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First published online 14 August 2007
doi: 10.1242/jcs.009977

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Regulation of meiotic cohesion and chromosome core morphogenesis during pachytene in Drosophila oocytes

Department of Biological Sciences, Dartmouth College, Hanover, NH 03755, USA

View larger version (53K): [in this window] [in a new window]   Fig. 1. Cohesin SMC localization during early oogenesis. (A) Diagram of a single ovariole with the youngest stage at the top. Each ovariole contains `cysts' composed of 16 interconnected germ cells, one of which is the oocyte (red). Meiosis initiates at the anterior tip of the ovariole in the germarium. The remainder of the ovariole is called the vitellarium. As cysts progress through oogenesis, they move toward the posterior end of the ovariole. In stage 14, the oldest egg chamber in an ovariole, the oocyte is arrested at metaphase I. Passage through the oviduct triggers the resumption of the meiotic divisions. (B) The germarium is made up of four regions: region 1, region 2A, region 2B and region 3 at the posterior end. Individual cysts are depicted in blue. On the far right is a diagram showing the assembly of SC (red) in a subset of cells within region 2A cysts. As cysts mature and move to the posterior end through the germarium, the SC becomes restricted to the ooctye. (C) Bright foci as well as diffuse SMC1 signal (green) is visible within region 1. The fusome localization pattern (white) suggests this is either an 8-cell cyst or an early 16-cell cyst. Bar, 4 µm. (D) Simultaneous staining with antibodies against SMC1 and SMC3 shows localization of cohesin SMCs (green) coincident with the SC protein C(3)G (magenta) in different regions of the germarium. In region 2A, two cysts are visible, with two to three cells per cyst containing thread-like SMC1/3 signal (arrow). In region 2B, SMC1/3 threads are restricted to two nuclei per cyst, and by region 3 long stretches of SMC1/3 signal are visible only within the oocyte. Bar, 5 µm. All panels represent projections of deconvolved Z-series using whole-mount germaria.

View larger version (46K): [in this window] [in a new window]   Fig. 2. Cohesin SMCs localize along chromosome cores of meiotic chromosomes. A chromosome spread slide was treated with buffer alone (top panels) or with DNase I (bottom panels) and immunostained for SMC1/3 (green), ORD (orange), histone (blue) and DNA (white). Although histone and DAPI staining is lost when chromosome loops are digested with DNase I, SMC1/3 and ORD signals remain visible along chromosome cores. For each fluorophore, identical exposure times were used to capture cells treated with DNase and not treated with DNase. Bar, 2 µm.

View larger version (30K): [in this window] [in a new window]   Fig. 3. Cohesin SMCs are associated with centromeres and chromosome arms in all 16 cells of each germ-line cyst. (A) A partial region 2A cyst from a wild-type chromosome spread preparation is stained for C(3)G (orange), SMC1/3 (white), DNA (blue) and CID (pink). SMC1/3 is enriched at the centromeres of all nuclei (solid arrows). In nuclei that contain C(3)G staining, SMC1/3 signal is thread-like. In pro-nurse cell nuclei that do not build SC, diffuse SMC1/3 staining indicates that cohesin subunits are associated with chromatin throughout the nuclei (open arrows). Bar, 5 µm. (B) Wild-type chromosome spread preparation shows that when the SC disassembles in a pro-nurse cell (left nucleus with only short linear stretches of C(3)G staining remaining), the diffuse SMC1/3 signal is similar in pattern and intensity to a nearby nucleus that does not contain appreciable C(3)G signal. Compare SMC1/3 staining within the two circles. Bar, 2 µm.

View larger version (50K): [in this window] [in a new window]   Fig. 4. ORD is required for centromeric localization of cohesin SMCs. (Top panels) Wild-type chromosome spread preparation immunostained for SMC1/3 (green), CID (orange) and C(3)G (magenta). Note that the two bright SMC1/3 patches overlap with the CID signal at the centromeres and thread-like SMC and C(3)G signals are visible along chromosome arms. Insets contain an enlarged view of the pericentric region (arrow) and show that enrichment of SMC1/3 staining extends beyond the CID signal. (Bottom panels) Single nucleus from a ordnull chromosome spread preparation immunostained for SMC1/3 (green), CID (orange) and C(3)G (magenta). Distinct gaps in the thread-like SMC signal correspond to CID foci, indicating that – in the absence of ORD – cohesin SMCs fail to accumulate at meiotic centromeres. Bar, 2 µm.

