Fig. 3: Areal differences in chromatin state of progenitor cells foreshadow the emergence of area-specific types of excitatory neurons.

a, Differentiation trajectories for excitatory neurons from the PFC (left) and V1 (right). b, UMAP projection of PFC and V1 scATAC-seq cells (n = 3 individuals) coloured by cell type predictions. Cells from the excitatory lineage are outlined. IN-STR, striatal interneurons. Suffixes 1–3 denote subclusters from Nowakowski et al.². c, d, UMAP projections of PFC and V1 scATAC-seq excitatory lineage cells coloured by area of origin (c) and pseudotime value (d). e, f, UMAP projections of PFC and V1 scRNA-seq excitatory lineage cells (n = 2 individuals) coloured by area of origin (e) and pseudotime value (f). g, h, Left, PFC and V1 scATAC-seq (g) and scRNA-seq (h) excitatory lineage cells ordered from bottom to top by pseudotime value with PFC–V1 divergence branch point shown (Methods). Cells coloured by EOMES gene activity score (g) or expression (h), highlighting IPCs. Right, schematic illustrating the excitatory neuron differentiation trajectory based on chromatin accessibility, in which PFC–V1 divergence becomes apparent at the level of IPCs (g), or gene expression, in which PFC–V1 divergence is not apparent in IPCs (h). i, Pile-ups of PFC and V1 signal in PFC and V1 differentially accessible (DA) peak sets. Pileups are centred on peaks and show ±10-kb flanking regions. j, Transcription factor motif enrichments of RA-related transcription factors in set of 4,176 PFC-specific peaks (Fisher’s exact, two-sided, FDR < 0.05). k, UMAP projection of deviation scores of motif enrichment for TGIF1. l, Experimental design to test role of RA in organoid area identity. m, UMAP projection of scRNA-seq data from day 70 organoids (n = 11,415 cells). Cells coloured by treatment. VA, vitamin A. n, Left, schematic of expected expression patterns of BCL11B, SATB2, AUTS2, and NR2F1 in primary human cortex. Right, images of primary developing human cortex from the PFC (left) and V1 (right) immunostained for CTIP2 and SATB2 (top) or AUTS2 and NR2F1 (bottom). Representative images shown from n = 2 specimens. o, Left, UMAP projection of cells coloured by expression of NEUROD2, TBR1, SATB2, and NR2F2. Right, images of organoids cultured without (left) or with vitamin A (right) immunostained for CTIP2 and SATB2 (top) or AUTS2 and NR2F1 (bottom). Representative images shown from n = 3 lines.