Unbiased intestinal single cell transcriptomics reveals previously uncharacterized enteric nervous system populations in larval zebrafish

The enteric nervous system (ENS) regulates many gastrointestinal functions including peristalsis, immune regulation and uptake of nutrients. Defects in the ENS can lead to severe enteric neuropathies such as Hirschsprung disease (HSCR), which is caused by defective ENS development. Zebrafish have proven to be fruitful in the identification of novel genes involved in ENS development and HSCR pathology. However, the composition and specification of enteric neurons and glial subtypes of the larval zebrafish at a single cell level, remains mainly unexplored. Here, we performed single cell RNA sequencing of zebrafish ENS at 5 days post-fertilization. We identified both vagal neural crest progenitors and Schwann cell precursors, as well as four clusters of early differentiated neurons. Interestingly, since we took an unbiased approach where we sequenced total intestines, an elavl3+/phox2bb- population of neurons and the presence of cx43+/phox2bb- enteric glia were identified in larval zebrafish. These populations have not been described before. Pseudotime analysis supported binary neurogenic branching of ENS differentiation, which happens via a notch-responsive state. Together, our data revealed previously unrecognized ENS populations and serve as a resource to gain new insights on ENS development and specification, proving that the zebrafish is a valuable model organism in the quest towards understanding and treating congenital enteric neuropathies.