_:b616264684 . _:b616264687 . . _:b616264686 . _:b616264717 . _:b616264681 . _:b56983350 "Intestinal epithelial cells were isolated as described previously (Sato et al., >>2009<<), with modifications. Small intestine was dissected out of mice of the appropriate genotype, opened longitudinally, and washed twice with cold PBS." . _:b616264680 . . . _:b616264683 . _:b616264682 . _:b56983311 . _:b616264719 . . _:b616264693 . _:b616264754 . _:b56983364 . _:b616264692 . _:b616264695 . . _:b56983347 "crossed with mice harboring tamoxifen-inducible Cre recombinases (Villin-, Hopx-, Bmi1-, and Lgr5-CreER mice) for conditional deletion of floxed alleles or activation of fluorescent reporter (el Marjou et al., 2004; Barker et al., >>2007<<; Sangiorgi and Capecchi, 2008; Takeda et al., 2011)." . . _:b616264694 . . . . . _:b616264752 . _:b616264689 . . . . _:b616264688 . _:b56983328 "RNA-binding proteins are expressed in the stem cell compartments of several tissues including the brain, intestine, and blood and are up-regulated in cancers arising from these tissues (Park et al., 2014; Li et al., 2015; Wang et al., >>2015<<). Msi proteins act primarily as translational regulators binding to messenger RNAs, and known target transcripts are involved in the regulation of cell cycle progression, metabolism, and stem cell self-renewal (Park et al., 2014; Li et al." . _:b56983398 "redundantly by binding to several transcripts that encode known negative regulators of mTORC1, including Pten, Bmpr1a, and Lrig1, and aberrant Msi activation leads to Pten repression and induction of the AKT\u2013mTORC1 axis (Li et al., >>2015<<; Wang et al., 2015)." . _:b616264691 . _:b616264706 . _:b56983356 "Msi proteins to intestinal homeostasis and stem cell self-renewal, we intercrossed Msi1 and Msi2 conditional knockout alleles previously generated in our laboratory (Katz et al., 2014; Park et al., 2014; Li et al., 2015; Wang et al., >>2015<<) with mice harboring a Villin-CreER transgene that drives robust, inducible recombination throughout all epithelial cells in the small intestine and colon (el Marjou et al., 2004; Li et al., 2015)." . . _:b616264690 . _:b56983336 "Ectopic induction of Msi2 increases LT-HSC numbers, which is associated with a reduction in stem cell quiescence and a concomitant increase in the percentage of actively cycling LT-HSCs (Kharas et al., >>2010<<). Conversely, Msi2 deletion results in loss of engraftment potential of LT-HSCs in transplantation assays, demonstrating the importance of Msi2 in this largely dormant stem cell population (Park et al., 2014)." . _:b616264701 . _:b56983329 "Msi proteins act primarily as translational regulators binding to messenger RNAs, and known target transcripts are involved in the regulation of cell cycle progression, metabolism, and stem cell self-renewal (Park et al., >>2014<<; Li et al., 2015; Wang et al., 2015)." . _:b616264742 . . _:b616264700 . _:b56983325 . _:b616264703 . _:b56983376 . _:b616264702 . _:b56983383 . . _:b616264697 . . _:b56983345 . _:b616264696 . . . _:b56983379 "Under basal, noninjury conditions, Hopx-/Bmi1-CreER ISCs are known to give rise to Lgr5+ CBCs upon division (Takeda et al., >>2011<<; Tian et al., 2011; Li et al., 2014)." . . . . _:b616264699 . _:b616264747 . _:b616264712 . _:b616264698 . . _:b56983389 "To address this question, we used a mouse model in which a single copy of Msi1 is targeted into safe-haven chromatin and is under control of the doxycycline-inducible tetracycline-responsive element (TRE-Msi1; Li et al., >>2015<<). Administration of doxycycline to Hopx-/Bmi1-CreER::R26-Lox-Stop-Lox-tdTomato::TRE-Msi1 mice for as little as 36 h resulted in robust exit of reserve ISCs from G0 and subsequent entry into the cell cycle (Fig. 5 A). This ectopic Msi" . _:b56983382 "Four days after initiation of lineage tracing (when about half of reserve ISCs have divided to form clusters of two or more daughter cells; Takeda et al., >>2011<<; Li et al., 2014), reserve ISC progeny began activating Msi genes and canonical Wnt target genes (Fig." . _:b616264723 . . _:b616264739 . _:b616264673 . . _:b56983333 . _:b56983349 "Cre recombinases (Villin-, Hopx-, Bmi1-, and Lgr5-CreER mice) for conditional deletion of floxed alleles or activation of fluorescent reporter (el Marjou et al., 2004; Barker et al., 2007; Sangiorgi and Capecchi, 2008; Takeda et al., >>2011<<)." . . . _:b616264677 . _:b56983406 "Interestingly, the phenotype of Msi loss of function is reminiscent of inactivation of mTORC1: both result in failed intestinal regeneration after injury and in G1 cell cycle arrest (Ashton et al., 2010; Kalaitzidis et al., >>2012<<; Faller et al., 2015)." . _:b616264702 . _:b56983405 . . _:b56983384 . _:b616264681 . _:b616264645 . _:b56983317 "At least some of these reserve ISCs can be marked by CreER reporter genes targeted to the endogenous Bmi1 and Hopx loci (Sangiorgi and Capecchi, >>2008<<; Takeda et al., 2011; Yan et al., 2012; Li et al., 2014); however, recent evidence suggests that more differentiated cells may also act as facultative stem cells upon ablation of CBCs (Tetteh et al., 2016)." . _:b56983404 . . _:b56983400 . . . _:b616264632 . _:b56983370 . _:b616264633 . . . _:b616264634 . _:b616264635 . _:b616264636 . _:b56983338 . _:b616264637 . _:b616264638 . _:b616264639 . _:b56983374 "of reserve intestinal ISCs that are marked, at least in part, by Hopx-CreER or Bmi1-CreER alleles (and possibly by other proxy reporter alleles; Montgomery et al., 2011; Takeda et al., 2011; Yan et al., 2012; Metcalfe et al., >>2014<<; Asfaha et al., 2015). Bmi1- or Hopx-CreER reporters are known to mark a largely overlapping population of rare reserve ISCs (although the Hopx-CreER population is more homogeneous; Li et al., 2014)." . _:b56983357 "2014; Park et al., 2014; Li et al., 2015; Wang et al., 2015) with mice harboring a Villin-CreER transgene that drives robust, inducible recombination throughout all epithelial cells in the small intestine and colon (el Marjou et al., >>2004<<; Li et al., 2015)." . . _:b56983366 . . _:b56983367 "of the mTORC1 complex in colorectal cancer (Li et al., 2015; Wang et al., 2015), and that mTORC1 is similarly dispensable for intestinal homeostasis but is required for epithelial regeneration in response to injury (Ashton et al., >>2010<<; Faller et al., 2015), we next sought to determine how Msi loss affects intestinal regeneration in response to