A conserved differentiation program facilitates inhibitory neuron production in the developing mouse and human cerebellum

Understanding the molecular mechanisms driving lineage decisions and differentiation during development is especially difficult in systems with a diverse progenitor pool, such as the mammalian cerebellum. Here, we used single-cell RNA-sequencing (scRNA-seq) to characterise 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 genomic experiments using primary NEPs showed that both ASCL1 and FOXO1 regulate neurogenesis genes during in vitro differentiation, while independently regulating proliferation and survival, respectively. Furthermore, we demonstrate that WNT is a regulator of the transition from an ASCL1+ to FOXO1+ state. Finally, we show that the ASCL1-FOXO1 axis and role of WNT signalling is conserved in primary human NEPs. Through understanding how cerebellar interneurons differentiate, our findings may inform therapeutic strategies for cerebellar disorders such as spinocerebellar ataxia, where cerebellar interneurons are overproduced. Overall design: Targeted DamID of Dam methylase, FOXO1- and ASCL1-Dam during in vitro differentiation of primary nestin-expressing progenitors.