Single Cell Profiling in the Sox10Dom/+ Hirschsprung Mouse Implicates Hoxa6 in Enteric Neuron Lineage Allocation

Abstract

Background & Aims Enteric nervous system (ENS) development requires migration, proliferation, and appropriate neuronal diversification from progenitors to enable normal gastrointestinal (GI) motility. Sox10 deficit causes aganglionosis, modeling Hirschsprung disease, and disrupts ratios of postnatal enteric neurons in proximal ganglionated bowel. How Sox10 deficiency alters ratios of enteric neuron subtypes is unclear. Sox10's prominent expression in enteric neural crest-derived progenitors (ENCP) and lack of this gene in enteric neurons led us to examine Sox10^Dom effects on ENS progenitors and early differentiating enteric neurons. Methods ENS progenitors, developing neurons, and enteric glia were isolated from Sox10+/+ and Sox10^Dom/+ 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 Hox gene regulatory networks (GRN) as progenitors transition to neuronal fates. Sox10^Dom/+ scRNA-seq profiles exhibited a novel progenitor cluster, decreased abundance of cells in transitional states, and shifts in cell distributions between two neuronal trajectories. Hoxa6 was differentially expressed in the neuronal lineages impacted in Sox10^Dom/+ mutants and HCR identified altered Hoxa6 expression in early developing neurons of Sox10^Dom/+ ENS. Conclusions Sox10^Dom/+ mutation shifts enteric neuron types by altering neuronal trajectories during early ENS lineage segregation. Multiple neurogenic transcription factors are reduced in Sox10^Dom/+ scRNA-seq profiles including multiple Hox genes. This is the first report that implicates Hox genes in lineage diversification of enteric neurons.