radiation injury." . _:b616264631 . _:b56983319 "At least some of these reserve ISCs can be marked by CreER reporter genes targeted to the endogenous Bmi1 and Hopx loci (Sangiorgi and Capecchi, 2008; Takeda et al., 2011; Yan et al., >>2012<<; Li et al., 2014); however, recent evidence suggests that more differentiated cells may also act as facultative stem cells upon ablation of CBCs (Tetteh et al., 2016)." . . . _:b56983402 "no adverse effects on homeostasis, active CBC self-renewal, or Wnt pathway target gene expression, despite the high level of both Msi family members in the Wnthigh CBC stem cells (Potten et al., 2003; Cambuli et al., 2013; Li et al., >>2015<<; Wang et al., 2015)." . _:b56983390 _:b56983391 . . _:b56983365 "Given prior findings demonstrating that Msi1/2 activity potentiates the activity of the mTORC1 complex in colorectal cancer (Li et al., >>2015<<; Wang et al., 2015), and that mTORC1 is similarly dispensable for intestinal homeostasis but is required for epithelial regeneration in response to injury (Ashton et al., 2010; Faller et al., 2015), we next sought to determine how Msi" . _:b616264674 . . . . _:b56983385 "to cells with a dormant genome and low metabolic activity, reflected by low RNA levels in a diploid cell (G0), rather than simply the absence of cycling, which can also result from G1 arrest (H\u00FCttmann et al., 2001; Fukada et al., >>2007<<). We first examined reserve ISC populations marked by Hopx-/Bmi1-CreER::R26-Lox-Stop-Lox-tdTomato and found that the majority of these populations reside in G0 (Fig." . _:b616264736 . _:b616264640 . _:b56983324 "Unlike Lgr5+ CBCs, the population of reserve ISCs is largely quiescent (in G0 and metabolically inactive) rather than activated (metabolically active and within the cell cycle; Li et al., >>2016<<). It has been postulated that the low metabolic activity of quiescent stem cells discourages genetic lesions induced by reactive oxygen species (Pazhanisamy, 2009). However, knowledge of the molecular mechanisms governing their" . . _:b616264707 . _:b56983318 "At least some of these reserve ISCs can be marked by CreER reporter genes targeted to the endogenous Bmi1 and Hopx loci (Sangiorgi and Capecchi, 2008; Takeda et al., >>2011<<; Yan et al., 2012; Li et al., 2014); however, recent evidence suggests that more differentiated cells may also act as facultative stem cells upon ablation of CBCs (Tetteh et al., 2016)." . . _:b616264635 . _:b616264692 . . _:b616264733 . . _:b616264696 . _:b616264697 . _:b616264698 . _:b616264699 . _:b616264700 . _:b56983373 . _:b616264634 . _:b616264701 . _:b56983390 _:b56983408 . _:b56983390 _:b56983409 . _:b56983397 . _:b56983364 "Ultimately, we tested the effects of Msi loss on the activity of CBCs marked by an Lgr5-eGFP-CreER reporter allele (Barker et al., >>2007<<). Msi loss had no effect on the frequency or proliferation of CBCs (Fig. S2, F and G)." . _:b56983359 . _:b56983347 . _:b616264702 . . _:b56983390 _:b56983410 . _:b616264703 . _:b616264688 . _:b56983390 _:b56983404 . _:b56983358 "2014; Li et al., 2015; Wang et al., 2015) with mice harboring a Villin-CreER transgene that drives robust, inducible recombination throughout all epithelial cells in the small intestine and colon (el Marjou et al., 2004; Li et al., >>2015<<). Ablation of either Msi1 or Msi2 individually throughout the intestinal epithelium and colon had no effect on tissue homeostasis, proliferation, or differentiation (not depicted), consistent with published studies demonstrating their" . _:b616264757 . _:b616264660 . _:b616264689 . _:b56983390 _:b56983405 . . _:b616264690 . _:b56983390 _:b56983406 . _:b56983367 . _:b616264691 . _:b56983390 _:b56983407 . _:b616264692 . _:b56983390 _:b56983400 . _:b616264664 . _:b616264693 . _:b56983390 _:b56983401 . _:b616264694 . _:b56983390 _:b56983402 . _:b56983308 . _:b616264695 . _:b56983390 _:b56983403 . _:b616264680 . _:b56983390 _:b56983396 . _:b56983400 "intestinal epithelium and colon, had no adverse effects on homeostasis, active CBC self-renewal, or Wnt pathway target gene expression, despite the high level of both Msi family members in the Wnthigh CBC stem cells (Potten et al., >>2003<<; Cambuli et al., 2013; Li et al., 2015; Wang et al., 2015)." . _:b616264681 . _:b56983390 _:b56983397 . _:b616264682 . _:b56983390 _:b56983398 . _:b616264683 . _:b56983390 _:b56983399 . _:b616264684 . _:b56983390 _:b56983392 . . _:b616264685 . _:b616264685 . _:b56983390 _:b56983393 . _:b616264686 . _:b56983390 _:b56983394 . _:b616264687 . . _:b56983390 _:b56983395 . _:b616264672 . _:b56983351 _:b56983352 . _:b616264673 . _:b56983351 _:b56983353 . _:b56983378 . _:b56983310 . _:b616264674 . _:b56983351 _:b56983354 . _:b616264675 . _:b56983351 _:b56983355 . _:b56983320 "loci (Sangiorgi and Capecchi, 2008; Takeda et al., 2011; Yan et al., 2012; Li et al., 2014); however, recent evidence suggests that more differentiated cells may also act as facultative stem cells upon ablation of CBCs (Tetteh et al., >>2016<<). Despite the lack of consensus on the precise populations contributing to regeneration after injury, clear evidence demonstrates that under basal conditions in the absence of injury, Bmi1-/Hopx-CreER\u2013marked reserve ISCs give rise to CBCs." . _:b616264745 . _:b616264676 . _:b56983351 _:b56983356 . _:b616264677 . _:b56983351 _:b56983357 . _:b616264678 . _:b56983351 _:b56983358 . _:b616264679 . . _:b616264664 . _:b616264708 . _:b56983351 _:b56983359 . _:b616264665 . _:b56983403 . _:b616264722 . . _:b616264666 . . _:b616264667 . _:b616264668 . . _:b616264695 . _:b616264669 . _:b616264670 . _:b616264671 . _:b616264656 . . _:b616264657 . _:b616264650 . _:b616264658 . . _:b616264659 . _:b616264660 . _:b616264682 . . _:b616264661 . _:b616264662 . . _:b616264663 . _:b616264648 . _:b616264649 . _:b616264650 . _:b616264651 . _:b616264753 . _:b616264637 . _:b616264652 . _:b616264653 . _:b616264654 . _:b616264655 . . _:b616264640 . _:b616264641 . _:b616264642 . _:b56983348 . . _:b616264643 . _:b616264669 . . _:b616264644 . _:b616264645 . _:b616264646 . _:b616264647 . _:b616264760 . . _:b616264761 . _:b616264762 . _:b616264763 . _:b616264726 . _:b616264764 . _:b616264765 . _:b616264766 . . . _:b616264767 . _:b616264752 . _:b616264753 . . _:b616264754 . _:b616264755 . _:b616264756 . . _:b616264757 . _:b56983390 . _:b616264758 . _:b56983375 . _:b616264759 . _:b616264744 . _:b616264745 . _:b56983399 . _:b616264746 . . _:b616264747 . _:b616264748 . _:b616264749 . _:b616264750 . . _:b616264751 . _:b616264736 . _:b56983326 "Musashi (Msi) RNA-binding proteins are expressed in the stem cell compartments of several tissues including the brain, intestine, and blood and are up-regulated in cancers arising from these tissues (Park et al., >>2014<<; Li et al., 2015; Wang et al., 2015)." . _:b616264724 . _:b616264737 . _:b616264738 . _:b616264739 . _:b56983387 . _:b616264740 . _:b616264741 . _:b56983343 . _:b616264742 . _:b616264743 . _:b616264728 . _:b616264729 . _:b616264730 . _:b616264731 . . _:b616264732 . _:b616264643 . _:b616264733 . _:b616264734 . _:b616264735 . . _:b616264720 . _:b616264721 . _:b616264722 . _:b616264749 . _:b616264723 . _:b616264724 . _:b616264725 . _:b616264726 . _:b616264727 . _:b56983380 "Under basal, noninjury conditions, Hopx-/Bmi1-CreER ISCs are known to give rise to Lgr5+ CBCs upon division (Takeda et al., 2011; Tian et al., >>2011<<; Li et al., 2014)." . _:b616264712 . _:b616264713 . _:b616264714 . _:b616264715 . _:b616264716 . _:b616264717 . _:b56983351 . _:b616264718 . _:b616264665 . _:b56983395 . _:b616264719 . _:b616264704 . _:b56983351 _:b56983384 . _:b616264767 . _:b616264705 . _:b56983306 . . _:b616264706 . _:b56983351 _:b56983385 . _:b56983351 _:b56983386 . _:b616264707 . _:b56983351 _:b56983387 . _:b616264708 . _:b56983351 _:b56983388 . . _:b616264709 . _:b616264700 . _:b56983351 _:b56983389 . _:b616264710 . _:b616264711 . . . _:b56983308 . _:b56983343 _:b56983344 . _:b56983351 _:b56983376 . _:b56983351 _:b56983377 . _:b56983343 _:b56983345 . _:b56983343 _:b56983346 . _:b56983351 _:b56983378 . _:b56983343 _:b56983347 . _:b616264729 . _:b56983351 _:b56983379 . _:b56983343 _:b56983348 . _:b56983309 . _:b616264709 "2"^^ . _:b56983351 _:b56983380 . _:b56983343 _:b56983349 . _:b56983351 _:b56983381 . _:b56983351 _:b56983382 . _:b56983343 _:b56983350 . _:b56983351 _:b56983383 . . _:b56983351 _:b56983368 . _:b56983310 . _:b616264708 "2"^^ . _:b56983351 _:b56983369 . _:b56983351 _:b56983370 . . _:b56983351 _:b56983371 . _:b56983351 _:b56983372 . _:b56983311 . _:b616264711 "2"^^ . _:b56983351 _:b56983373 . _:b56983351 _:b56983374 . _:b56983351 _:b56983375 . _:b56983363 "activity of the canonical Wnt pathway and are in contrast to in vitro studies and in vivo gain-of-function assays suggesting that Msi potentiates activity of this pathway (Rezza et al., 2010; Spears and Neufeld, 2011; Cambuli et al., >>2015<<)." . _:b56983334 "In the hematopoietic system, Msi2 is an important modulator of long-term hematopoietic stem cell (LT-HSC) proliferation and self-renewal (Hope et al., 2010; Ito et al., 2010; Kharas et al., >>2010<<; Park et al., 2014)." . _:b56983351 _:b56983360 . _:b616264710 "2"^^ . _:b56983351 _:b56983361 . _:b616264704 . _:b56983351 _:b56983362 . _:b56983351 _:b56983363 . _:b56983351 _:b56983364 . _:b616264705 "2"^^ . _:b56983351 _:b56983365 . _:b56983351 _:b56983366 . _:b56983351 _:b56983367 . _:b616264772 . . _:b616264704 "2"^^ . _:b56983306 . _:b616264707 "2"^^ . _:b56983307 . _:b56983307 . _:b56983323 "population of Wnt\u2212 reserve ISCs that give rise to active, Wnthigh Lgr5+ CBCs upon division, and consequently all functional cell types of the epithelium over long periods of time (Takeda et al., 2011; Tian et al., 2011; Yan et al., >>2012<<; Li et al., 2014). Unlike Lgr5+ CBCs, the population of reserve ISCs is largely quiescent (in G0 and metabolically inactive) rather than activated (metabolically active and within the cell cycle; Li et al., 2016)." . _:b616264655 . _:b56983396 "to an actively proliferating crypt base columnar cell, whose self-renewal is driven by high Wnt pathway activity (Li and Clevers, 2010; Montgomery et al., 2011; Takeda et al., 2011; Tian et al., 2011; Li et al., 2014; Asfaha et al., >>2015<<). There has been considerable interest in gaining a better understanding of radioresistant intestinal cells that contribute to regeneration after damage by radiation/chemotherapy because of the relevance in the context of traditional" . _:b616264706 "2"^^ . _:b56983316 . _:b56983361 "findings demonstrate that Msi does not support activity of the canonical Wnt pathway and are in contrast to in vitro studies and in vivo gain-of-function assays suggesting that Msi potentiates activity of this pathway (Rezza et al., >>2010<<; Spears and Neufeld, 2011; Cambuli et al., 2015)." . . _:b616264717 "2"^^ . _:b56983317 . _:b616264731 . _:b616264716 "2"^^ . _:b56983318 . _:b56983408 "marked with proxy reporter alleles driven by the mTert promoter (a population that likely overlaps with the Hopx-/Bmi1-CreER-marked population in the current study based on their functional and molecular similarities; Richmond et al., >>2015<<). These findings are entirely consistent with observations in hematopoietic and muscle tissues that the mTORC1 complex similarly governs stem cell quiescence (Kalaitzidis et al., 2012; Rodgers et al., 2014)." . . _:b616264719 "2"^^ . _:b56983319 . _:b616264718 "2"^^ . _:b56983312 . _:b616264713 "2"^^ . _:b56983313 . . _:b616264768 . _:b616264712 "2"^^ . _:b56983314 . _:b616264769 . . _:b616264770 . _:b616264709 . _:b616264771 . _:b56983380 . _:b616264772 . _:b616264715 "2"^^ . _:b56983325 "It has been postulated that the low metabolic activity of quiescent stem cells discourages genetic lesions induced by reactive oxygen species (Pazhanisamy, >>2009<<). However, knowledge of the molecular mechanisms governing their radioresistance and subsequent exit from the quiescent state in response to \u03B3-IR injury is lacking." . _:b56983315 . . _:b56983317 . _:b616264708 . _:b616264714 "2"^^ . _:b56983324 . _:b616264699 . _:b616264711 . _:b616264725 "2"^^ . _:b56983325 . _:b616264710 . _:b56983309 "CBCs are characterized by high activity of the canonical Wnt pathway, and activity of the Wnt target gene Lgr5 is commonly used for their identification and prospective isolation (Cheng and Leblond, >>1974<<; Barker et al., 2007)." . _:b616264724 "2"^^ . _:b56983326 . _:b616264705 . _:b56983352 "1 A; Li et al., >>2015<<). To unequivocally test the functional contribution of Msi proteins to intestinal homeostasis and stem cell self-renewal, we intercrossed Msi1 and Msi2 conditional knockout alleles previously generated in our laboratory (Katz et al.," . _:b56983323 . _:b616264727 "2"^^ . _:b56983327 . _:b616264704 . _:b56983369 . _:b616264726 "2"^^ . _:b56983320 . _:b616264707 . _:b56983373 "\u03B3-IR resides in a population of reserve intestinal ISCs that are marked, at least in part, by Hopx-CreER or Bmi1-CreER alleles (and possibly by other proxy reporter alleles; Montgomery et al., 2011; Takeda et al., 2011; Yan et al., >>2012<<; Metcalfe et al., 2014; Asfaha et al., 2015). Bmi1- or Hopx-CreER reporters are known to mark a largely overlapping population of rare reserve ISCs (although the Hopx-CreER population is more homogeneous; Li et al., 2014)." . _:b616264770 . . _:b616264721 "2"^^ . _:b56983321 . _:b616264706 . _:b56983306 _:b56983308 . _:b56983322 . _:b616264720 "2"^^ . _:b616264717 . _:b56983306 _:b56983309 . . _:b56983306 _:b56983310 . "10.1083%2Fjcb.201604119" . _:b56983319 . _:b56983306 _:b56983311 . _:b616264723 "2"^^ . _:b56983323 . _:b616264716 . _:b56983306 _:b56983307 . _:b56983355 "contribution of Msi proteins to intestinal homeostasis and stem cell self-renewal, we intercrossed Msi1 and Msi2 conditional knockout alleles previously generated in our laboratory (Katz et al., 2014; Park et al., 2014; Li et al., >>2015<<; Wang et al., 2015) with mice harboring a Villin-CreER transgene that drives robust, inducible recombination throughout all epithelial cells in the small intestine and colon (el Marjou et al., 2004; Li et al., 2015)." . _:b56983338 "In the intestinal epithelium, Msi proteins are expressed throughout the crypt, including in the active CBC stem cell compartment (Itzkovitz et al., >>2011<<; Li et al., 2014, 2015; Wang et al., 2015)." . _:b616264722 "2"^^ . _:b56983394 "a radioresistant, slow-cycling reserve ISC giving rise to an actively proliferating crypt base columnar cell, whose self-renewal is driven by high Wnt pathway activity (Li and Clevers, 2010; Montgomery et al., 2011; Takeda et al., >>2011<<; Tian et al., 2011; Li et al., 2014; Asfaha et al., 2015)." . _:b56983332 . _:b616264719 . _:b56983346 . _:b616264733 "2"^^ . _:b56983333 . _:b616264718 . _:b56983343 "materials and methods" . _:b616264657 . _:b56983363 . _:b56983334 . _:b616264732 "2"^^ . _:b616264638 . _:b56983306 _:b56983324 . _:b616264713 . _:b56983306 _:b56983325 . _:b56983306 _:b56983326 . _:b56983306 _:b56983327 . _:b56983306 _:b56983320 . _:b56983335 . _:b616264735 "2"^^ . _:b616264712 . _:b56983306 _:b56983321 . _:b616264683 . . _:b56983306 _:b56983322 . _:b56983306 _:b56983323 . _:b56983312 . _:b56983306 _:b56983316 . _:b56983376 "Lgr5-CreER had no significant effect on regeneration, although a modest, nonsignificant decrease in regeneration was observed, possibly reflecting the function of an Lgr5low cell previously described as radioresistant (Tao et al., >>2015<<). In contrast, Msi loss in rare Hopx-CreER reserve ISCs (making up less than 0.5% of the crypt epithelium; Takeda et al., 2011; Li et al., 2014) and some of their immediate progeny (as some reserve ISCs must divide during the time" . _:b56983362 . _:b56983328 . _:b616264734 "2"^^ . _:b616264715 . _:b56983306 _:b56983317 . . _:b56983306 _:b56983318 . _:b56983306 _:b56983319 . _:b56983306 _:b56983312 . _:b56983329 . _:b616264729 "2"^^ . _:b616264714 . _:b56983306 _:b56983313 . _:b56983306 _:b56983314 . _:b56983357 . _:b56983306 _:b56983315 . _:b56983306 _:b56983340 . _:b56983330 . _:b616264728 "2"^^ . _:b56983306 _:b56983341 . _:b56983396 . _:b616264725 . _:b56983306 _:b56983342 . . _:b56983345 "Generation of the TRE-Msi1 doxycycline-inducible mouse model and Msi1 and Msi2 conditional alleles was previously described (Li et al., 2015; Park et al., >>2015<<). They were crossed with mice harboring tamoxifen-inducible Cre recombinases (Villin-, Hopx-, Bmi1-, and Lgr5-CreER mice) for conditional deletion of floxed alleles or activation of fluorescent reporter (el Marjou et al., 2004; Barker et" . _:b56983306 _:b56983336 . _:b56983331 . _:b616264731 "2"^^ . _:b56983306 _:b56983337 . _:b616264724 . _:b56983306 _:b56983338 . _:b56983306 _:b56983339 . _:b56983306 _:b56983332 . _:b56983340 . _:b616264730 "2"^^ . _:b56983306 _:b56983333 . _:b56983322 "largely overlapping population of Wnt\u2212 reserve ISCs that give rise to active, Wnthigh Lgr5+ CBCs upon division, and consequently all functional cell types of the epithelium over long periods of time (Takeda et al., 2011; Tian et al., >>2011<<; Yan et al., 2012; Li et al., 2014). Unlike Lgr5+ CBCs, the population of reserve ISCs is largely quiescent (in G0 and metabolically inactive) rather than activated (metabolically active and within the cell cycle; Li et al., 2016)." . _:b56983320 . _:b616264727 . _:b56983306 _:b56983334 . _:b56983306 _:b56983335 . _:b56983306 _:b56983328 . _:b56983341 . _:b616264741 "2"^^ . _:b56983306 _:b56983329 . _:b616264726 . _:b56983306 _:b56983330 . _:b56983306 _:b56983331 . _:b616264740 "2"^^ . _:b56983342 . _:b616264721 . _:b56983334 . _:b616264743 "2"^^ . . _:b616264720 . _:b56983343 . . _:b616264742 "2"^^ . _:b56983336 . . _:b56983336 . _:b616264723 . _:b56983311 "burden of the high-turnover epithelium, they are sensitive to DNA damage\u2013causing agents such as high-dose \u03B3-irradiation (\u03B3-IR), and several independent studies have demonstrated that CBCs are largely ablated after \u03B3-IR (Hua et al., >>2012<<; Yan et al., 2012; Metcalfe et al., 2014; Asfaha et al., 2015). Recently, Tao et al. (2015) showed that high Wnt pathway activity and basal crypt positioning sensitize CBCs to DNA damage, leading to their preferential depletion." . _:b616264737 "2"^^ . _:b56983337 . . _:b616264722 . _:b616264736 "2"^^ . _:b56983338 . . _:b616264733 . _:b616264632 . _:b616264661 . _:b56983408 . _:b56983339 . _:b616264739 "2"^^ . _:b616264732 . . _:b616264694 . _:b56983348 . _:b56983344 "Generation of the TRE-Msi1 doxycycline-inducible mouse model and Msi1 and Msi2 conditional alleles was previously described (Li et al., >>2015<<; Park et al., 2015). They were crossed with mice harboring tamoxifen-inducible Cre recombinases (Villin-, Hopx-, Bmi1-, and Lgr5-CreER mice) for conditional deletion of floxed alleles or activation of fluorescent reporter (el Marjou et al." . _:b616264738 "2"^^ . _:b616264735 . . _:b616264749 "2"^^ . _:b56983349 . _:b56983340 . _:b616264734 . _:b616264667 . _:b56983391 "clear that several tissues in adult mammals, most notably the hematopoietic system, bifurcate their stem cell compartments into a slow-cycling, quiescent stem cell capable of giving rise to a cycling, active stem cell (Li and Clevers, >>2010<<). The benefit of such an