Intestinal epithelial cells comprise diverse cells with specific functions, e.g., chemosensory tuft cells are important for anti-helminthic and -protozoan immunity and mucus secretion by goblet cells provides mucosal protection. Cholinergic signaling via M₁ and M₃ muscarinic receptors (M₁R and M₃R) modulates these functions; global M₃R deficiency reduces cell proliferation and mucus production and increases T_H1/T_H17 cytokines. Intestinal epithelial cell-specific ablation of M₁R and M₃R in mice reportedly expands the jejunal tuft cell population, but little is known about the effects of M₁R and M₃R deficiency on epithelial cell distribution in different regions of the small intestine and colon. Aim: To measure the effects of selective intestinal epithelial cell depletion of M₁R and M₃R on the numbers of specialized epithelial cells in different regions of the small intestine and colon. Methods: Using the villin-Cre approach, we created conditional knockout (CKO) mice (C56BL/6 genetic background) with intestinal epithelial cell-selective deficiency of M₁R and M₃R. With a combination of anti-DCAMKL1 antibody, Alcian blue-periodic acid Schiff, hematoxylin & eosin, and chromogranin-A staining, we measured the relative numbers of tuft, goblet, Paneth, and enteroendocrine cells along the intestinal axis from the pylorus to the anus of 9- to 12-week-old mice. We compared results in tissues from M₁R and M₃R CKO mice to those from littermate controls. Results: M₁R and M₃R CKO mice were viable and fertile. RT-PCR of full thickness and mucosal extracts of the small intestine and colon confirmed mucosal deletion of M₁R and M₃R mRNA in CKO but not control mice; brain, stomach, and liver M₁R and M₃R mRNA levels were the same in CKO and control mice. Age/sex-matched M₁R and M₃R CKO mice were grossly indistinguishable from controls. Interestingly, compared to littermate controls, we detected twice as many tuft cells per villus in the middle third of M₃R CKO mouse small intestine (2.9±0.3 vs 1.5±0.2 tuft cells/villus; p<0.005; Fig. 1A). Also, in M₃R CKO vs control mice, we found fewer goblet cells in the proximal third of the small intestine (2.9±0.6 vs 4.9±0.9; Fig. 2A) and in distal colon crypts (10.9±0.8 vs 12.3±1.1) - both p<0.05. We observed no differences in either Paneth or enteroendocrine cell numbers, or in cell counts comparing M₁R CKO and littermate control mice. Conclusions: Our data highlight the importance of assessing cellular diversity regionally throughout the small and large intestine - we were surprised to find that M₃R deletion resulted in intestinal region-dependent alterations in tuft and goblet cell numbers, whereas M₁R deletion had no impact on these features. Further research is needed to elucidate the mechanisms whereby changes in M₃R expression alter epithelial cell composition along the GI axis and how this impacts intestinal function.

<b>Figure 1. Increased numbers of tuft cells in the middle third of the small intestine in M<sub>3</sub>R CKO mice. A.</b> Comparison of the numbers of tuft cells per villus-crypt unit in the middle third of the small intestine of M<sub>1</sub>R and M<sub>3</sub>R CKO compared to littermate control mice. Bars represent mean ± SEM; symbols represent individual data points. <b>B.</b> Image showing representative tuft cells (arrows) stained with anti-DCAMKL1 antibody (red). Green background staining is for the cell adhesion protein e-cadherin.

Figure 1. Increased numbers of tuft cells in the middle third of the small intestine in M₃R CKO mice. A. Comparison of the numbers of tuft cells per villus-crypt unit in the middle third of the small intestine of M₁R and M₃R CKO compared to littermate control mice. Bars represent mean ± SEM; symbols represent individual data points. B. Image showing representative tuft cells (arrows) stained with anti-DCAMKL1 antibody (red). Green background staining is for the cell adhesion protein e-cadherin.

<b>Figure 2. Reduced numbers of goblet cells in the proximal third of the small intestine in M<sub>3</sub>R CKO mice. A.</b> Comparison of the numbers of goblet cells per villus in the proximal third of the small intestine of M<sub>1</sub>R and M<sub>3</sub>R CKO compared to littermate control mice. Bars represent mean ± SEM; symbols represent individual data points. <b>B.</b> Image showing representative goblet cells (arrows) stained with Alcian blue-periodic acid Schiff.

Figure 2. Reduced numbers of goblet cells in the proximal third of the small intestine in M₃R CKO mice. A. Comparison of the numbers of goblet cells per villus in the proximal third of the small intestine of M₁R and M₃R CKO compared to littermate control mice. Bars represent mean ± SEM; symbols represent individual data points. B. Image showing representative goblet cells (arrows) stained with Alcian blue-periodic acid Schiff.

Benjamin Raufman

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