Engrailed transcription factors direct embryonic medial excitatory cerebellar nuclei neuron identity and survival

In the cerebellum, the excitatory neurons of the three cerebellar nuclei (eCN) form the primary output for the circuit. 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 eCNm using scRNA-seq and spatial expression analysis during embryogenesis. Our analysis shows that the eCNm are transcriptionally divergent from the rest of the eCN by E14.5. We previously showed that conditional loss of the homeobox genesEn1andEn2leads to death of a subset of embryonic eCNm. We demonstrate that loss ofEn1/2in embryonic eCNm results in cell death of a specific posterior eCNm molecular subdomain and loss of TBR2 expression in an anterior 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 two other cerebellar excitatory neuron types. Thus, our work defines embryonic eCNm molecular diversity and reveals conserved roles for EN1/2 in the cerebellar excitatory neuron lineage.