Evolution of new cerebellar nuclei by excitatory progenitor diversification in the early rhombic lip
The cerebellar nuclei, the output regions of the cerebellum, have evolved via repeated duplication of a conserved cell type set to produce different numbers of nuclei across species. Here, we investigate the mechanism underlying this process using developmental single-cell and spatial transcriptomics time courses in mouse and chicken. We show that new nuclei formation is governed by excitatory neurons born from nucleus-specific progenitors in the early and late rhombic lip (RL), with inhibitory neurons incorporating into established nuclear territories. Evolutionarily newer canonical cerebellar nuclei with increasingly higher-order functions are produced by diversification of the early RL, where nuclear identity and spatial organization are established in part by co-option of border formation programs in conserved progenitor cell types. In contrast, the late RL generates the higher-order subnuclei of the medial nucleus and forms a non-canonical olivocerebellar circuit. Together, our findings suggest that new brain regions can evolve through developmental diversification of excitatory progenitors and spatial segregation of conserved sister cell types with generic inhibitory neurons filling in after.