organizational structure is the capacity to promote tissue regeneration after damage while maintaining the proliferative output necessary to keep up with the demands of high-turnover tissues using a relatively" . _:b56983350 . _:b616264748 "2"^^ . _:b616264729 . _:b56983405 "Interestingly, the phenotype of Msi loss of function is reminiscent of inactivation of mTORC1: both result in failed intestinal regeneration after injury and in G1 cell cycle arrest (Ashton et al., >>2010<<; Kalaitzidis et al., 2012; Faller et al., 2015)." . _:b56983384 "quiescence usually refers to cells with a dormant genome and low metabolic activity, reflected by low RNA levels in a diploid cell (G0), rather than simply the absence of cycling, which can also result from G1 arrest (H\u00FCttmann et al., >>2001<<; Fukada et al., 2007). We first examined reserve ISC populations marked by Hopx-/Bmi1-CreER:" . _:b616264751 "2"^^ . _:b56983342 . _:b56983351 . _:b616264735 . _:b616264728 . . _:b616264750 "2"^^ . _:b56983386 "Interestingly, these findings are consistent with previous studies on the mTORC1 complex, inactivation of which similarly results in G1 arrest (Kalaitzidis et al., >>2012<<) as well as failure of the intestinal epithelium to regenerate in response to high dose \u03B3-IR injury (Ashton et al., 2010; Faller et al., 2015)." . _:b56983344 . _:b616264731 . _:b616264693 . _:b616264745 "2"^^ . _:b56983345 . _:b56983328 . _:b616264730 . _:b616264744 "2"^^ . _:b56983346 . _:b616264741 . . _:b616264747 "2"^^ . _:b56983347 . _:b616264740 . _:b56983327 . _:b616264746 "2"^^ . _:b56983356 . _:b56983331 . _:b616264743 . _:b616264678 . _:b616264757 "2"^^ . _:b56983357 . _:b616264742 . . _:b616264756 "2"^^ . _:b56983358 . _:b616264737 . _:b616264671 . _:b56983359 . _:b616264759 "2"^^ . _:b616264654 . _:b616264736 . _:b616264734 . _:b616264758 "2"^^ . _:b56983352 . _:b616264739 . . _:b616264727 . _:b56983331 "as translational regulators binding to messenger RNAs, and known target transcripts are involved in the regulation of cell cycle progression, metabolism, and stem cell self-renewal (Park et al., 2014; Li et al., 2015; Wang et al., >>2015<<). In the hematopoietic system, Msi2 is an important modulator of long-term hematopoietic stem cell (LT-HSC) proliferation and self-renewal (Hope et al., 2010; Ito et al., 2010; Kharas et al., 2010; Park et al., 2014)." . _:b616264753 "2"^^ . _:b56983353 . _:b616264738 . _:b616264752 "2"^^ . _:b56983354 . _:b616264749 . _:b616264755 "2"^^ . _:b56983355 . _:b616264684 . _:b616264748 . . _:b56983364 . _:b56983350 . _:b616264754 "2"^^ . _:b616264751 . _:b616264765 "2"^^ . _:b56983365 . _:b56983356 . _:b616264750 . . _:b616264764 "2"^^ . _:b56983366 . _:b616264745 . _:b56983327 "Musashi (Msi) RNA-binding proteins are expressed in the stem cell compartments of several tissues including the brain, intestine, and blood and are up-regulated in cancers arising from these tissues (Park et al., 2014; Li et al., >>2015<<; Wang et al., 2015)." . _:b616264767 "2"^^ . _:b56983367 . _:b56983340 "In the intestinal epithelium, Msi proteins are expressed throughout the crypt, including in the active CBC stem cell compartment (Itzkovitz et al., 2011; Li et al., 2014, 2015; Wang et al., >>2015<<). We have previously established that Msi proteins are obligate and functionally redundant intestinal oncoproteins that drive epithelial transformation in large part through inhibition of intestinal tumor suppressors including Pten," . _:b616264744 . _:b616264631 "12"^^ . _:b616264766 "2"^^ . _:b56983360 . _:b616264747 . . _:b56983361 . _:b616264761 "2"^^ . _:b616264746 . _:b616264760 "2"^^ . _:b56983362 . _:b616264757 . _:b616264763 "2"^^ . _:b56983363 . _:b616264756 . . _:b616264713 . _:b616264711 . _:b616264750 . . _:b56983372 . _:b616264762 "2"^^ . _:b616264759 . . _:b56983373 . _:b616264758 . _:b616264772 "2"^^ . _:b56983374 . _:b616264738 . _:b56983366 "Given prior findings demonstrating that Msi1/2 activity potentiates the activity of the mTORC1 complex in colorectal cancer (Li et al., 2015; Wang et al., >>2015<<), and that mTORC1 is similarly dispensable for intestinal homeostasis but is required for epithelial regeneration in response to injury (Ashton et al., 2010; Faller et al., 2015), we next sought to determine how Msi loss affects" . _:b616264753 . _:b56983332 . . _:b56983337 "Conversely, Msi2 deletion results in loss of engraftment potential of LT-HSCs in transplantation assays, demonstrating the importance of Msi2 in this largely dormant stem cell population (Park et al., >>2014<<)." . _:b56983335 "In the hematopoietic system, Msi2 is an important modulator of long-term hematopoietic stem cell (LT-HSC) proliferation and self-renewal (Hope et al., 2010; Ito et al., 2010; Kharas et al., 2010; Park et al., >>2014<<). Ectopic induction of Msi2 increases LT-HSC numbers, which is associated with a reduction in stem cell quiescence and a concomitant increase in the percentage of actively cycling LT-HSCs (Kharas et al., 2010). Conversely, Msi2 deletion" . _:b56983375 . _:b616264752 . . _:b56983368 . _:b616264638 "9"^^ . _:b616264675 . _:b616264755 . _:b56983322 . _:b616264639 "8"^^ . _:b616264769 "2"^^ . _:b56983369 . . _:b616264754 . _:b616264636 "9"^^ . _:b56983370 . _:b616264768 "2"^^ . _:b616264765 . _:b616264637 "9"^^ . _:b56983371 . _:b616264771 "2"^^ . _:b616264764 . _:b616264770 "2"^^ . _:b56983380 . _:b616264689 . _:b616264767 . _:b616264634 "10"^^ . _:b616264701 . _:b616264651 . _:b616264764 . _:b616264635 "9"^^ . _:b56983381 . _:b616264766 . _:b616264633 "11"^^ . "PMC0" . _:b56983382 . _:b616264761 . _:b616264632 "11"^^ . . _:b616264769 . _:b56983383 . _:b616264646 "6"^^ . _:b616264760 . . _:b56983376 . _:b616264647 "6"^^ . _:b616264763 . _:b616264662 . _:b56983377 . . _:b616264762 . _:b616264644 "7"^^ . . _:b56983378 . . _:b616264680 . _:b56983344 . _:b616264645 "7"^^ . _:b616264644 . _:b56983379 . _:b616264772 . . _:b616264642 "7"^^ . _:b616264687 . _:b56983388 . . . _:b56983358 . _:b616264643 "7"^^ . _:b56983307 "Intestinal stem cells (ISCs), which are crucial for physiological tissue homeostasis and regeneration after injury, are thought to play a critical role in this process (Potten, >>2004<<; Ch\u2019ang et al., 2005)." . _:b56983389 . _:b616264672 . _:b616264640 "8"^^ . _:b56983390 . _:b616264769 . . _:b616264728 . _:b616264641 "7"^^ . _:b56983370 "or control Msi1flx/flx::Msi2flx/flx mice were treated with five