Einleitung The enteric nervous system, an essential component of the intestinal stem cell (ISC) niche, regulates epithelial homeostasis with its main transmitter acetylcholine (Ach) predominantly via G-protein coupled receptors M3R and M1R. Epithelial-specific ablation of M3R in the murine small intestine results in a significant reduction of Lgr5+ ISC activity and the selective expansion of Dclk1+ tuft cells.
Ziele Since regional differences in ISC and tuft cells have been described, we aimed to investigate the effect of M3R ablation on colonic ISC and epithelial differentiation.
Methodik Immunohistochemical analysis of tissues from Vil-Cre x M3 fl/fl mice compared to WT mice was performed by staining for markers such as DCLK1, ChgA and p-EGFR. Further, we performed RT-PCR analysis of mucosal isolates of Vil-Cre x M3R fl/fl mice compared to WT mice employing markers for ISC activity and regulation (Lgr5, Ascl2, Olfm4, Prom1, Cdca7, Bmi1) and genes encoding EGF receptors (Egfr, Erbb2, Erbb3). Further experiments will include Western Blot, ELISA and in vitro organoids.
Ergebnis Immunohistochemical analysis of small intestinal tissues of Vil-Cre x M3R fl/fl mice confirmed the significant expansion of DCLK1+ tuft cells. Unexpectedly, the number of DCLK1+ cells in colonic tissues decreased. In contrast, ChgA+ cells showed a similarly decreased cell count in small intestinal and colonic tissues. Simultaneously, we observed a significant increase of p-EGFR+ cells in colonic tissues. RT-PCR analysis revealed significant decreases of Lgr5 and Olfm4 in small intestinal isolates, while colonic isolates showed strong reduction in Lgr5 and Ascl2. Genes encoding EGF receptors did not show differences in colonic isolates, yet colonic crypt depth was significantly decreased.
Schlussfolgerung Colonic epithelial ablation of M3R appears to confer similarly evident modulations to epithelial ISC behavior as previously observed in the jejunum. Yet, the compensatory mechanisms appear to differ as we observed the significant expansion of p-EGFR+, and not Dclk1+, epithelial cells in colonic Vil-Cre x M3R fl/fl isolates. These observations reveal significant regional differences in intestinal ISC differentiation and support the importance of the cholinergic niche in ISC modulation.
