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. 2022 May 27;50(10):5652–5671. doi: 10.1093/nar/gkac408

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© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 5. — Reduced loading of MSH-5 into recombination foci in top-3-ZnF and rmh-2. (A) Upper panel: representative images of GFP::MSH-5 foci localization at different stages of meiotic prophase I in C. elegans gonads of the indicated genotypes. Zone 1 = transition zone; zone 2 = early pachynema; zone 3 = mid pachynema; zone 4 = late pachynema. Images of gonads show DAPI staining (magenta) and endogenous GFP (green). Bottom: Quantification of GFP::MSH-5 foci in the meiotic part of the gonad. Scatter plots indicate the number of foci per nucleus. For each genotype, the mean ± SD number of foci in each zone are indicated, with n = number of nuclei assessed. gfp::msh-5: zone 1, 0.05 ± 0.3, n = 157; zone 2, 8.6 ± 5.8, n = 149; zone 3, 10.82 ± 4.2, n = 122; and zone 4, 6.1 ± 0.7, n = 92. gfp::msh-5; top-3-ZnF: zone 1, 0.0 ± 0.0, n = 151; zone 2, 2.7 ± 3.2, n = 135; zone 3, 7.3 ± 1.6, n = 133; and zone 4, 6.1 ± 0.7, n = 72. gfp::msh-5; rmh-2(jf94): zone 1, 0.02 ± 0.1, n = 125; zone 2, 2.2 ± 2.9, n = 107; zone 3, 8.5 ± 2.8, n = 93; and zone 4, 6 ± 0.8, n = 84. gfp::msh-5; top-3-ZnF; rmh-2(jf94): zone 1, 0 ± 0, n = 120; zone 2, 1 ± 1.7, n = 110; zone 3, 5.3 ± 2.7, n = 70; and zone 4, 5.7 ± 0.8, n = 58. Statistical significance was determined using the Mann–Whitney test (see Supplementary table 1 for more details of the statistical analysis). (B) Upper panel: representative images of nuclei in early and mid pachynema in the indicated genotypes after staining for RAD-51 (blue), GFP (magenta) and DAPI (yellow). Lower panel: the percentage of nuclei in early and mid pachynema that were positive for the indicated meiotic markers (RAD-51 only, blue; MSH-5 only, orange; and both RAD-51 and MSH-5, gray). (C) Upper panel: representative images of GFP::MSH-5 and OLLAS::COSA-1 co-localization in recombination foci in late pachynema in the indicated genotypes. Gonads were stained with DAPI (blue), anti-GFP (yellow) and anti-OLLAS (magenta) antibodies. Dashed circles highlight nuclei with clear co-localization. Lower panel: quantification of OLLAS::COSA-1 foci in late pachynema in the indicated genotypes. The scatter plot indicates the number of foci per nucleus. Mean ± SD are indicated, n = number of nuclei assessed. ollas::cosa-1; gfp::msh-5, 6 ± 0.6, n = 63; ollas::cosa-1; gfp::msh-5; top-3-ZnF, 5.8 ± 0.7, n = 67; ollas::cosa-1; gfp::msh-5; rmh-2, 5.9 ± 0.6, n = 52; ollas::cosa-1; gfp::msh-5; top-3-ZnF; rmh-2, 6 ± 0.8, n = 41. There is no significant difference between the indicated genotypes. Statistical significance was determined using the Mann–Whitney test.