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. 2024 Feb 26;13:e94694. doi: 10.7554/eLife.94694

Figure 1. Mutation in mouse Sass6 activates the 53BP1-USP28-p53 mitotic surveillance pathway.

(A) Left-side views of wild-type (WT), Sass6em4/em4, Sass6em4/em4 Trp53−/−, Sass6em4/em4 Trp53bp1−/−, and Sass6em4/em4 Usp28−/− embryos at E9.5. Anterior is up in all images. At least three embryos per genotype showed similar phenotypes. Scale bar = 500 µm. (B) Immunostaining for p53 on transverse sections of WT, Sass6em4/em4, Sass6em4/em4 Trp53−/−, Sass6em4/em4 Trp53bp1−/−, and Sass6em4/em4 Usp28−/− embryos at E8.5. The sections shown encompass the neural plate (top) and mesenchyme (bottom), demarcated by the dashed line. Dorsal is up in all images. Scale bars = 25 µm. (C) Immunostaining for Cleaved-Caspase3 (Cl. CASP3) as mentioned in (B). Arrowheads indicate Cl. CASP3-positive cells, while asterisks mark non-specific staining of blood cells. (D) Quantification of the nuclear p53 in (B). Values were normalized to WT. Error bars represent mean ± SD WT: 1.00 ± 0.06 (n=2582 cells from three embryos); Sass6em4/em4: 2.4 ± 0.5 (n=2372 from four embryos); Sass6em4/em4 Trp53−/−: 0.8 ± 0.3 (n=2379 from three embryos); Sass6em4/em4 Usp28−/−: 1.4 ± 0.3 (n=2775 from four embryos); Sass6em4/em4 Trp53bp1−/−: 1.2 ± 0.1 (n=1840 from two embryos). *p<0.05, **p<0.01, ***p<0.001 (one-way ANOVA with Tukey’s multiple comparisons). (E) Quantification of Cl. CASP3 in (C) as mentioned in (D). WT: 1.00 ± 0.1 (n=2582 cells from three embryos); Sass6em4/em4: 2.3 ± 0.3 (n=2372 from four embryos); Sass6em4/em4 Trp53−/−: 1.2 ± 0.1 (n=2379 from three embryos); Sass6em4/em4 Usp28−/−: 1.4 ± 0.1 (n=2775 from four embryos); Sass6em4/em4 Trp53bp1−/−: 1 ± 0.1 (n=1840 from two embryos). ***p<0.001, ****p<0.0001 (one-way ANOVA with Tukey’s multiple comparisons).

Figure 1.

Figure 1—figure supplement 1. Mutations in Sass6 lead to an increase in the mitotic index in mouse embryos.

Figure 1—figure supplement 1.

The percentage of phospho-Histone H3-positive neural and mesenchymal cells of wild-type (WT), Sass6em4/em4, and Sass6em5/em5 at E8.5. Three embryos per genotype were used for the quantifications. Error bars represent mean ± SD WT: 5 ± 1% (n=1449 cells); Sass6em4/em4: 10 ± 1% (n=896); Sass6em5/em5: 11 ± 2% (n=941). *p<0.05, ns = not significant with p>0.05 (one-way ANOVA with Tukey’s multiple comparisons).