AP orientation of center MTs, osk is transported toward the posterior.

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