Synergistic effects of coding and enhancer loss-of-function alleles cause progressive loss of inhibitory motor neurons in the enteric nervous system.

Coding and enhancer variants of the receptor tyrosine kinase gene contribute to ~50% of Hirschsprung disease (HSCR) risk, a congenital disorder of disrupted enteric nervous system (ENS) development. The greatest contribution of this risk is from a common variant (rs2435357) in an ENS-active, SOX10-bound enhancer (MCS+9.7) that reduces gene expression and triggers expression changes in other ENS genes in the human fetal gut. To uncover the cellular basis of -mediated aganglionosis, we used CRISPR/Cas9 to delete (Δ) the homologous mouse enhancer (mcs+9.7). We used single cell RNA sequencing and high-resolution immunofluorescence to demonstrate four significant features of the developing E14.5 gut of Δmcs+9.7/Δmcs+9.7 embryos: (1) a small (5%) yet significant reduction in gene expression in only two major cell types - early differentiating neurons and fate-restricted inhibitory motor neurons; (2) no significant cellular loss in the ENS; and, (3) loss of expression of 19 cell cycle regulator genes suggesting a proliferative defect. To identify the functional threshold for normal ENS development, we also generated, in combination with the CFP null allele, (4) Δmcs+9.7/CFP double heterozygote mice which reduced gene expression in the ENS to 42% with severe loss of inhibitory motor neurons, an effect restricted to the hindgut and driven by proliferative loss. Thus, gene expression drives proliferation of ENS progenitor cells and hindgut-specific inhibitory motor neuron development, and that HSCR aganglionosis arises from a cascade of cellular defects triggered by >50% loss of function.