Spatiotemporal dynamics of the developing zebrafish enteric nervous system at the whole-organ level

Neural crest cells give rise to the neurons of the enteric nervous system (ENS) that innervate the gastrointestinal (GI) tract to regulate gut motility. The immense size and distinct subregions of the gut present a challenge to understanding the spatial organization and sequential differentiation of different neuronal subtypes. Here, we profile enteric neurons (ENs) and progenitors at single-cell resolution during zebrafish embryonic and larval development to provide a near-complete picture of transcriptional changes that accompany the emergence of ENS neurons throughout the GI tract. Multiplex spatial RNA transcript analysis identifies the temporal order and distinct localization patterns of neuronal subtypes along the length of the gut. Finally, we show that functional perturbation of select transcription factors Ebf1a, Gata3, and Satb2 alters the cell fate choice, respectively, of inhibitory, excitatory, and serotonergic neuronal subtypes in the developing ENS.