Human cortical organoids expose a differential function of GSK3 on direct and indirect neurogenesis

Summary The regulation of proliferation and polarity of neural progenitors is crucial for the development of the brain cortex, with modes and timings of cell division intimately related to the stereotypical acquisition of layer-specific neuronal identities. Animal studies have implicated glycogen synthase kinase 3 (GSK3) as a pivotal regulator of both proliferation and polarity, yet the functional relevance of its signaling for the unique features of human corticogenesis remain to be elucidated. Here we harness human cortical brain organoids to probe the longitudinal impact of GSK3 inhibition through multiple developmental stages. Our results indicate that chronic GSK3 inhibition increases the proliferation of neural progenitors and causes massive derangement of cortical tissue architecture. Surprisingly, single cell transcriptome profiling revealed only a discrete impact on early neurogenesis and uncovered a pivotal role of GSK3 in the regulation of NEUROD1/2 lineages and outer radial glia (oRG) output, without compromising direct neurogenic trajectories. Through this first single cell-level dissection of the GSK3 regulatory network in human corticogenesis, our work underscores the robustness of transcriptional programs in determining neuronal identity independent of tissue architecture.