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, or differentiation of enteric neural crest-derived cells (ENCDCs), with pathogenic variants at the RET locus accounting for a major fraction of cases. While coding variants in RET are known to impair enteric nervous system (ENS) development, the contribution of non-coding regulatory elements remains incompletely understood. The greatest contribution of this risk is from a common variant (rs2435357) in an ENS-active, SOX10-bound RET enhancer (MCS+9.7) that reduces RET expression in vivo and triggers expression changes in other ENS genes in the human fetal gut. Here, we investigate the role of this enhancer during ENS development using a targeted mouse model by deleting mcs9.7. Single-cell RNA-seq profiling of wildtype and homozygous delmcs9.7/ delmcs9.7 embryos revealed an ~8% decrease in RET expression within ENS 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 also generated a compound heterozygous mouse (+/delmcs9.7/;+/CFP) that showed additive reductions in Ret expression in these same cell types and bidirectional feedback between Ret and its upstream transcription factor Sox10. We observed reduced expression of 19 cell cycle genes and significant loss of nNOS+ and VIP+ neurons, consistent with decreased inhibitory neuron network driven by loss of proliferation, a hallmark of HSCR. These findings establish a biphasic, cell-type-specific role for the mcs9.7 enhancer in modulating Ret dosage and reveal 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.