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

Hirschsprung disease (HSCR) is a congenital enteric neuropathy caused by disrupted migration, proliferation, and/or differentiation of enteric neural crest-derived cells (ENCDCs), with pathogenic variants at RET accounting for a major fraction of cases. While coding variants in RET are known to impair enteric nervous system (ENS) development, the causal effects of pathogenic regulatory variants remain poorly understood. The largest contribution to HSCR risk is from a common variant (rs2435357) in an ENS-active, SOX10-bound RET enhancer (MCS+9.7) that reduces RET gene expression in vivo and triggers expression changes in other ENS genes in the human fetal gut. Here, we investigate the role of this enhancer in ENS development using a targeted mouse model by deleting this enhancer ({Delta}mcs9.7). Single-cell RNA-seq profiling of wildtype and {Delta}mcs9.7/ {Delta}mcs9.7 homozygote embryos at the developmental stage E14.5 revealed an [~]8% decrease in Ret expression within ENS cell clusters, with no gross shifts in ENS cell type composition. Ret expression loss was limited to early differentiating neurons and inhibitory motor neurons, indicating cell type-specific enhancer control. To identify the Ret functional threshold for normal ENS development, we next generated a compound heterozygous mouse (+/{Delta}mcs9.7; +/CFP), heterozygous null for both the enhancer and coding (CFP) alleles, that showed additive reductions in Ret expression in these same cell types and bidirectional feedback between Ret and its transcription factor Sox10. We observed reduced expression of multiple cell cycle genes and significant loss of nNOS+ and VIP+ neurons in the E14.5 gut, consistent with decreased inhibitory neuron network function driven by loss of proliferation, the hallmark of HSCR. These findings established a cell type-specific role for the mcs9.7 enhancer in modulating Ret dosage and revealed how subtle enhancer perturbations alter neural subtype specification without overt aganglionosis. Together, our work uncovers the functional consequences of a common RET enhancer variant offering new insights into gene regulatory mechanisms driving HSCR.