Single Cell RNA-sequencing analysis of the juvenile and postnatal mouse enteric nervous system of the small intestine

The study aimed to achieve a molecular definition of submucosal neuron classes in the mouse small intestine and describe their differentiation from enteric stem cells during postnatal stages. Method: We performed single cell RNA-sequencing of enteric neurons from small intestine at the postnatal day (P)24; (Baf53b-CrexR26RtdTom mice) and ENS cells from the small intestine at P7 (Wnt1-CrexR26RtdTom). Results: 3 unique cardinal submucosal enteric neuron classes (smENC1-3) were identified and confirmed in native tissue by histochemical methods and class-specific Cre mice mated with reporter mice. Analysis of postnatal transcriptomes revealed that enteric stem cells differentiate into two neuronal branches, that successively diversify into smENC3 (Branch A) or smENC1 and smENC2 (Branch B). Conclusion: Utilizing single cell RNA-sequencing we conclude that murine small intestine consists of 3 cardinal submucosal classes that arise from binary neurogenic branching and postmitotic identity conversions. Overall design: We isolated single submucosal neurons from pan-neuronal Baf53bCre;R26R-Tomato mice (P24) and single ENS cells (including enteric stem cells, glia and neurons) from Wnt1-Cre;R26RtdTom postnatal animals (P7) using flow cytometry and obtained transcriptomes by single cell RNA-sequencing (10xGenomics).