Intrinsic and non-cell-autonomous roles for the neurodevelopmental syndrome-linked transcription factor UNC-3/EBF.

Transcription factors are central to neuronal development, yet their functions beyond the cells in which they are expressed remain poorly understood. Here, we uncover unexpected non-cell-autonomous roles for UNC-3, a terminal selector of cholinergic motor neuron identity in C. elegans, whose human ortholog (EBF3) is linked to a neurodevelopmental syndrome. Single-cell RNA-sequencing reveals unc-3 loss in cholinergic motor neurons elicits pronounced transcriptional changes in GABAergic motor neurons that do not express unc-3, which can be rescued by cholinergic-specific UNC-3 restoration. Mechanistically, gene network analysis identifies the pro-regenerative bZIP factor CEBP-1/CEBPB as a key driver of these transcriptional changes. At the circuit level, unc-3 loss causes synaptic and axon pathfinding defects in GABA motor neurons alongside misregulation of neurite development genes. Finally, UNC-3 not only acts as a direct transcriptional activator but also suppresses inappropriate gene expression through indirect mechanisms. Together, these findings broaden terminal selectors as both intrinsic and extrinsic regulators of neuronal identity and circuit assembly, providing a mechanistic framework for understanding EBF3-associated neurodevelopmental disease.