Distinct DNA methylation profile between E11.5NSph and E14.5NSph. (A) Schematic of the analysis in this study. E11.5NSph and E14.5NSph were cultured from telencephalons of E11.5 and E14.5 mouse embryos and used as models of NPCs. Comparative analysis of D-REAM data was performed to identify NSph-T-DMRs. Immunocytochemical analysis of differentiated NSphs (E11.5NSph-diff and E14.5NSph-diff) was conducted using antibodies against βIII-tubulin (TUBB3) and glial fibrillary acidic protein (GFAP). TUBB3-positive and GFAP-positive cells are indicated in red and green, respectively, and DAPI-stained nuclei are indicated in blue. (B) Distinct characteristics of E11Hypo-T-DMRs and E14Hypo-T-DMRs. The proportion of CGI genes (upper bar charts) and the distributions of NSph-T-DMRs to TSSs (lower panels) are displayed. E11Hypo-T-DMRs (left) and E14Hypo-T-DMRs (right) were mapped in 208 and 604 genes, respectively. The y-axis represents the proportions of each fraction to the whole as 1. The width of the histogram is 250 bp. (C) Integrated Genome Browser (IGB) images of Nrcam and Kat5 gene loci (Ensembl Transcripts) showing comparative MATscores of E14.5NSph to E11.5NSph. Filled and open arrowheads indicate E11Hypo-T-DMRs and E14Hypo-T-DMRs, respectively. Regions analyzed by COBRA (D) are represented by gray rectangles. (D) COBRA representing DNA methylation status of HpyCH4IV sites in Nrcam and Kat5 gene regions. Bisulfite PCR products using genomic DNA from E11.5NSph and E14.5NSph were not treated (−) or treated with HpyCH4IV (+) and electrophoresed. (E) DNA methylation status of the indicated regions located in 4 disease-associated genes (gray rectangles of the upper panels) was analyzed by bisulfite sequencing. Each open, filled, and gray circle represents unmethylated, methylated CpG, and CpG with an undetermined methylation state, respectively. Percentages of methylated CpGs are indicated.