Christensen, J. B.; Donovan, A. P. A.; Moradi, M.; Vanacore, G.; Helmy, M.; Reid, A. J.; Lee, J. T. H.; Bayraktar, O. A.; Brand, A. H.; Bayin, N. S.

Understanding the molecular mechanisms driving lineage decisions and differentiation during development is challenging in complex systems with a diverse progenitor pool, such as the mammalian cerebellum. Importantly, how different transcription factors cooperate to generate neural diversity and the gene regulatory mechanisms that drive neuron production, especially during the late stages of cerebellar development are poorly understood. Here, we used single cell RNA-sequencing (scRNA-seq) to investigate the developmental trajectories of Nestin-expressing progenitors (NEPs) in the neonatal mouse cerebellum. We identified FOXO1 as a key regulator of NEP-to-inhibitory neuron differentiation, acting directly downstream of ASCL1. Genome occupancy and functional experiments using primary NEPs showed that both ASCL1 and FOXO1 regulate neurogenesis genes during differentiation while independently regulating proliferation and survival, respectively. Furthermore, we demonstrated that WNT signalling promotes the transition from an ASCL1+ to a FOXO1+ cellular state. Finally, we showed that the role of WNT signalling in promoting neuron production via FOXO1 is conserved in primary human NEPs. By resolving how cerebellar interneurons differentiate, our findings could have implications for cerebellar disorders such as spinocerebellar ataxia, where cerebellar interneurons are overproduced.