Fig. 1: Mammalian-specific changes in the ZBTB18-associated CREs and TF expression in neocortical ExNs.

a, Immunolabelling for Arpp21–Gfp⁺ IT (IT neurons onwards) and Fezf2–Gfp⁺ ET (ET neurons onwards) neurons and BCL11B in mouse PD 0 neocortex. b, Schematic for isolation and processing of Arpp21–Gfp-positive and Fezf2–Gfp-positive cells from neocortex for RNA-seq and H3K27ac ChIP–seq. c, TFBS for 69 TFs enriched among both IT-biased H3K27ac peaks (red circle) and H3K27ac peaks near IT-biased genes (blue circle) compared with ET-biased peaks and genes, respectively. TFBS enrichments were tested using Fisher’s exact test. Significant TF motifs had a Benjamini–Hochberg-corrected P value < 0.05 and odds ratio > 1. For RNA-seq, n = 3 biological replicates per condition. For ChIP–seq, n = 2 biological replicates per condition. d, Schematic showing two dorsal pallial regions (H and M) microdissected from E17 chicken embryo. e, Venn diagram showing the co-occurrence of peaks derived from ZBTB18-HA ChIP–seq and H3K27ac peaks preferably found in IT and ET neurons and in chicken dorsal pallium. X are peaks in both IT and ET neurons excluded from analysis. f, Heat map showing pairwise alignment distances among vertebrates for six putative CREs overlapping ZBTB18 ChIP–seq peaks linked to IT neurons and axon guidance. The left grid indicates ChIP–seq peak identification method. Columns on the right show if the regions (H3K27ac or ZBTB18 peaks) are orthologous and active in the chicken embryonic dorsal pallium. Grey columns mark non-orthologous regions; red and black columns show the presence or absence of H3K27ac peaks in orthologous regions. Distances are relative to mice. g, Dot plot of Zbtb18-expressing neurons in the dorsal pallium showing cell percentages and co-expression of genes with the ZBTB18 binding motif in IT neurons across mammalian (mouse) and non-mammalian species (chicken, lizard and turtle). h,i, Coronal sections of chicken (h) and mouse (i) brains showing co-localization of BCL11B and SATB2 with ZBTB18. j, Bar plots showing the co-localization percentage for BCL11B and ZBTB18 over total BCL11B⁺ cells and SATB2 and ZBTB18 over total SATB2⁺ cells in chicken and mouse. A standard unpaired two-tailed t-test was applied. The graph shows the mean ± s.e.m. **P = 0.0011; ***P = 0.0004 (n = 3 per species). E, entopallium; fL, fetal (immature) layer; H, hyperpallium; HA, apical hyperpallium; Hp, hippocampus; M, mesopallium; N, nidopallium; RPKM, reads per kilobase of transcript per million mapped reads; Str, striatum. Scale bars, 100 µm (a), 1 mm (h,i (mouse brain)), 150 µm (h,i (mouse inset)), 1 mm (h,i (chicken brain)), 300 µm (h,i (chicken inset)), 250 µm (h,i (Cux2)).