Engrailed transcription factors direct excitatory cerebellar neuron diversity and survival.

The excitatory neurons of the three cerebellar nuclei (eCN) form the primary output for the cerebellum. The medial eCN (eCNm) were recently divided into molecularly defined subdomains in the adult, however how they are established during development is not known. We define molecular subdomains of the embryonic eCNm using scRNA-seq and spatial expression analysis, showing they evolve during embryogenesis to prefigure the adult. Furthermore, the medial eCN are transcriptionally divergent from the other nuclei by E14.5. We previously showed that loss of the homeobox genes En1 and En2 leads to loss of approximately half of embryonic eCNm. We demonstrate that mutation of En1/2 in embryonic eCNm results in death of specific posterior eCNm molecular subdomains and down regulation of TBR2 (EOMES) in an anterior embryonic subdomain, as well as reduced synaptic gene expression. We further reveal a similar function for EN1/2 in mediating TBR2 expression, neuron differentiation and survival in the other excitatory neurons (granule and unipolar brush cells). Thus, our work defines embryonic eCNm molecular diversity and reveals conserved roles for EN1/2 in the cerebellar excitatory neuron lineage.