High density of REC8 constrains sister-chromatid axes and prevents illegitimate synaptonemal complex formation (2016)
Figure 1

Figure 1. Visualization of sister-chromatid axes and sites of local separation of axial elements with super-resolution microscopy.

A) Representative nuclear spreads of wild-type pachytene and Rec8−/− pachytene-like spermatocytes. Nuclear spreads were immunostained for SYCP3 and ACA. Magnified views are indicated by dashed areas. Schematic representation on magnified chromosomes represents tightly associated sister-AEs (separated for easier representation) in paired wild-type homologs, and the appearance of two distinguishable/separated sister-AEs once REC8-mediated cohesion is lost in Rec8−/− univalents. Bars, 10 μm in spreads and 1 μm on insets.

B) Representative nuclear spread of zygotene-like Stag3 mutant spermatocytes. Nuclear spreads were immunostained for SYCP3 and ACA. Magnified views are indicated by dashed areas. Schematic representation on magnified univalents represents: separation of sister-AEs (centre) and close association of sister-AEs (right). Bellow: graph showing the percentages of axes with: separation of AEs (dark grey area) and closely associated AEs (light grey area). 414 axes analysed, from 11 nuclei. Bars, 10 μm in spreads and 1 μm on insets.

C) Magnified views of zygotene-like Stag3 mutant univalents with LSAEs. Nuclear spreads were immunostained for SYCP3 and ACA. Schematic representation indicates sites of LSAEs. Bellow: graph showing the percentages of axes with: LSAEs (red area), extensive separation of AEs (dark grey area) and closely associated AEs (light grey area). 414 axes analysed, from 11 nuclei. Bars, 1 μm.

D) Representative nuclear spread of zygotene-like Smc1β−/− spermatocytes. Nuclear spreads were immunostained for SYCP3 and ACA. Magnified views are indicated by dashed areas. Schematic representation on magnified univalents represents sites of LSAEs. Bellow: graph showing the percentages of axes with: LSAEs (red area), extensive separation of AEs (dark grey area) and closely associated AEs (light grey area). 404 axes analysed, from 10 nuclei. Bars, 10 μm in spreads and 1 μm on insets.

E) STED images of representative zygotene-like Stag3 mutant and Smc1β−/− univalents displaying LSAEs. Nuclear spreads were immunostained for SYCP3. Bars, 1μm.

F) Graph indicating the inter-axis distances measured in wild-type pachytene bivalents; pachytene-like Rec8−/− and zygotene-like Stag3 mutant and Smc1β−/− univalents. Each measurement in wild-type and Rec8−/− corresponds to the median of 3 distances measured along one homolog (n=60). Each measurement in Stag3 mutant and Smc1β−/− mutant axes correspond to 1 distance measured at sites of local separation of AEs (n=40 and n=20, respectively). Horizontal lines indicate median.

G) Graph indicating the inter-axis distances measured at terminally and centrally located sites of LSAEs in zygotene-like Stag3 mutant univalents. Each measurement corresponds to 1 distance measured at terminally (n=20) and centrally (n=20) located sites of local separation of axial elements. Horizontal lines indicate median.

H) Schematic representation of the axis architecture observed in the different backgrounds analysed: close association of sister-AEs rendering a single AE per homolog (wild-type), separation of sister-AEs, with appearance of two individual sister-AEs (Rec8−/−), and LSAEs rendering local “axial openings” with two individual sister-AEs (Stag3 mutant and Smc1β−/−). Centromeres are shown in red. For easier comparison of distance between axes, a red bar of equal length was added to each chromosome drawing.