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  • br Material and methods br

    2018-10-20


    Material and methods
    Results Quiescent cells can be identified in vivo by virtue of their ability to retain label. Historically, this was achieved by treating animals with nucleotide analogues, for example 5-bromo-2′-deoxyuridine (BrdU) or tritiated thymidine, that are incorporated into DNA during replication (Morris & Potten, 1994). More recently, transgenic systems have been developed in which enhanced green fluorescent protein (Egfp)-labeled histones are inducibly expressed and then incorporated into chromatin (Tumbar et al., 2004). We utilized a mouse strain that allows for ubiquitous, doxycycline (DOX)-inducible expression of an H2B-Egfp fusion protein (Foudi et al., 2009) (Fig. 1A). Immediately following a pulse of DOX, the entire intestinal epithelium is Egfp-positive. As the cells at the muscarinic antagonist of the crypt divide and migrate up toward the lumen, Egfp signal is progressively diluted. Within a short period of time, around 25days, a single Egfp-positive cell typically remains near the bottom of the crypt (Fig. 1B). To validate that the Egfp-positive quiescent cells are really stem cells, we used qRT-PCR to measure the expression of both stem cell (Lgr5, Olmf4, Ascl2, and Axin2) and differentiation markers (Muc2, Alph, and Chga) in epithelial cells isolated from Rosa26-M2-rtTA; ColA1-H2B-Egfp animals 25days post DOX induction. Egfp+ epithelial cells from these animals were enriched for stem cell markers relative to Egfp− epithelial cells, while the Egfp− cells were enriched for markers of differentiation (Fig. 1C). As a control, we performed the same experiment on Lgr5-Egfp-IRES-CreERT2 mice, where the Egfp+ cells represent bona fide intestinal epithelial stem cells. In these animals, as in the Rosa26-M2-rtTA; ColA1-H2B-Egfp animals, Egfp+ epithelial cells were enriched for stem cell markers (Fig. 1C). These data indicate that the isolated label retaining cells present at the bases of the crypts of Rosa26-M2-rtTA; ColA1-H2B-Egfp animals 25days after DOX stimulation are indeed intestinal stem cells. In order to determine whether mutant K-Ras (K-RasG12D) affects label retention, we crossed mice carrying the inducible histone H2B-Egfp transgene to animals expressing K-RasG12D in the distal small intestinal and colonic epithelia (Haigis et al., 2008). Both control and K-Ras mutant animals were exposed to DOX in the drinking water for 14days to ensure that all cells of the colonic epithelium were labeled. After this initial pulse, DOX was removed from the drinking water and intestinal epithelia were analyzed at different time points in order to detect cells that retained the H2B-Egfp label. Egfp-positive epithelial cells were quantified and tabulated as the number of positive cells per crypt. These experiments were used to determine the “half-life” of the H2B-Egfp label in both control and K-RasG12D epithelium, which we defined as the time-point at which 50% of the colonic crypts retain at least a single labeled cell. As expected, control and mutant animals illustrated little difference in Egfp half-life at the proximal small intestine, since K-RasG12D is expressed only in the distal small intestine and colon (Fig. 2A). By contrast, the retention of label was significantly different between control and mutant animals in the distal small intestinal (a half-life difference of 1.2days) and colonic epithelia (a half-life difference of 3.7days), with K-RasG12D tissues exhibiting a significantly reduced Egfp half-life (Fig. 2B,C). While mutational activation of K-Ras altered the kinetics of label retention in the colonic epithelium, it did not alter the distribution of Egfp+ cells, the majority of which were found in positions 1–5 at the bottom of the crypt (Fig. 2D). Consistent with the idea that mutant K-Ras alters the kinetics of label retention through its effect on proliferation, animals expressing K-RasG12D exhibited an increased frequency of mitotic stem cells relative to controls (Fig. 2E). Taken together, these data indicate that K-RasG12D promotes hyperproliferation of normally quiescent intestinal stem cells.