RET Signaling Persists in the Adult Intestine and Stimulates Motility by Limiting PYY Release from Enteroendocrine Cells

Abstract

Background & Aims: RET receptor tyrosine kinase is necessary for enteric nervous system (ENS) development. Loss-of-function RET mutations cause Hirschsprung disease (HSCR), in which infants are born with aganglionic bowel. Despite surgical correction, HSCR patients often experience chronic defecatory dysfunction and enterocolitis, suggesting that RET is important after development. To test this hypothesis, we determined the location of postnatal RET and its significance in gastrointestinal (GI) motility. Methods: RetCFP/+ mice and human transcriptional profiling data were studied to identify the enteric neuronal and epithelial cells that express RET. To determine whether RET signaling in these cells regulates adult gut motility in vivo, genetic and pharmacologic approaches were used to disrupt RET in either all RET-expressing cells, a major subset of enteric neurons, or intestinal epithelial cells. Results: Distinct subsets of enteric neurons and enteroendocrine cells expressed RET in the adult intestine. RET disruption in the intestinal epithelium, rather than in enteric neurons, slowed GI motility selectively in adult male mice. This effect was phenocopied by RET kinase inhibition. Most RET+ epithelial cells were either enterochromaffin cells that release serotonin (5-HT) or L-cells that release peptide YY (PYY), both of which can alter motility. RET kinase inhibition exaggerated PYY release in a nutrient-dependent manner without altering 5-HT secretion. PYY receptor blockade fully rescued dysmotility in mice lacking epithelial RET. Conclusion: RET signaling normally limits nutrient-dependent PYY release from L-cells and this activity is necessary for normal intestinal motility in male mice. These effects could contribute to post-operative dysmotility in HSCR, which predominantly affects males, and uncovers a mechanism that could be targeted to treat post-prandial GI dysfunction.

A New Transgenic Tool to Study the Ret Signaling Pathway in the Enteric Nervous System

“…Female and male guts had a comparable distribution of GFP + ENS neurons suggesting that there is no sex difference connected to the expression of ret in the adult. This is consistent with reports that found no difference in Ret expression in ENS neurons in adult female or male mice [39].…”

“…From larval stages and in adulthood, we find a small population of GFP -ENS neurons along the gut length. This is consistent with what has been observed in adult human and mouse ENS, which also contain Ret + and Retpopulations of ENS neurons in the adult [22,39,40,46]. Interestingly, the percentage of GFP -ENS neurons increases in the adult gut compared to larval stages suggesting that in the adult Ret-independent ENS neurogenesis is more prevalent than in larval zebrafish.…”

“…In mice and human, single-cell RNA-sequencing analysis found that Ret is expressed in most ENS neurons in juveniles and adults [22,[38][39][40]. Accordingly, a ret:EGFP transgenic mouse line showed EGFP expression in differentiating neurons [21].…”

“…In mammals, Ret continues to be expressed in a population of ENS neurons in the adult and contributes to synapse formation and neuronal plasticity by regulating neurite outgrowth [38,39,[42][43][44][45]. Recently, it has been shown that ENS neurogenesis continues into adulthood in zebrafish [31], but which signals regulate neuronal development at that stage remains unknown.…”

A New Transgenic Tool to Study the Ret Signaling Pathway in the Enteric Nervous System

“…In mice and human, single-cell RNA-sequencing analysis found that Ret is expressed in most ENS neurons in juveniles and adults [ 22 , 42 , 43 , 44 ]. Accordingly, a ret:EGFP transgenic mouse line showed EGFP expression in differentiating neurons [ 21 ].…”