daily doses of tamoxifen and subjected to 12 Gy of ionizing \u03B3-IR 1 wk later to ablate proliferative cells including CBCs (Tian et al., 2011; Tao et al., >>2015<<). In this context, intestinal regeneration (quantified by the number of clonal regenerative crypt foci per unit length 72 h after injury) was severely compromised in the absence of Msi activity (Fig." . _:b56983391 . _:b616264768 . _:b56983359 "throughout the intestinal epithelium and colon had no effect on tissue homeostasis, proliferation, or differentiation (not depicted), consistent with published studies demonstrating their functional redundancy (Sakakibara et al., >>2002<<; Li et al., 2015)." . _:b56983355 . _:b616264759 . _:b616264653 "5"^^ . _:b56983384 . _:b616264771 . . . _:b616264652 "5"^^ . _:b56983385 . . _:b616264770 . _:b616264655 "4"^^ . _:b56983386 . _:b56983395 "slow-cycling reserve ISC giving rise to an actively proliferating crypt base columnar cell, whose self-renewal is driven by high Wnt pathway activity (Li and Clevers, 2010; Montgomery et al., 2011; Takeda et al., 2011; Tian et al., >>2011<<; Li et al., 2014; Asfaha et al., 2015)." . _:b56983352 . . _:b616264654 "5"^^ . _:b56983387 . _:b616264650 "6"^^ . _:b616264748 . . _:b56983396 . _:b616264651 "6"^^ . _:b56983397 . _:b616264648 "6"^^ . _:b56983362 "Msi does not support activity of the canonical Wnt pathway and are in contrast to in vitro studies and in vivo gain-of-function assays suggesting that Msi potentiates activity of this pathway (Rezza et al., 2010; Spears and Neufeld, >>2011<<; Cambuli et al., 2015)." . _:b616264652 . . _:b616264709 . _:b56983398 . _:b616264649 "6"^^ . _:b616264730 . _:b56983342 "redundant intestinal oncoproteins that drive epithelial transformation in large part through inhibition of intestinal tumor suppressors including Pten, resulting in downstream mTORC1 complex activation (Li et al., 2015; Wang et al., >>2015<<); however, their role in intestinal homeostasis, regeneration, and stem cell self-renewal in vivo is entirely unknown." . _:b56983399 . _:b616264661 "4"^^ . . _:b56983392 . _:b56983392 . _:b616264670 . _:b56983330 . _:b616264660 "4"^^ . _:b56983393 . _:b56983409 "These findings are entirely consistent with observations in hematopoietic and muscle tissues that the mTORC1 complex similarly governs stem cell quiescence (Kalaitzidis et al., >>2012<<; Rodgers et al., 2014)." . _:b616264688 . _:b616264691 . _:b616264663 "4"^^ . _:b616264743 . _:b56983394 . _:b616264662 "4"^^ . _:b56983395 . _:b616264657 "4"^^ . . _:b56983404 . _:b616264656 "4"^^ . _:b56983394 . _:b56983405 . _:b56983341 . _:b616264659 "4"^^ . . _:b56983406 . _:b56983381 "As predicted, in the resting state, reserve ISCs marked by Hopx-/Bmi-CreER::R26-Lox-Stop-Lox-tdTomato reporter activity 18 h after a single tamoxifen dose (Takeda et al., >>2011<<; Li et al., 2014) expressed low levels of Msi genes and existed in a Wntoff/low state, with little to no expression of canonical Wnt targets such as Lgr5, Ascl2, and Ccnd1, along with high levels of the cell cycle inhibitor Cdkn1a, in" . _:b616264653 . _:b616264658 "4"^^ . _:b56983407 . _:b616264669 "3"^^ . _:b56983339 . . _:b56983400 . _:b56983410 "These findings are entirely consistent with observations in hematopoietic and muscle tissues that the mTORC1 complex similarly governs stem cell quiescence (Kalaitzidis et al., 2012; Rodgers et al., >>2014<<)." . . _:b616264703 . _:b616264668 "3"^^ . . _:b56983401 . . _:b616264671 "3"^^ . _:b616264746 . _:b56983402 . _:b616264670 "3"^^ . _:b56983403 . _:b616264665 "4"^^ . _:b616264690 . _:b616264664 "4"^^ . _:b616264758 . _:b56983375 "ISCs that are marked, at least in part, by Hopx-CreER or Bmi1-CreER alleles (and possibly by other proxy reporter alleles; Montgomery et al., 2011; Takeda et al., 2011; Yan et al., 2012; Metcalfe et al., 2014; Asfaha et al., >>2015<<). Bmi1- or Hopx-CreER reporters are known to mark a largely overlapping population of rare reserve ISCs (although the Hopx-CreER population is more homogeneous; Li et al., 2014)." . _:b616264667 "3"^^ . . _:b56983377 "In contrast, Msi loss in rare Hopx-CreER reserve ISCs (making up less than 0.5% of the crypt epithelium; Takeda et al., >>2011<<; Li et al., 2014) and some of their immediate progeny (as some reserve ISCs must divide during the time required for multiple tamoxifen injections before irradiation) resulted in failed epithelial regeneration to an extent" . _:b56983333 "In the hematopoietic system, Msi2 is an important modulator of long-term hematopoietic stem cell (LT-HSC) proliferation and self-renewal (Hope et al., 2010; Ito et al., >>2010<<; Kharas et al., 2010; Park et al., 2014)." . _:b616264666 "3"^^ . _:b56983330 "proteins act primarily as translational regulators binding to messenger RNAs, and known target transcripts are involved in the regulation of cell cycle progression, metabolism, and stem cell self-renewal (Park et al., 2014; Li et al., >>2015<<; Wang et al., 2015). In the hematopoietic system, Msi2 is an important modulator of long-term hematopoietic stem cell (LT-HSC) proliferation and self-renewal (Hope et al., 2010; Ito et al., 2010; Kharas et al., 2010; Park et al., 2014)." . _:b616264676 . _:b616264633 . _:b616264718 . _:b56983391 . . _:b616264762 . _:b616264677 "3"^^ . . _:b56983408 . . _:b616264676 "3"^^ . _:b56983368 "in colorectal cancer (Li et al., 2015; Wang et al., 2015), and that mTORC1 is similarly dispensable for intestinal homeostasis but is required for epithelial regeneration in response to injury (Ashton et al., 2010; Faller et al., >>2015<<), we next sought to determine how Msi loss affects intestinal regeneration in response to radiation injury." . _:b56983393 "system, with a radioresistant, slow-cycling reserve ISC giving rise to an actively proliferating crypt base columnar cell, whose self-renewal is driven by high Wnt pathway activity (Li and Clevers, 2010; Montgomery et al., >>2011<<; Takeda et al., 2011; Tian et al., 2011; Li et al., 2014; Asfaha et al., 2015)." . _:b616264751 . _:b56983409 . _:b56983382 . _:b616264679 "3"^^ . _:b56983410 . _:b616264678 "3"^^ . _:b616264658 . _:b616264642 . _:b616264673 "3"^^ . _:b56983381 . _:b616264672 "3"^^ . . _:b616264675 "3"^^ . _:b56983389 . _:b56983379 . _:b616264674 "3"^^ . _:b56983354 "test the functional contribution of Msi proteins to intestinal homeostasis and stem cell self-renewal, we intercrossed Msi1 and Msi2 conditional