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. 2016 Feb 4;5:e10851. doi: 10.7554/eLife.10851

Figure 6. WAPL-1 antagonizes COH-3/4 cohesion in diakinesis oocytes.

(A) Projections of diakinesis oocytes of the indicated genotypes stained with DAPI. 6 DAPI-stained bodies (WT) indicates presence of 6 bivalents, 12 DAPI-stained bodies indicates absence of chiasmata, and the presence of more than 12 DAPI-stained bodies indicates separation of sister chromatids, with 24 demonstrating separation of all sisters. The average number of DAPI-stained bodies observed in each genotype is indicated on the bottom right of each panel, number of nuclei analyzed: WT (20), rec-8 (29), coh-3 coh-4 (16), rec-8 coh-3 coh-4 (17), rec-8 spo-11 (94), coh-3 coh-4 spo-11 (26), and coh-3 coh-4 syp-2 (26). Arrowheads in rec-8 syp-1 panel point to chromosome fragments. Note that removal of SPO-11 or SYP-1/2 causes loss of cohesion in rec-8 mutants but not in coh-3 coh-4 double mutants. (B) Projections of diakinesis oocytes of the indicated genotypes stained with DAPI, quantification shown in (C). Note the reduction of DAPI-stained bodies in rec-8 spo-11 wapl-1 compared with three other genotypes (p<0.0001 in all cases, t-test). Error bars = standard deviation. Number of nuclei analyzed: rec-8 spo-11 (94), rec-8 spo-11 wapl-1 (85), rec-8 spo-11 wapl-1 coh-3 coh-4 (29), rec-8 spo-11 wapl-1GFP::wapl-1 (27). (D) Automated quantification (CellProfiler) of area sizes corresponding to chromatin bodies in projections of diakinesis oocytes of indicated genotypes stained with DAPI. Values on the X axis represent area in pixels and binning of the different categories was adjusted using oocytes of known phenotypes: WT (bivalents), coh-3 coh-4 (univalents) spo-11 rec-8 (detached sisters). Number of oocytes analyzed: WT (40), coh-3 coh-4 (61) spo-11 rec-8 (116). (E) Automated quantification of area sizes corresponding to chromatin bodies in projections of diakinesis oocytes, note that removing WAPL-1 from rec-8 syp-1 double mutants causes a large decrease of chromosome fragments and an increase in univalents. Number of nuclei analyzed: rec-8 syp-1 (52), rec-8 syp-1 wapl-1 (36). (F) Example of DAPI-stained oocyte from rec-8 syp-1 wapl-1 demonstrating absence of chromosome fragments, compare with rec-8 syp-1 example shown in A. (G) Projections of diakinesis oocytes stained with DAPI, note that chromosome fragments are present in rec-8 coh-4 (arrowheads) but not in rec-8 coh-4 wapl-1 oocytes. (H) Automated quantification of area sizes corresponding to chromatin bodies in projections of diakinesis oocytes, note that removing WAPL-1 from rec-8 coh-4 double mutants causes a large decrease of chromosome fragments. 26 diakinesis oocytes were analyzed for both rec-8 coh-4 and rec-8 coh-4 wapl-1.

DOI: http://dx.doi.org/10.7554/eLife.10851.025

Figure 6.

Figure 6—figure supplement 1. Control demonstrating that unscheduled DSBs are not formed in wapl-1 spo-11 rec-8 triple mutants.

Figure 6—figure supplement 1.

Projections of pachytene nuclei from wapl-1 spo-11 rec-8 triple mutants stained with anti-RAD-51 antibodies and DAPI, note the absence of RAD-51 foci (compare with RAD-51 staining in the wapl-1 panel shown in Figure 2B). Scale bar = 5 µm.
DOI: http://dx.doi.org/10.7554/eLife.10851.026
Figure 6—figure supplement 2. Examples of automated area analysis using CellProfiler.

Figure 6—figure supplement 2.

Images show projections of diakinesis oocytes of the indicated genotypes stained with DAPI. Red lines represent the boundaries of chromatin bodies as determined by CellProfiler.
DOI: http://dx.doi.org/10.7554/eLife.10851.027