GENERATION OF MOUSE NEOCORTICAL ORGANOIDS TO MODEL THE IMPACT OF CIS-REGULATORY VARIATION ON CORTICAL NEUROGENESIS ACROSS EVOLUTIONARY TIMESCALES (scRNA-Seq)

Natural selection has shaped the gene regulatory networks that orchestrate the development of the neocortex, leading to diverse neocortical structure and function across mammals, but the molecular and cellular mechanisms driving phenotypic changes have proven difficult to characterize. Here, we develop a reproducible protocol to generate cortical organoids from mouse epiblast stem cells that enable in depth mechanistic studies of cortical developmental in vitro. Cortical organoids develop with similar kinetics to the mouse cortex in vivo, recapitulate the cellular diversity present in the embryonic neocortex, and undergo relatively rapid maturation compared to human organoids. We generated cortical organoids from F1 hybrid epiblast stem cell lines from crosses between standard laboratory mice (C57Bl/6J) and four wild-derived inbred mouse strains from distinct sub-species that span ~1 million years of evolutionary divergence. Using scRNA-seq and allele-specific expression analysis we identified hundreds of genes that exhibit differential cis-regulation during cortical neurogenesis. These experimental methods and cellular resources represent a powerful new platform for investigating gene regulatory mechanisms across evolutionary timescales.