Abstract
Cerebellar Purkinje cells (PCs), the sole output neurons of the cerebellar cortex, are essential for motor coordination, learning and circuit formation. While functionally diverse, the extent of PC heterogeneity and the molecular drivers of this diversity remain unclear. Using single-cell RNA sequencing, we identified at least 11 molecularly distinct PC subtypes in the embryonic mouse cerebellum. Spatial reconstruction revealed that these subtypes are organized in embryonic patterns that predict key features of adult cerebellar architecture, including longitudinal stripes and lobular specificities. PC-subtype identity is defined by the combinatorial expression of Foxp1, Foxp2 and Foxp4. Genetic deletion of Foxp1 and Foxp2 disrupts PC diversification and cerebellar patterning, including the loss of a Foxp1(+) subtype and the failure of cerebellar hemisphere formation. Foxp1(+) PCs are enriched in the fetal human cerebellum but are rare in chick, suggesting a role in cerebellar evolution. These findings uncover early PC diversification and identify Foxp1(+) PCs as critical regulators of cerebellar hemispheric development.