The m5C reader Ybx1 regulates embryonic cortical neurogenesis by promoting progenitor cell cycle progression
Fig 1
Ybx1 is highly expressed in the progenitor cells of earlier embryonic mouse cortex and conditional ablation of Ybx1 causes impaired cortical development.
(A) In situ hybridization using DIG-labeled RNA probes (antisense and sense) targeting Ybx1 was performed on coronal sections of mouse brains at E11.5 to P0. Representative cortical regions were shown as magnified images. Scale bars, 500 μm. (B) Immunostaining for Ybx1 protein was done on coronal sections of mouse brains at E11.5, E13.5, E15.5, E17.5, and P0. Representative cortical regions were shown. Scale bars, 100 μm. (C) Representative images of P0 neonatal mice from the same litter with different genotypes were shown at 2 and 12 h after birth, respectively. (D) Quantitative analysis of neonatal body weight at P0 was shown as box and whisker plots. Ybx1fl/fl, Nestin-Cre, Ybx1+/fl, and Nestin-Cre, Ybx1fl/fl were designated as control (Ctrl), heterozygous (Het), and cKO, respectively. Ctrl (n = 39 pups) vs. Het (n = 26 pups), p = 0.37; Ctrl vs. cKO (n = 28 pups), **p = 0.0014; Het vs. cKO, *p = 0.021; ns, not significant; all by one-way ANOVA followed by Tukey’s multiple comparison test. (E) Representative images of the P0 brains from cKO (#1), Het (#2), and Ctrl (#3) pups shown in (D). Black arrowed lines indicate the cortical length and width. (F) Quantitative analysis of cortical length and width at P0 was shown as box and whisker plots. n = 21 pups for Ctrl, n = 15 pups for Het, n = 16 pups for cKO: p = 0.65 for width, p = 0.80 for length; ns, not significant; all by one-way ANOVA followed by Tukey’s multiple comparison test. (G) Representative images of P0 coronal brain sections stained with an antibody against Ybx1 protein demonstrated efficient ablation of Ybx1 in the cKO brain. The asterisk indicates the enlarged ventricle in the cKO brain. Scale bar, 1,000 μm. (H) Quantification of lateral ventricle area of P0 coronal brain sections was presented as box and whisker plots: Ctrl (n = 21 confocal fields) vs. Het (n = 22 confocal fields), *p = 0.035; Ctrl vs. cKO (n = 21 confocal fields), ****p = 2.03E−11; Het vs. cKO, ****p = 2.03E−011; all by one-way ANOVA followed by Tukey’s multiple comparison test. (I) Quantification of P0 cortex thickness was presented as box and whisker plots: Ctrl (n = 20 confocal fields) vs. cKO (n = 21 confocal fields), ****p = 6.73-05; Het (n = 19 confocal fields) vs. cKO, **p = 0.0014; ns, not significant; all by one-way ANOVA followed by Tukey’s multiple comparison test. The data underlying all the graphs shown in the figure are included in S1 Data.