Fig. 4: BRA-2 is essential for normal establishment of synapsis.
a Left: nuclei from the indicated mutants and stage stained for axial (HTP-3, red) and central (SYP-1, green) elements of the SC, counterstained by DAPI (blue). Note extensive regions of HTP-3 devoid of SYP-1 signal, indicating the absence of synapsis. Scale bar 5 μm. Right: schematic representation depicting zoning of the germ line used to quantify synapsis. The chart shows quantification of nuclei with full SC in the indicated zones and genetic backgrounds. Bars show S.E.M., and asterisks denote statistical significance assessed by two-sided Χ2 test (ns= non-significant, **p = 0.004, ****p < 0.0001). At least three germ lines were analyzed and the number of nuclei scored in WT, bra-1 and bra-2 mutants respectively was: zone 1 (229, 224, 306), zone 2 (235, 301, 249), zone 3 (204, 243, 235), zone 4 (199, 227, 230), zone 5 (167, 186, 180), zone 6 (143, 146, 134). b In vivo imaging of GFP::SYP-3 in untreated worms and after exposure to auxin indicates slightly reduced formation of SYP-3 foci coupled with (c) impaired elongation proficiency and starkly reduced SYP-3 polymerization speed. 100 nuclei were analysed for each condition. Statistical significance was calculated by the Mann-Whitney test (two-sided). d Left: representative images of nuclei from HA::AID::bra-2; TIR1::mRuby animals stained for HTP-3 (red)/SYP-1 (green) at the indicated stages (TZ=Transition Zone, EP=Early Pachytene, LP=Late Pachytene) and conditions of exposure to auxin. Scale bar 5 μm. Right: Quantification of nuclei displaying full synapsis across the germ line in the indicated backgrounds and exposure conditions to auxin. 2-3 germ lines were analyzed and the number of nuclei scored in -auxin, 19 h auxin and 16 h recovery conditions respectively, was: zone 1 (254, 113, 269), zone 2 (295, 111, 332), zone 3 (248, 146, 289), zone 4 (270, 172, 231), zone 5 (229, 147, 214), zone 6 (194, 118, 177). Bars show S.E.M. and asterisks denote statistical significance assessed by two-sided Χ2 test (ns= non-significant, ****p < 0.0001). e Schematization of the assay performed to track stage-specific cells before and after BRA-2 depletion. (PMT= Premeiotic Tip, TZ=Tranistion Zone). f Co-staining of SYP-1 (green)/pHTP-3S285 (red) with EdU (gray) in the indicated conditions of exposure to auxin. Arrowheads point to nuclei that are positive for EdU incorporation and show incomplete SC. Scale bar 10 μm. Analysis was performed in biological duplicates.
