Single Cell Profiling in the Hirschsprung Mouse Implicates in Enteric Neuron Lineage Allocation.

BACKGROUND & AIMS: Enteric nervous system (ENS) development requires migration, proliferation, and appropriate neuronal diversification from progenitors to enable normal gastrointestinal (GI) motility. deficit causes aganglionosis, modeling Hirschsprung disease, and disrupts ratios of postnatal enteric neurons in proximal ganglionated bowel. How deficiency alters ratios of enteric neuron subtypes is unclear. prominent expression in enteric neural crest-derived progenitors (ENCP) and lack of this gene in enteric neurons led us to examine effects ENS progenitors and early differentiating enteric neurons.

METHODS

ENS progenitors, developing neurons, and enteric glia were isolated from and littermates for single-cell RNA sequencing (scRNA-seq). scRNA-seq data was processed to identify cell type-specific markers, differentially expressed genes, cell fate trajectories, and gene regulatory network activity between genotypes. Hybridization chain reaction (HCR) validated expression changes detected in scRNA-seq.

RESULTS

scRNA-seq profiles revealed three neuronal lineages emerging from cycling progenitors via two transition pathways accompanied by elevated activity of gene regulatory networks (GRN) as progenitors transition to neuronal fates. scRNA-seq profiles exhibited a novel progenitor cluster, decreased abundance of cells in transitional states, and shifts in cell distributions between two neuronal trajectories. was differentially expressed in the neuronal lineages impacted in mutants and HCR identified altered expression in early developing neurons of ENS.

CONCLUSIONS

mutation shifts enteric neuron types by altering neuronal trajectories during early ENS lineage segregation. Multiple neurogenic transcription factors are reduced in scRNA-seq profiles including multiple genes. This is the first report that implicates genes in lineage diversification of enteric neurons.