Fig. 1. Hopx labels ISCs at the +4 position in intestinal epithelial crypts.

(A) Whole mount X-gal stained small intestine from a Hopx^LacZ/+ mouse. (B) Double staining of Hopx^LacZ/+ intestine for LacZ and BrdU using a post-irradiation regeneration labeling protocol. (C) Quantification of BrdU positive cells at each crypt position. (D) Composite image (9 images combined into 1) of X-gal stained Hopx^ERCre/+;R26^LacZ/+ small intestine 2 months after a 5 day tamoxifen pulse. (E and F) X-gal staining 13 months after tamoxifen induction; whole-mount image (E) and eosin counter-stained section (F). (G) Immunohistochemical GFP staining in crypts of Hopx^ERCre/+;R26^mT-mG/+ mice 18h after tamoxifen induction. GFP-positive cells are at the +4 position. (H) Time course of Hopx lineage tracing (Hopx^ERCre/+;R26^LacZ/+) after a single tamoxifen pulse. (I) X-gal staining of Lgr5^EGFP-ERCre/+;R26^LacZ/+ intestine 18 hours after tamoxifen induction. Lgr5 cells are found between Paneth cells at the crypt base. (J) Comparison of the location of Lgr5 and Hopx positive cells in the small intestine. Lgr5-expressing cells are predominantly at the 1' or 2' position while most Hopx-expressing cells are at the +4 or +5 position (5). (Analysis performed 18 hours after a single pulse of tamoxifen.) (K and L) The pattern of X-gal staining was analyzed in Hopx^ERCre/+;R26^LacZ/+ (K) and Lgr5^EGFP-ERCre/+;R26^LacZ/+ (L) mice after a 5 day tamoxifen induction (chase periods as indicated, M=months). Staining patterns were scored according to the key below the graphs. Scale bars: 10 μm (B, H, I), 25 μm (G), 100 μm (E and F), 250 μm (A), and 1000 μm (D).