Interindividual Variation in Gut Nitrergic Neuron Density Is Regulated By GDNF Levels and ETV1

Background & Aims: The size and function of the enteric nervous system (ENS) can vary substantially between individuals. Because ENS function is involved in the etiology of a growing number of common human diseases, understanding mechanisms that regulate ENS variation is important. Methods: We analyzed RNAseq data from 41 normal adult human colon biopsies and single-cell RNA-seq data from human and mouse developing gut. To establish cause-consequence relationship we used alleles in mice that allow levels change of the candidate effector molecule in the comparable range to human samples. We used siRNA and primary neuronal cultures to define downstream molecular events and characterized gut functional changes in mice where molecular phenotypes paralleled findings in humans. Results: We found that glial cell line–derived neurotrophic factor (GDNF) levels in the human colon vary about 5-fold and correlate strongly with nitrergic marker expression. In mice, we defined that GDNF levels are regulated via its 3’ untranslated region (3’ UTR) in the gastrointestinal tract and observed similar correlation between GDNF levels and nitrergic lineage development. We identified miR-9 and miR-133 as evolutionarily conserved candidates for negative regulation of GDNF expression in the gastrointestinal tract. Functionally, an increase in inhibitory nitrergic innervation results in an increase in gastrointestinal tract transit time, stool size, and water content accompanied with modestly reduced epithelial barrier function. Mechanistically, we found that GDNF levels regulate nitrergic lineage development via induction of transcription factor ETV1, corroborated by single-cell gene expression data in human and mouse developing enteric neurons. Conclusions: Our results reveal how normal variation in GDNF levels influence ENS size, composition, and gut function, suggesting a mechanism for well-known interindividual variation among those parameters.