An early cell shape transition drives evolutionary expansion of human forebrain
The human brain has undergone rapid expansion since humans diverged from other great apes, but the mechanism of this human-specific enlargement is still unknown. Here, we use cerebral organoids derived from human, gorilla and chimpanzee cells to study developmental mechanisms driving evolutionary brain expansion. We find that the differentiation of neuroepithelial cells to neurogenic radial glia is a protracted process in apes, involving a previously unrecognized transition state characterized by a change in cell shape. Furthermore, we show that human organoids are larger due to a delay in this transition. Temporally resolved RNA-seq from human and gorilla organoids reveals differences in gene expression patterns associated with cell morphogenesis, and in particular highlight ZEB2, a known regulator of epithelial-mesenchymal transition and cell shape. We show, through loss- and gain-of-function experiments, that ZEB2 promotes the progression of neuroepithelial differentiation, and its ectopic overexpression in human is sufficient to trigger a premature transition. By mimicking the nonhuman ape expression of this human evolution gene, we are able to force the acquisition of nonhuman ape architecture in human organoids, establishing for the first time, an instructive role of neuroepithelial cell shape in human brain expansion. Overall design: Organoids generated from 3 replicate batches of H9 and G1 cell lines were collected at 7 time points for bulk RNA-seq analysis: 0, 2, 3, 5, 10, 15 and 25 days post neural induction. ~300 organoids per replicate were collected at the time points ranging between day 0 and 5, ~150 organoids at day 10, ~100 organoids at day 15, and ~50 organoids at day 25. RNA was isolated using Direct-zol-96 RNA kit. cDNA libraries were generated according to NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB #E7760) and barcoded with NEBNext Multiplex Oligos for Illumina. All 42 bulk RNA samples were pooled and sequenced with single-end 50 base mode using 3 lanes of an Illumina HiSeq 4000. Human and gorilla reads were aligned to GRCh38 and Kamilah_GGO_v0 reference genomes respectively. GTF gene annotation files for both reference genomes were filtered down to 16,763 genes that are annotated with identical gene names in both species. Read counts were normalized to transcripts per million (TPM) within each sample and can be found in the supplementary file, 'hum_gor_combined_tpms_HISAT2.txt'.