View larger version (61K): [in this window] [in a new window]   Fig. 5. Cohesin SMCs exhibit a distinct localization pattern at the centromeres of nurse cell chromosomes that is not dependent on ORD. Vitellarial stage-3 egg chambers (see schematic on right) from a whole-mount ord+ ovariole (top panels) and an ordnull ovariole (bottom panels) were immunostained for SMC1/3 (green) and DNA (white). Note that SMC staining in both wild-type and mutant nurse cell nuclei coincides with bright DAPI-stained regions (solid arrows). Insets correspond to a magnified view of each region indicated by a solid arrow. Note the SMC1/3 finger-like projections. Open arrows indicate SMC1/3 staining in the oocyte nucleus. All images are projections of deconvolved Z-series. The insets include only a subset of sections shown for the entire egg chamber. Bar, 10 µm. The schematic on the right indicates the stage of the cysts shown.

View larger version (80K): [in this window] [in a new window]   Fig. 6. Chromosome cores disassemble in the absence of ORD activity. SMC1 (top two rows of panels) and SMC3 (bottom two rows panels) immunostaining are shown for single ord+ and ordnull nuclei within indicated regions of the germarium. In early region 2A, continuous SMC1 and SMC3 threads are visible in both mutant and wild type. As cysts mature, thread-like SMC staining becomes severely disrupted in the mutant. Only dim fragments or puncta are observed in region 2B ordnull oocytes, and by region 3 mutant nuclei often contain no visible signal. Note that ordnull nuclei lack bright SMC1 and SMC3 foci. Images are projections of deconvolved Z-series of whole-mount preparations. For each antibody, wild-type and mutant images for each stage were captured and processed identically. Bar, 1 µm.

View larger version (34K): [in this window] [in a new window]   Fig. 7. Cohesin SMCs remain associated with chromosome arms when cores break down prematurely in the absence of ORD activity. Shown is a chromosome spread from the ovary of an ordnull female stained for SMC1/3 (white), C(3)G (magenta) and DNA (blue). In a nucleus that contains fragmented SMC1/3 and C(3)G staining (left), diffuse chromatin-associated SMC1/3 signal is still visible, indicating that SMCs remain associated with chromosome arms when cores break down in the absence of ORD activity. Note that diffuse cohesin SMC signal in this nucleus is similar in intensity to the SMC1/3 staining in the adjacent pro-nurse cell that did not build SC (compare signal intensity within the two circles). Bar, 5 µm.

View larger version (48K): [in this window] [in a new window]   Fig. 8. Fragmentation of chromosome cores is detectable before C(3)G localization defects become apparent in ordnull germaria. (A) Graph comparing SMC1 and SMC3 localization defects in ordnull germaria (see Tables 1 and 2) with those observed for C(3)G (Webber et al., 2004). (B) Individual nuclei from whole-mount preparations of ordnull germaria were simultaneously immunostained for SMC1/3 (green) and C(3)G (magenta). In early region 2A, cohesin SMC and C(3)G signals coincide extensively. Although long stretches of thread-like C(3)G staining are still visible in late region 2A cysts, SMC1/3 staining is severely fragmented. When fragmentation of C(3)G becomes apparent, SMC staining is limited to very short fragments and puncta. Images are projections of deconvolved Z-series. SMC images from different stages were captured and processed identically as were the C(3)G images from late region 2A and region 2B. Bar, 2 µm.

View larger version (57K): [in this window] [in a new window]   Fig. 9. C(2)M activity is not required for association of cohesin SMCs with chromosome arms or centromeres. (A) Single nuclei from wild-type (WT) and c(2)MEP[2115] whole-mount preparations immunostained for SMC1/3 (green), CID (orange), and C(3)G (magenta). In wild-type region 2A and region 3 nuclei, the brightest spots of SMC1/3 staining correspond to the CID signal that marks the centromeres (open arrows). Although chromosome cores are absent in c(2)M mutant germaria, patches/foci of SMC1/3 still coincide with CID (solid arrows), indicating that cohesin SMCs localize to centromeres in the absence of C(2)M function. Images are projections of a deconvolved Z-series. Bar, 2 µm. (B) Germarial chromosome spread preparations from wild-type (WT) and c(2)MEP[2115] mutant (c(2)M–/–) females expressing GFP-ORD were immunostained for SMC1/3 (green), GFP-ORD (orange), and C(3)G (magenta). In c(2)M mutants, all nuclei display diffuse SMC1/3 and ORD signal that is similar to that of WT pro-nurse cell nuclei that do not assemble SC. Note also that all c(2)M mutant nuclei exhibit enrichment of SMC1/3 and ORD signal at the centromeres of pro-oocytes and pro-nurse cells (open and solid arrows, respectively). Bar, 10 µm.

View larger version (45K): [in this window] [in a new window]   Fig. 10. Model for chromosome core assembly and maintenance. In wild-type meiotic prophase, cohesin proteins localize along the axes of pairs of sister chromatids. Cohesin complexes are brought together by C(2)M, resulting in the formation of visible chromosome cores that provide a scaffold for SC assembly. In the absence of ORD activity, cohesin SMC subunits still associate stably with chromosome arms and chromosome cores can assembly transiently, but are not maintained.