Selective modulation of murine intestinal M ₁ and M ₃ muscarinic receptor expression has divergent effects on specialized epithelial cells and body weight

M₁ and M₃ muscarinic receptors encoded by <i>CHRM1</i> and <i>CHRM3</i> mediate neuronal and non-neuronal cholinergic signaling. Mice with global M₃R deficiency reportedly weigh less than controls, but the cell type(s) involved are unknown. As the intestinal epithelium modulates nutrient absorption, we asked whether deleting M₁R and M₃R only from intestinal epithelial cells would alter the distribution of specialized small intestinal epithelial cells or body weight. We reviewed reports of global M₁R and M₃R deficiency and body weight, used single-cell RNA sequencing (scRNA-Seq) to assess <i>Chrm1</i> and <i>Chrm3</i> expression by small intestinal epithelial cells, created mice with conditional intestinal epithelial cell M₁R and M₃R deletion (CKO mice), and compared the distribution of specialized intestinal epithelial cells and body weights of CKO and control mice, and the development of enteroids. Prior weight comparisons commonly used only male mice, frequently without comparison with littermate controls. scRNA-Seq analysis of tissues from M₁R and M₃R floxed mice revealed robust <i>Chrm1</i> and <i>Chrm3</i> expression by enteric goblet cells. CKO mice with selective mucosal depletion of <i>Chrm1</i> and <i>Chrm3</i> RNA were viable, fertile, and had fewer small intestinal goblet cells than controls. M₃R CKO mice had more tuft cells than controls. Although female mice weighed ∼20% less than males, we detected no weight differences between M₁R and M₃R CKO and control mice; enteroids derived from these mice developed at the same pace. Intestinal epithelial cell M₁R and M₃R deficiency impacts the distribution of specialized intestinal epithelial cells but not murine body weight.<b>NEW & NOTEWORTHY</b> Small intestinal goblet cells robustly express both <i>Chrm1</i> and <i>Chrm3</i>; mucosal immune cells express primarily <i>Chrm3</i>. Mice with intestinal epithelial cell (IEC) <i>Chrm3</i>/M₃R deletion have fewer small intestinal goblet cells but more tuft cells. The increase in tuft cells, which produce acetylcholine, may represent a feedback mechanism to compensate for reduced muscarinic receptor (MR) expression. IEC-selective deletion of MRs does not impact murine weight or enteroid development, suggesting that MRs beyond the intestinal epithelium regulate weight.