Project Summary The neocortex, a structure covering mammalian brains, computes high-order sensory, motor, and cognitive processes. Over the course of evolution, the neocortex of certain species expanded dramatically and folded, thereby conferring superior sensorimotor and cognitive abilities. The immense expansion of the neocortex in humans made possible the complex behavior, cognition, and intellect unique to our species. Neocortical expansion and folding reflect an increase in the number of neural cells and, therefore, depend on the number of neural progenitors (NPs). The long-term goal of this project is to understand the mechanisms regulating NP expansion and neocortical growth and folding. Sonic hedgehog (SHH) signaling promotes NP expansion. In mice, ectopically elevated SHH signaling activity causes the neocortex to expand and fold. SHH signaling also increases the number of NPs in the ferret, a species with a naturally folded neocortex, and in human cerebral organoids, miniature models of the developing brain grown from human pluripotent stem cells. SHH signaling expands NPs by regulating transcription. The short-term goal of this project is to understand how transcriptional programs downstream of SHH signaling expand NPs. Preliminary data show that cyclin-dependent kinase 6 (CDK6) regulates a transcriptional program that drives NP expansion downstream of SHH signaling and that a histone H3 mutation (H3pG34R) that is recurrent in pediatric brain cancer disrupts the SHH signaling–driven transcriptional program driving NP expansion. Mutations in CDK6 and histone H3 are associated with neurodevelopmental disorders and brain tumors. Based on these findings, this project will investigate transcriptional programs driving NP expansion and mechanisms regulating the transcriptional programs by using mouse models and human NP models that have gain- or loss-of-function mutations in SHH signaling, CDK6, and histone H3. This project has two main aims. Aim 1 is to investigate how CDK6 regulates transcriptional programs downstream of SHH signaling to expand NPs. For this aim, we will investigate (1.1) how SHH signaling and CDK6 affect the transcriptome of each NP type in situ by employing a novel spatial transcriptomic method with subcellular resolution and high transcriptome output and (1.2) how SHH signaling and CDK6 affect histone modifications and chromatin structure to regulate transcription. Aim 2 is to investigate how the H3pG34R mutation affects NPs. For this aim, we will investigate (2.1 and 2.3) the effects of the H3pG34R mutation on the behavior and transcriptional program of NPs and (2.2) how mutations that co-occur with H3pG34R in pediatric brain cancer modulate the effects of H3pG34R on NPs. The proposed study will provide important insights into the transcriptional regulation of NP expansion and how CDK6 and H3 mutations affect such transcriptional programs, thereby deepening our mechanistic understanding of brain development, evolution, ...