Selective modulation of murine intestinal M1 and M3 muscarinic receptor expression has divergent effects on specialized epithelial cells and body weight

M1 and M3 muscarinic receptors encoded by CHRM1 and CHRM3 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 if 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 Chrm1 and Chrm3 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 to littermate controls. scRNA-Seq analysis of tissues from M1R and M3R floxed mice revealed robust Chrm1 and Chrm3 expression by enteric goblet cells. CKO mice with selective mucosal depletion of Chrm1 and Chrm3 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. Overall design: C57BL/6 mice with selective knockdown of two different muscarinic receptor subtypes were used for the generation of organoids. Control mice were used for the generation of single cell suspensions. Additional analysis methods to validate the models and analyze cell subtype differences included included qPCR, immunohistochemistry, and single cell RNA sequencing