Msi is up-regulated during activation of reserve ISCs and is required for lineage tracing from these cells under basal conditions. (A) Representative immunofluorescence micrographs of Hopx-CreER reserve ISCs and their progeny in Hopx-CreER::LSL-tdTomato mice 18 h (top) and 4 d (bottom) after a single tamoxifen injection. TdTomato (red), E-cadherin (white), and DAPI (blue). Bar, 50 µM. (B) Violin plots showing expression levels of Msi1, Msi2, Lgr5, Ascl2, Cdkn1a, and Ccnd1 in single, FACS-purified Bmi1- and Hopx-CreER ISCs, their daughter cells (progeny), and Lgr5-eGFP⁺ CBCs. Bmi1- and Hopx-CreER⁺ ISCs were marked by a single dose of tamoxifen in Bmi1- or Hopx-CreER::LSL-tdTomato mice and were purified by FACS 18 h later. Hopx-CreER progeny were isolated by FACS purification of tdTomato⁺ cells 96 h after activation of the tdTomato reporter. The width of the violin plot represents the number of single cells at the given expression level on the y-axis. The white dot within the violin plot represents the mean expression value for the group of cells. (C) Immunofluorescence staining for tdTomato (red) and E-cadherin (white) and quantification of lineage tracing events (ribbons of tdTomato⁺ cells with contiguous tracing from crypts, through crypt–villus junction, and into villi) from Bmi1- and Hopx-CreER ISCs 14 d after marking reserve ISCs with a single tamoxifen injection to Hopx-CreER::LSL-tdTomato::Msi1/2^flx/flx or Bmi1-CreER::LSL-tdTomato::Msi1/2^flx/flx mice and their control counterparts (n = 3). Bar, 50 µM. Arrowheads point to single MsiDKO cells that are not able to contribute in lineage tracing. All data are expressed as mean ± SD. *, P < 0.05; ***, P < 0.0005, Student’s t test.