Figure 1.

Validation of Lgr5+ stem cell loss in dextran sulfate sodium (DSS) colitis. Compared with uninjured water-treated mice, those treated with 3% DSS for 6 days exhibit body weight loss (A), thickening of the colon (B), and histological damage as scored by a pathologist (C) and demonstrated in hematoxylin-eosin (H&E) stained images (D) (n = 5 male mice per group). In situ hybridization (ISH) with probes targeted to stem cell marker genes Lgr5 (E), Ephb3 (F), and Lrig1 (G) shows localization of expression to the crypt base during homeostasis. However, after DSS treatment, the expression of these markers appears greatly reduced, in both atrophic regions (“affected”) and in regions of the distal colon that appear histologically normal (“unaffected”). ISH with probes targeted to the interferon-responsive gene Ly6a (H) shows the highest levels of expression in the affected region. A counterstain with Cdh1 is provided because Ly6a expression is not exclusive to the epithelium. I: densitometry of the ISH stainings in the epithelial layer of colonic crypts quantifies their epithelial-specific expression patterns. Ten contiguous distal colonic crypts were chosen for image quantification. Lgr5, Ephb3, and Lrig1 exhibit near-total loss of expression, whereas Lrig1 shows strong epithelial upregulation. J: the density of ISH-recorded Lgr5 staining depended on the dose of DSS given (n = 5 mice per dose). At lower doses (e.g., 0.5% vs. 3% DSS), many Lgr5+ stem cells persisted. Scale bars: 50 µm. Statistics: *P < 0.05; **P < 0.01; ***P < 0.001. Two-tailed t test. Error bars: means ± SE.