Diversification of molecularly defined myenteric neuron classes revealed by single cell RNA-sequencing

Abstract

Autonomous functions of the gastrointestinal tract require the combined activity of functionally distinct neurons of the enteric nervous system (ENS). However, the range of enteric neuron diversity and how it emerges during development remain largely unknown.We here make a novel molecular definition of 12 enteric neuron classes (ENCs) within the myenteric plexus of the mouse small intestine. We identify communication features and provide histochemical markers for discrete motor, sensory, and interneurons together with genetic tools for class-specific targeting. Transcriptome analysis of embryonic ENS reveals a largely post-mitotic principle of diversification, where only ENC1 or ENC8 phenotypic traits arise through a binary neurogenic trajectory, and other identities form through subsequent differentiation. We propose generic and class-specific transcriptional regulators and functionally connect the transcription factor Pbx3 to one post-mitotic identity conversion.Our results offers a conceptual and molecular resource for dissecting ENS circuits, and predicting key regulators for the directed differentiation of distinct enteric neuron classes.

Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypesin situ

“…Analysis of IPANs is of critical interest given the prominent role played by this subtype in the ENS. Our findings confirm the existence of multiple IPAN subtypes previously indicated for mice 6,10 and expand the marker toolkit available to localize While stem cell transplantation studies have successfully generated ENs that integrate into aganglionic bowel 21,22 , it has not been possible to assess whether integrated cells accurately reflected adult EN subtypes. Our profiling of adult EN provides the first benchmark resource of its kind that can be used to assess whether EN generated by directed differentiation mimic their in vivo counterparts.…”

“…Among rodents, Nmu is known to label guinea pig IPANs and a recent study has confirmed this in mice based on co-localization with the established IPAN marker Calb2 6,10 . Our finding of Nmu in mouse IPANs is consistent with these studies, although to our knowledge NMU has been untested in the human ENS.…”

“…Type II ENs in mouse cluster 5 (Nmu+) and in humans appear to both share similar morphology 1,6 and selective marker genes. However, mouse clusters 6 and 7 were not morphologically conserved 1,6,10 , despite sharing similar markers genes across species (KLHL1, KCNH7, NXPH2, and CDH9).…”

“…Recent advances have been made in profiling pools of EN and glia using bulk sequencing from fetal mice 8 , when neuronal processes are less-developed and cells survive tissue dissociation. Most recently, single-cell gene expression profiles of ~1100 ENs have been generated from postnatal mice around weaning 9,10 . Because neuronal differentiation is still ongoing at these stages, such profiles are unlikely to fully capture adult EN gene diversity 11 .…”

Age-associated changes in lineage composition of the enteric nervous system regulate gut health and disease

“…Prior scRNAseq analysis of postnatal ENS did not detect and characterize the true identity of the MEN lineage. This is because previous studies were performed either exclusively on neural crest-derived ENS cells (Zeisel et al, 2018), or when done in a more agnostic manner, only applied known canonical neural markers to identify the ENS population (Drokhlyansky et al, 2019;Morarach et al, 2020). In either case, the discovery of the distinct identity and derivation of MENs would not have been possible.…”