Fig. 6
Repression of p53 target genes by CHD8 is necessary for normal brain growth. a Volcano plot of RNA-seq data illustrating in red genes that are differentially expressed (FDR < 0.05) in E10.5 cKO telencephalon, with p53 pathway genes labelled. b Pathway enrichment analysis of differentially expressed genes, with the top KEGG pathway terms shown [43]. See also Additional file 5: Table S4. c qRT-PCR validation of a selection of p53 pathway genes identified by RNA-seq (n = 4 for each condition. Mean ± SEM; *p < 0.05, ***p < 0.001, student’s t test). d Wholemount brains from E18.5 Ctrl, cKO and cKO-p53het mice are shown, anterior to the top. Data are representative of 6 embryos per genotype. Scale bar = 1 mm. e–g Cleaved caspase 3 (CC3) immunohistochemistry (brown) on frontal sections through the telencephalon of E12.5 embryos. Scale bar = 200 μm. h Quantification of CC3+ cells/μm2 of the pallium in embryos of each genotype (Ctrl, n = 4; cKO, n = 3; cKO-p53het, n = 3; Mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ANOVA followed by Tukey’s multiple comparisons test). i Quantification of CC3+ cells/μm2 of the subpallium (medial ganglionic eminence) in embryos of each genotype (Ctrl, n = 4; cKO, n = 3; cKO-p53het, n = 3; Mean ± SEM; *p < 0.05, **p < 0.01, ANOVA followed by Tukey’s multiple comparisons test). j–l Immunohistochemistry to detect BrdU + (red) and phospho-histone 3B (PH3B)+ nuclei in frontal sections through the telencephalon of E12.5 embryos. Scale bar = 100 μm. j′–l′ Magnified images of the boxed neocortical regions in j–l, with the ventricular zone (vz) at the bottom and pial surface at the top. Scale bar = 25 μm. m Quantification of PH3B+ cells/μm of neocortical ventricular surface (Ctrl, n = 4; cKO, n = 3; cKO-p53het, n = 3; Mean ± SEM; *p < 0.05, **p < 0.01, ANOVA followed by Tukey’s multiple comparisons test). n, p Immunostaining of frontal E12.5 sections for DCX (green) to label differentiating neurons, with nuclei counterstained with Hoechst 33,342. Scale bar = 200 μm. n′, p′ Magnified images of n, p, with the ventricular zone (vz) at the bottom and pial surface (pp) at the top. Scale bar = 25 μm