knockout alleles previously generated in our laboratory (Katz et al., 2014; Park et al., >>2014<<; Li et al., 2015; Wang et al., 2015) with mice harboring a Villin-CreER transgene that drives robust, inducible recombination throughout all epithelial cells in the small intestine and colon (el Marjou et al., 2004; Li et al., 2015)." . . _:b616264685 "3"^^ . _:b616264755 . _:b56983341 "and functionally redundant intestinal oncoproteins that drive epithelial transformation in large part through inhibition of intestinal tumor suppressors including Pten, resulting in downstream mTORC1 complex activation (Li et al., >>2015<<; Wang et al., 2015); however, their role in intestinal homeostasis, regeneration, and stem cell self-renewal in vivo is entirely unknown." . _:b56983377 . _:b616264684 "3"^^ . _:b616264687 "3"^^ . _:b56983397 "Recently, we have demonstrated that the Msi2 RNA-binding protein is capable of driving long-term hematopoietic stem cells out of quiescence and into the cell cycle (Kharas et al., >>2010<<). In the intestinal epithelium, Msi1 and Msi2 function redundantly by binding to several transcripts that encode known negative regulators of mTORC1, including Pten, Bmpr1a, and Lrig1, and aberrant Msi activation leads to Pten repression" . . . _:b616264686 "3"^^ . _:b56983372 . _:b616264631 . _:b616264681 "3"^^ . . _:b616264680 "3"^^ . _:b616264646 . _:b616264683 "3"^^ . _:b56983365 . . . _:b56983385 . . _:b616264682 "3"^^ . _:b616264693 "3"^^ . _:b616264663 . _:b616264692 "3"^^ . _:b56983313 "they are sensitive to DNA damage\u2013causing agents such as high-dose \u03B3-irradiation (\u03B3-IR), and several independent studies have demonstrated that CBCs are largely ablated after \u03B3-IR (Hua et al., 2012; Yan et al., 2012; Metcalfe et al., >>2014<<; Asfaha et al., 2015). Recently, Tao et al. (2015) showed that high Wnt pathway activity and basal crypt positioning sensitize CBCs to DNA damage, leading to their preferential depletion." . _:b616264732 . . _:b616264695 "2"^^ . . _:b616264694 "2"^^ . _:b56983399 "to several transcripts that encode known negative regulators of mTORC1, including Pten, Bmpr1a, and Lrig1, and aberrant Msi activation leads to Pten repression and induction of the AKT\u2013mTORC1 axis (Li et al., 2015; Wang et al., >>2015<<)." . _:b56983374 . _:b56983360 . _:b616264697 . _:b616264740 . _:b616264689 "3"^^ . _:b56983308 "Intestinal stem cells (ISCs), which are crucial for physiological tissue homeostasis and regeneration after injury, are thought to play a critical role in this process (Potten, 2004; Ch\u2019ang et al., >>2005<<)." . _:b616264688 "3"^^ . _:b616264691 "3"^^ . _:b616264649 . _:b616264744 . _:b616264690 "3"^^ . _:b616264701 "2"^^ . _:b616264700 "2"^^ . _:b616264710 . _:b616264686 . _:b616264703 "2"^^ . _:b56983371 "epithelium in response to such high-dose \u03B3-IR resides in a population of reserve intestinal ISCs that are marked, at least in part, by Hopx-CreER or Bmi1-CreER alleles (and possibly by other proxy reporter alleles; Montgomery et al., >>2011<<; Takeda et al., 2011; Yan et al., 2012; Metcalfe et al., 2014; Asfaha et al., 2015)." . _:b56983310 "CBCs are characterized by high activity of the canonical Wnt pathway, and activity of the Wnt target gene Lgr5 is commonly used for their identification and prospective isolation (Cheng and Leblond, 1974; Barker et al., >>2007<<). Although CBCs are widely believed to maintain the daily proliferative burden of the high-turnover epithelium, they are sensitive to DNA damage\u2013causing agents such as high-dose \u03B3-irradiation (\u03B3-IR), and several independent studies have" . _:b56983360 "intestinal epithelium and colon had no effect on tissue homeostasis, proliferation, or differentiation (not depicted), consistent with published studies demonstrating their functional redundancy (Sakakibara et al., 2002; Li et al., >>2015<<)." . _:b616264702 "2"^^ . _:b616264697 "2"^^ . _:b616264696 . _:b616264696 "2"^^ . _:b616264699 "2"^^ . _:b616264698 "2"^^ . _:b56983407 . _:b56983401 . . . _:b616264737 . _:b56983314 "to DNA damage\u2013causing agents such as high-dose \u03B3-irradiation (\u03B3-IR), and several independent studies have demonstrated that CBCs are largely ablated after \u03B3-IR (Hua et al., 2012; Yan et al., 2012; Metcalfe et al., 2014; Asfaha et al., >>2015<<). Recently, Tao et al. (2015) showed that high Wnt pathway activity and basal crypt positioning sensitize CBCs to DNA damage, leading to their preferential depletion." . _:b616264765 . _:b56983353 "To unequivocally test the functional contribution of Msi proteins to intestinal homeostasis and stem cell self-renewal, we intercrossed Msi1 and Msi2 conditional knockout alleles previously generated in our laboratory (Katz et al., >>2014<<; Park et al., 2014; Li et al., 2015; Wang et al., 2015) with mice harboring a Villin-CreER transgene that drives robust, inducible recombination throughout all epithelial cells in the small intestine and colon (el Marjou et al., 2004; Li" . _:b56983348 "tamoxifen-inducible Cre recombinases (Villin-, Hopx-, Bmi1-, and Lgr5-CreER mice) for conditional deletion of floxed alleles or activation of fluorescent reporter (el Marjou et al., 2004; Barker et al., 2007; Sangiorgi and Capecchi, >>2008<<; Takeda et al., 2011)." . . . . _:b56983349 . . . _:b616264771 . _:b56983306 "introduction" . _:b56983321 "alleles mark a largely overlapping population of Wnt\u2212 reserve ISCs that give rise to active, Wnthigh Lgr5+ CBCs upon division, and consequently all functional cell types of the epithelium over long periods of time (Takeda et al., >>2011<<; Tian et al., 2011; Yan et al., 2012; Li et al., 2014)." . _:b56983387 "previous studies on the mTORC1 complex, inactivation of which similarly results in G1 arrest (Kalaitzidis et al., 2012) as well as failure of the intestinal epithelium to regenerate in response to high dose \u03B3-IR injury (Ashton et al., >>2010<<; Faller et al., 2015)." . _:b56983372 "to such high-dose \u03B3-IR resides in a population of reserve intestinal ISCs that are marked, at least in part, by Hopx-CreER or Bmi1-CreER alleles (and possibly by other proxy reporter alleles; Montgomery et al., 2011; Takeda et al., >>2011<<; Yan et al., 2012; Metcalfe et al., 2014; Asfaha et al., 2015)." . _:b616264715 . . _:b616264639 . _:b616264768 . _:b56983390 "discussion" . . _:b56983393 . _:b616264647 . _:b616264705 . _:b616264641 . _:b56983398 . _:b616264631 . . _:b56983386 . . . _:b616264761 . _:b56983388 . _:b616264763 . _:b616264756 . . _:b56983315 . _:b616264637 . _:b616264721 . _:b616264636 . _:b56983401 "and colon, had no adverse effects on homeostasis, active CBC self-renewal, or Wnt pathway target gene expression, despite the high level of both Msi family members in the Wnthigh CBC stem cells (Potten et al., 2003; Cambuli et al., >>2013<<; Li et al., 2015; Wang et al., 2015)." . . _:b616264666 . _:b616264639 . . _:b56983313 . _:b616264725 . _:b616264638 . _:b56983346 "They were crossed with mice harboring tamoxifen-inducible Cre recombinases (Villin-, Hopx-, Bmi1-, and Lgr5-CreER mice) for conditional deletion of floxed alleles or activation of fluorescent reporter (el Marjou et al., >>2004<<; Barker et al., 2007; Sangiorgi and Capecchi, 2008; Takeda et al., 2011)." . _:b616264720 . _:b616264633 . _:b616264632 . _:b56983316 . _:b56983402 . _:b616264635 . . . _:b56983406 . . _:b616264714 . . _:b56983309 . _:b616264634 . . _:b56983403 "on homeostasis, active CBC self-renewal, or Wnt pathway target gene expression, despite the high level of both Msi family members in the Wnthigh CBC stem cells (Potten et al., 2003; Cambuli et al., 2013; Li et al., 2015; Wang et al., >>2015<<)." . _:b616264645 . _:b56983368 . . _:b56983361 . _:b616264644 . _:b616264647 . . _:b56983369 "or control Msi1flx/flx::Msi2flx/flx mice were treated with five daily doses of tamoxifen and subjected to 12 Gy of ionizing \u03B3-IR 1 wk later to ablate proliferative cells including CBCs (Tian et al., >>2011<<; Tao et al., 2015). In this context, intestinal regeneration (quantified by the number of clonal regenerative crypt foci per unit length 72 h after injury) was severely compromised in the absence of Msi activity (Fig." . _:b56983392 "that observed in the hematopoietic system, with a radioresistant, slow-cycling reserve ISC giving rise to an actively proliferating crypt base columnar cell, whose self-renewal is driven by high Wnt pathway activity (Li and Clevers, >>2010<<; Montgomery et al., 2011; Takeda et al., 2011; Tian et al., 2011; Li et al., 2014; Asfaha et al., 2015)." . _:b616264646 . _:b56983315 "Recently, Tao et al. (>>2015<<) showed that high Wnt pathway activity and basal crypt positioning sensitize CBCs to DNA damage, leading to their preferential depletion." . _:b616264641 . _:b616264668 . _:b616264716 . _:b56983326 . _:b616264640 . _:b56983321 . . _:b56983353 . _:b56983371 . _:b616264643 . _:b56983318 . . _:b616264642 . . . _:b56983409 . _:b616264653 . _:b616264698 . _:b616264648 . _:b616264652 . . _:b616264655 . _:b56983316 "after radiation injury, although the timing of diphtheria toxin administration makes it difficult to dissect the contribution of radioresistant versus de novo\u2013generated Lgr5+ cells to the regenerative process (Metcalfe et al., >>2014<<)." . _:b616264654 . _:b56983312 "epithelium, they are sensitive to DNA damage\u2013causing agents such as high-dose \u03B3-irradiation (\u03B3-IR), and several independent studies have demonstrated that CBCs are largely ablated after \u03B3-IR (Hua et al., 2012; Yan et al., >>2012<<; Metcalfe et al., 2014; Asfaha et al., 2015). Recently, Tao et al. (2015) showed that high Wnt pathway activity and basal crypt positioning sensitize CBCs to DNA damage, leading to their preferential depletion." . _:b616264649 . . _:b616264648 . _:b56983337 . _:b616264651 . . _:b616264650 . . _:b616264661 . . _:b616264660 . _:b616264659 . _:b56983339 "In the intestinal epithelium, Msi proteins are expressed throughout the crypt, including in the active CBC stem cell compartment (Itzkovitz et al., 2011; Li et al., 2014, >>2015<<; Wang et al., 2015)." . _:b616264663 . _:b56983332 "In the hematopoietic system, Msi2 is an important modulator of long-term hematopoietic stem cell (LT-HSC) proliferation and self-renewal (Hope et al., >>2010<<; Ito et al., 2010; Kharas et al., 2010; Park et al., 2014)." . _:b616264662 . _:b616264657 . . _:b616264656 . . _:b56983329 . _:b616264659 . _:b616264741 . . . _:b616264658 . . _:b616264766 . _:b56983410 . _:b616264669 . _:b616264668 . _:b616264760 . _:b616264679 . . _:b616264671 . _:b56983378 "epithelial regeneration after injury, recent data also suggest that the more differentiated progeny of Lgr5+ CBCs can revert to the CBC state and reacquire stem cell activity after CBC ablation with diphtheria toxin (Tetteh et al., >>2016<<). Under basal, noninjury conditions, Hopx-/Bmi1-CreER ISCs are known to give rise to Lgr5+ CBCs upon division (Takeda et al., 2011; Tian et al., 2011; Li et al., 2014)." . _:b616264670 . _:b56983335 . . _:b616264665 . _:b56983383 "Recent findings that high Wnt pathway activity and cell cycling render CBCs susceptible to DNA damage (Tao et al., >>2015<<) support a model in which a more dormant pool of reserve stem cells remains resistant to injury to regenerate the epithelium after damage." . _:b616264664 . . _:b616264667 . _:b56983407 "the phenotype of Msi loss of function is reminiscent of inactivation of mTORC1: both result in failed intestinal regeneration after injury and in G1 cell cycle arrest (Ashton et al., 2010; Kalaitzidis et al., 2012; Faller et al., >>2015<<). We have previously established that in the context of colorectal cancer, mTORC1 is a functionally important target of Msi activity, and both mTORC1 and Msi are required for transformation of the epithelium downstream of APC loss." . _:b616264656 . . _:b56983404 "Reserve ISCs are often referred to as being quiescent or residing in G0, as this state is thought of as being protective to stem cells in unfavorable environments (Cheung and Rando, >>2013<<). Indeed, we observed that the majority of ISCs marked by Hopx-CreER or Bmi1-CreER reside in G0, whereas the remainder of the population cycles actively. We demonstrate that Msi activity controls the exit of reserve ISCs from the G0 state" . _:b616264666 . . _:b616264677 . _:b56983354 . _:b616264676 . _:b616264679 . _:b616264678 . _:b56983351 "results" . . _:b616264673 . . _:b56983388 "mTORC1 complex, inactivation of which similarly results in G1 arrest (Kalaitzidis et al., 2012) as well as failure of the intestinal epithelium to regenerate in response to high dose \u03B3-IR injury (Ashton et al., 2010; Faller et al., >>2015<<)." . . _:b616264672 . _:b616264675 . _:b616264636 . _:b56983324 . _:b616264674 . . . . _:b616264685 . . _:b56983314 . .