Fig. 4: Folate deficiency increases H3K4me3 levels in NTD mice.

A Morphology of CD-1 mouse embryos with low-folate diet and MTX induced from E9.5, representative images were shown, left panel is control mouse fetus(n = 100), middle panel is anencephalous(n = 26), and right panel is spina bifida (n = 18). The arrowhead is where the NTDs located. Scale bar, 1 mm. B Immunostaining for KDM5A and H3K4me3 in the neural tubes in E13.5 folate-deficient NTD mouse embryos(n = 6) and control embryos(n = 6). Images were acquired with a confocal microscope, and nuclei were stained with DAPI. C RT‒qPCR analysis of neurodevelopment-related gene expression in the brains of folate-deficient NTD model mice at E9.5(n = 6) and control mice(n = 6). Gapdh was used as a loading control. *p < 0.05, **p < 0.01, ***p < 0.001. D H3K4me3 ChIP assays were performed in brain tissue from E9.5 folate-deficient NTD model mice(n = 12) and control mice(n = 12). Mouse IgG was used as a control. The enrichment of H3K4me3 in the promoters of the neurodevelopment-related genes Axin2, Bcl9l, Atoh1, Nkx2.2, Sox1 and Isl1 was measured via RT‒qPCR. *p < 0.05, **p < 0.01, ***p < 0.001. E KDM5A and H3K4me3 levels in the folate-deficient NTD mouse model(n = 12) and control mice(n = 12) brains and spines at E9.5 were measured via western blotting. GAPDH and H3 were used as loading controls for KDM5A and H3K4me3, respectively. F GO enrichment analysis of genes with increased H3K4me3 peaks identified by Cut&Tag in E9.5 folate-deficient NTD model mice. The x-axis shows the GO terms, and the Y-axis shows the percentage of genes. Fig. 4C and 4D are presented as the means ± SEM from three biological replicates.