Project Summary The formation of the central nervous system (CNS) involves a fundamental cell fate diversification—the generation of neurons and glia. The timed, sequential appearance of neurons followed by glia is critical for properly organizing CNS cytoarchitecture and physiology. In support of this, perturbations of the neurogenic- to-gliogenic timer have been demonstrated in multiple neurodevelopmental disorders. Thus, an understanding of the mechanisms that control this cell fate decision is of utmost importance. Although the gliogenic switch is a mechanism conserved from flies to humans, the primate cortex has undergone significant cellular and molecular evolution that has contributed to the >1000 fold increase in cortical surface area compared to rodents. These evolutionary changes include the expansion of a unique progenitor population called outer radial glia, along with significant changes in the molecular, functional, and morphological features of astrocytes. These observations support the hypothesis that outer radial glia are the cellular progenitors of human astrocytes and that this population exhibits human-specific gliogenic drivers. This proposal aims to address two key fundamental questions about human gliogenesis: (1) In which human progenitor cell(s) does the gliogenic switch occur, and (2) what are the intrinsic mechanisms that drive glial competence? In this proposal, we are utilizing human iPSC-derived cortical organoids as a reductionist model system to ask what drives human gliogenesis. The organoid model system is ideal for addressing this question because (a) gliogenesis occurs consistently and reproducibly within a narrow temporal window, and (b) it is genetically tractable and amenable to precise temporal studies that are not possible in other platofrms. We have developed two aims that systematically test (Aim 1) the origins and molecular drivers of human astrogenesis, and (Aim 2) the ability of human-specific pro-glial transcription factors to initiate gliogenesis. We propose a set of experimental approaches that will trace lineage trajectories of individual cells throughout the gliogenic swtich and also test the mechanistic impact of impaired gliogenic drivers. We also propose a set of innovative experiments to test distinct activation schemes of candidate transcription factors tailored to their specific temporal and spatial binding patterns during human development.