In our study, we investigated the effect of muscarinic receptor blockade on murine intestinal stem cell activity and differentiation. Following pharmacologic (scopolamine) and genetic muscarinic receptor interruption (M3R-KO, M1R-KO; Vil-Cre x M3R fl/fl, Vil-Cre x M1R fl/fl mice, respectively), we observe a selective, significant expansion of DCLK1-positive tuft cells. This is primarily sensed by endocrine Prox1-positive cells (Prox1-CreERT2 x M3R fl/fl mice), while Lgr5-positive ISCs respond with a reduction of their cellular activity (Lgr5-EGFP-IRES-CreERT2 x M3R fl/fl mice). To characterize expanding tuft cells in more detail, adult BAC transgenic DCLK1-DTR-ZSgreen reporter mice generated in our lab were treated with scopolamine or sodium chloride as controls (Sham) for 7d and live jejunal EPC-positive/ZSgreen-positive cells sorted. Extracted total RNA from sorted ZSgreen-positive cells of scopolamine- and sham-treated mice was sequenced. Further analysis revealed that expanding tuft cells orchestrate an increase in mucosal acetylcholine, which maintained intracellular signaling pathways such as p-ERK/ERK 1/2 or TCF-1/7. Finally, acute irradiation injury was employed to investigate the importance of this regulatory circuit for tissue homeostasis. Indeed, tissue injury in Vil-Cre x M3R fl/fl mice resulted in a severe reduction of epithelial DCLK1-positive tuft cells, concomitant to reduced mucosal Ach levels as well as intracellular PI3K-/p-ERK levels. Therefore, DCLK1-positive tuft cells appear essential for the maintenance of a regular intestinal cholinergic niche.

Chrm3, M3R, Trpm5, Pou2f3, Dclk1, Nkx6-3, Qpct, Abca2, Lmx1a, Nr4a1, Arc, Prss12