Volume 136, Issue 7, Pages 2187-2194.e1 (June 2009) Prominin-1/CD133 Marks Stem Cells and Early Progenitors in Mouse Small Intestine  Hugo J. Snippert, Johan H. van Es, Maaike van den Born, Harry Begthel, Daniel E. Stange, Nick Barker, Hans Clevers  Gastroenterology  Volume 136, Issue 7, Pages 2187-2194.e1 (June 2009) DOI: 10.1053/j.gastro.2009.03.002 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 Prominin-1 is not exclusive for Lgr5+ intestinal stem cells. (A) Confocal image of EGFP-positive intestinal stem cells with ToPro-3 counterstained nuclei in Lgr5-EGFP-IRES-CreERT2 KI mice. Lgr5 expression is restricted to the 6–8 slender cells between Paneth cells at the entire crypt bottom. Scale bar, 75 μm. (B) FACS diagram of isolated crypt cells from wild-type (WT) mice and Lgr5-EGFP-IRES-CreERT2 KI mice. Intestinal stem cells are sorted as Lgr5high versus early progenitors as Lgr5med versus late progenitors as Lgr5low. (C) qPCR on sorted cell fractions from panel B, respectively. Lgr5 expression levels confirm sorted EGFP levels and is dramatically enriched in intestinal stem cells (Lgr5high) versus progenitor cells. Prom1 however is not enriched in Lgr5high stem cells compared with early progenitors and shows approximately 5 times higher expression in stem cells versus late progenitors. Gastroenterology 2009 136, 2187-2194.e1DOI: (10.1053/j.gastro.2009.03.002) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 Prominin-1 expression pattern is restricted to Lgr5+ intestinal stem cells and progenitor cells. (A) In situ hybridization shows that Prom1 expression pattern is restricted to the bottom half of wild-type (WT) intestinal crypts, encompassing Lgr5+ stem cells and the earliest progenitor cells. (B) In situ hybridization of Prom1 on intestine of APCmin mice shows wide but not uniform expression within adenomas. (C) Immunofluorescence labeling of Prom1 in WT crypts counterstained with DNA dye DAPI shows location of Prom1 at the apical membranes of progenitors and Lgr5+ stem cells located between the Paneth cells at the bottom of the crypt. (D) Immunofluorescence labeling of Prom1 in crypts from Lgr5-EGFP-IRES-CreERT2 KI mice. Overlay with DNA dye DAPI (left) or bright field (right) shows that Prom1 is not exclusively expressed on Lgr5+ stem cells but is also present on early progenitors. All scale bars represent 50 μm. Gastroenterology 2009 136, 2187-2194.e1DOI: (10.1053/j.gastro.2009.03.002) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 Prom1-mCherry-IRES-CreERT2 KI mice faithfully recapitulate expression pattern and subcellular localization of endogenous Prom1. (A) mCherry is fused to the C-terminus of a Prom1 expression construct to test its subcellular localization in colorectal carcinoma cell line LS174 W4. On treatment with doxycycline, the LKB1-STRAD-Mo25 complex becomes stable and polarizes LS174 W4 cells. Prom1-mCherry becomes localized to the actin-rich apical sides of polarized cells. Scale bar, 10 μm. (B) The mCherry-IRES-CreERT2 construct was cloned just in front of the stop codon of the last coding exon of Prom1, thereby maintaining true endogenous expression levels because of the intact promoter region, intronic sequences, and the endogenous 3′ UTR. (C) Southern blot of targeted mouse ES cells shows heterozygous-targeted alleles in lane 1 to 3 and a homozygous wild-type allele in control lane 4. (D) Confocal imaging of mCherry confirms that Prom1-mCherry fusion protein is expressed at the lower half of intestinal crypts and localizes to the apical side of the plasma membrane. Scale bar, 75 μm. Gastroenterology 2009 136, 2187-2194.e1DOI: (10.1053/j.gastro.2009.03.002) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 Lineage tracing in the small intestine confirms that Prom1 is not exclusively restricted to Lgr5+ intestinal stem cells. (A) Histologic analysis of LacZ activity in small intestine 1 day after tamoxifen induction. Scale bar, 50 μm. (B) Frequency at which blue cells appeared at specific locations counted relative to the crypt bottom 1 day after induction of tracing. Results are depicted as means of 5 independent stretches of proximal small intestine totaling >400 crypts. Most Cre+ LacZ-labeled cells occurred just above the Lgr5+ intestinal stem cells (position 0, 1′, and 2′). Quantitative data on the start position of lineage tracing with the use of Lgr5-EGFP-IRES-CreERT2 KI mice were published recently.5 The graph shows a comparison between the initial tracing positions of both mouse models. (C) Histologic analysis of LacZ activity in small intestine 75 days after tamoxifen induction confirms that intestinal stem cells are positive for Prom1 and are able to produce LacZ-positive ribbons. Scale bar, 100 μm. (D) LacZ-positive cells 4 days after tamoxifen induction shows that enterocytes, goblet cells, and Paneth cells belong to progeny of Prom1+ cells. Scale bar, 12.5 μm. (E) Frequency at which blue tracing events were detected in small intestine at the most proximal part. Results are depicted as relative means, normalized for the size of the area, and compared with the relative amount of tracing events after 1 day, which is set at 100%. Error bars represent standard deviation. Gastroenterology 2009 136, 2187-2194.e1DOI: (10.1053/j.gastro.2009.03.002) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 1 Whole-mount pictures of lineage tracing in small intestine. (A) Whole-mount picture taken from the outside of a small intestine from Prom1-mCherry-IRES-CreERT2 KI mice crossed with Rosa26-LacZ reporter mice, showing lineage tracing events 24 hours after tamoxifen induction. Scale bar, 500 μm. (B) Whole-mount picture of a small intestine 7 days after lineage tracing was initiated in Prom1+ cells. Note that the present tracing events became bigger in size, but that the overall number of events became less over time. Scale bar, 500 μm. (C) Whole-mount magnification of a lineage tracing 7 days after initiation. Scale bar, 100 μm. Gastroenterology 2009 136, 2187-2194.e1DOI: (10.1053/j.gastro.2009.03.002) Copyright © 2009 AGA Institute Terms and Conditions