Identification of Subpopulations and Molecular Signatures of Enteric Neural Crest via Single Cell Transcriptomics

Abstract

The enteric nervous system (ENS) is crucial for digestion and gut homeostasis; it is formed by neural crest (NC)-derived progenitors during embryogenesis. Failed gut colonization by enteric crest cells (ENCCs) leads to severe congenital disorder named Hirschsprung disease (HSCR). A correct composition of ENCC-derived cell subpopulations decides the colonizing success. Here we show the molecular and population dynamics of mouse ENCC progenitors during ENS development. Colonization by ENCCs in the gut takes place in a limited time frame during embryogenesis, therefore the progenitor pool size, competency of sensing migration cue, and the degree of spatio-temporal differentiation must be tightly regulated by the gene regulatory network (GRN), which may undergo disrupted in HSCR. Single cell analysis of molecular signatures provides us the resolution to address the importance of lineage composition in ENS development and disease. By crossing R26tdTomato reporter with Wnt1-Cre mouse line, which specifically labels neural crest descendants, we generated single cell RNA library from 469 ENCC derivatives, covering development stages when ENCCs reach the foregut (E9.5), the proximal colon (E12.5), and the distal colon (E14.5). Bioinformatic analyses including clustering, t-SNE visualization, and public datasets of related tissues cross-comparison indicated the cell heterogeneity increases greatly with development progression. Based on the signature genes, we preliminarily identified 7 cell subpopulations belonging to different developmental stages. With unbiased algorithm, we identified around 200 to 300 of genes unique to each subpopulation of cells. Interestingly, as some recent studies suggest the pivotal role of Vitamin A and retinoids in ENS development, we found differential enrichment of retinoic acid (RA) metabolism-related genes in respect to different subpopulations, further implying the importance of RA signaling in regulating ENCC migration and differentiation during ENS genesis. To sum up, with single-cell transcriptomics, we delineated the molecular signatures of 469 NCC-derivatives belonging to 7 major subpopulations; and our data suggest RA signaling is essential during migration, lineage maintenance and commitment for normal ENS development.