Single-molecule transcript counting of stem-cell markers in the mouse intestine

Determining the molecular identities of adult stem cells requires technologies for sensitive transcript detection in tissues. In mouse intestinal crypts, lineage-tracing studies indicated that different genes uniquely mark spatially distinct stem-cell populations, residing either at crypt bases or at position +4, but adetailed analysis of their spatial co-expression has not been feasible. Here we apply three-colour single-molecule fluorescence in situhy bridization to study a comprehensive panel of intestinal stem-cell markers during homeostasis, ageing and regeneration.We find that the expression of all markers overlaps at crypt-base cells. This co-expression includes Lgr5, Bmi1 and mTert, genes previously suggested to mark distinct stem cells. Strikingly, Dcamkl1 tuft cells, distributed through out the cryptaxis, co-express Lgr5 and other stem-cell markers that are otherwise confined to crypt bases. We also detect significant changes in the expression of some of the markers following irradiation, indicating their potentialrole in the regeneration process. Our approach can enable the sensitive detection of putative stem cells in other tissues and intumours, guiding complementary function alstudies to evaluate their stem-cell properties.