A mouse organoid platform for modeling cerebral cortex development and cis-regulatory evolution in vitro

Natural selection has shaped the gene regulatory networks that orchestrate cortical development, leading to structural and functional variation across mammals, but the molecular and cellular mechanisms underpinning these changes have only begun to be characterized. Here, we develop a reproducible protocol for cerebral cortex organoid generation from mouse epiblast stem cells (EpiSCs), which recapitulates the timing and cellular differentiation programs of the embryonic cortex. We generated cortical organoids from F1 hybrid EpiSCs derived from crosses between laboratory mice (C57BL/6J) and four wild-derived inbred strains spanning ∼1 M years of evolutionary divergence to comprehensively map cis-acting transcriptional regulatory variation across developing cortical cell types, using single-cell RNA sequencing (scRNA-seq). We identify hundreds of genes that exhibit dynamic allelic imbalances, providing the first insight into the developmental mechanisms underpinning changes in cortical structure and function between subspecies. These experimental methods and cellular resources represent a powerful platform for investigating gene regulation in the developing cerebral cortex.