Microdissection and Visualization of Individual Hair Follicles for Lineage Tracing Studies

Abstract

Acetylcholine (ACh) is a neurotransmitter that is present in central, parasympathetic, and neuromuscular synapses of mammals. However, non-neuronal ACh is also predicted to function as a local cell signaling molecule. The physiological significance of the presence of non-neuronal ACh in the intestine remains unclear. Here, experiments using cultured crypt–villus organoids that lack nerve and immune cells led us to suggest that endogenous ACh is synthesized in the intestinal epithelium to evoke growth and differentia- tion of the organoids through activation of muscarinic ACh receptors (mAChRs). Extracts of cultured organoids exhibited a noticeable capacity for ACh synthesis that was sensitive to a potent inhibitor of choline acetyltransferase. Treatment of organoids with carbachol downregulated growth of organoids and expression of marker genes for each epithelial cell type. On the other hand, mAChR antagonists enhanced growth and differentiation of Lgr5-positive stem cells. Collectively, our data provide evidence that endoge- nous ACh released from mouse intestinal epithelium maintains the homeostasis of intestinal epithelial cell growth and differentiation via mAChRs.