Abstract
About 10-30% of people living in Western countries experience some form of gut dysmotility including constipation and diarrhea, which have been tied to disorders such as irritable bowel syndrome (IBS) and Hirschsprung disease1,2. Previous research showed that knocking out or inhibiting the RET receptor in mouse enteroendocrine cells (EECs), the hormone-secreting cells of the intestinal epithelium, leads to an increase in the release of neuropeptides PYY and GLP-1 in adult male mice, resulting in slowed gut motility. The RET receptor is activated by four co-receptors, the GFRa receptors, but it is unknown which ones are involved in the mechanism that regulates PYY and GLP-1 release. I hypothesized that GFRa3, which demonstrates the highest mRNA expression in mouse EECs, is responsible for the majority of RET signaling in EECs and would show a high level of co-expression in mature RET+ EECs. To determine this, I characterized the mRNA and protein expression of GFRa receptors in EECs along the murine GI tract. I stained for GFRa3 expression in the epithelium of both the mature EEC ChgA-GFP+ reporter mice and Ret-GFP+ reporter mice. Contrary to expectations, only about 29% of colonic ChgA+ EECs in male mice expressed GFRa3, while only 8.8% in female mice expressed GFRa3, with even lower percentages observed in the duodenum. GFRa3 co-expression with RET+ EECs was also relatively low. For all regions of the GI tract, excluding the duodenum, a range of only ~17-27% of Ret+ cells co-expressed GFRa3. I also re-analyzed publicly available single-cell RNA sequencing datasets including EECs from the small intestine and colon and determined which subtypes expressed Gfra1, Gfra2, Gfra3, Gfra4 and/or Ret and saw little overlap between Ret and any Gfra co-receptor. The low level of overlap indicates that intracellular GFRa3-RET signaling may not fully account for increased GLP-1 and PYY release observed after blocking all RET signaling in EECs. Further studies must be done on how RET signaling occurs in EECs, which could lead to additional drug targets to treat gut dysmotility.