Mouse Label-Retaining Cells are Molecularly and Functionally Distinct From Reserve Intestinal Stem Cells

BACKGROUND & AIMS: Intestinal homeostasis and regeneration after injury are controlled by 2 different types of cells: slow cycling, injury-resistant reserve intestinal stem cells (ISCs) and actively proliferative ISCs. Putative reserve ISCs have been identified using a variety of methods, including CreER insertions at Hopx or Bmi1 loci in mice and DNA label retention. Label-retaining cells (LRCs) include dormant stem cells in several tissues; in the intestine, LRCs appear to share some properties with reserve ISCs, which can be marked by reporter alleles. We investigated the relationships between these populations. METHODS: Studies were performed in Lgr5-EGFP-IRESCreERT2, Bmi1-CreERT2, Hopx-CreERT2, and TRE-H2BGFP::Hopx-CreERT2::lox-stop-lox-tdTomato mice. Intestinal epithelial cell populations were purified; we compared reporter allele-marked reserve ISCs and several LRC populations (marked by H2B-GFP retention) using histologic flow cytometry and functional and single-cell gene expression assays. RESULTS: LRCs were dynamic and their cellular composition changed with time. Short-term LRCs had properties of secretory progenitor cells undergoing commitment to the Paneth or enteroendocrine lineages, while retaining some stem cell activity. Long-term LRCs lost stem cell activity and were a homogenous population of terminally differentiated Paneth cells. Reserve ISCs marked with HopxCreER were primarily quiescent (in G0), with inactive Wnt signaling and robust stem cell activity. In contrast, most LRCs were in G1 arrest and expressed genes that are regulated by the Wnt pathway or are in the secretory lineage. CONCLUSIONS: LRCs are molecularly and functionally distinct from reporter-marked reserve ISCs. This information provides an important basis for future studies of relationships among ISC populations

Heterogeneity in Readouts of Canonical Wnt Pathway Activity within Intestinal Crypts

BACKGROUND: Canonical Wnt pathway signaling is necessary for maintaining the proliferative capacity of mammalian intestinal crypt base columnar stem cells (CBCs). Furthermore, dysregulation of the Wnt pathway is a major contributor to disease, including oncogenic transformation of the intestinal epithelium. Given the critical importance of this pathway, numerous tools have been used as proxy measures for Wnt pathway activity, yet the relationship between Wnt target gene expression and reporter allele activity within individual cells at the crypt base remains unclear. RESULTS: Here, we describe a novel Axin2-CreERT2-tdTomato allele that efficiently marks both WntHigh CBCs and radioresistant reserve intestinal stem cells. We analyze the molecular and functional identity of Axin2-CreERT2-tdTomato-marked cells using single cell gene expression profiling and tissue regeneration assays and find that Axin2 reporter activity does not necessarily correlate with expression of Wnt target genes and, furthermore, that Wnt target genes themselves vary in their expression patterns at the crypt base. CONCLUSIONS: Wnt target genes and reporter alleles can vary greatly in their cell-type specificity, demonstrating that these proxies cannot be used interchangeably. Furthermore, Axin2-CreERT2-tdTomato is a robust marker of both active and reserve intestinal stem cells and is thus useful for understanding the intestinal stem cell compartment

Single-Cell Analysis of Proxy Reporter Allele-Marked Epthelial Cells Establishes Intestinal Stem Cell Hierarchy

The recent development of targeted murine reporter alleles as proxies for intestinal stem cell activity has led to significant advances in our understanding of somatic stem cell hierarchies and dynamics. Analysis of these reporters has led to a model in which an indispensable reserve stem cell at the top of the hierarchy (marked by Bmi1 and Hopx reporters) gives rise to active intestinal stem cells (marked by an Lgr5 reporter). Despite these advances, controversy exists regarding the specificity and fidelity with which these alleles distinguish intestinal stem cell populations. Here, we undertake a comprehensive comparison of widely used proxy reporters including both CreERT2 and EGFP cassettes targeted to the Lgr5, Bmi1, and Hopx loci. Single-cell transcriptional profiling of these populations and their progeny reveals that reserve and active intestinal stem cells are molecularly and functionally distinct, supporting a two-stem-cell model for intestinal self-renewal

The Msi Family of RNA-Binding Proteins Function Redundantly as Intestinal Oncoproteins

Members of the Msi family of RNA-binding proteins have recently emerged as potent oncoproteins in a range of malignancies. MSI2 is highly expressed in hematopoietic cancers, where it is required for disease maintenance. In contrast to the hematopoietic system, colorectal cancers can express both Msi family members, MSI1 and MSI2. Here, we demonstrate that, in the intestinal epithelium, Msi1 and Msi2 have analogous oncogenic effects. Further, comparison of Msi1/2-induced gene expression programs and transcriptome-wide analyses of Msi1/2-RNA-binding targets reveal significant functional overlap, including induction of the PDK-Akt-mTORC1 axis. Ultimately, we demonstrate that concomitant loss of function of both MSI family members is sufficient to abrogate the growth of human colorectal cancer cells, and Msi gene deletion inhibits tumorigenesis in several mouse models of intestinal cancer. Our findings demonstrate that MSI1 and MSI2 act as functionally redundant oncoproteins required for the ontogeny of intestinal cancers

Calorie Restriction Governs Intestinal Epithelial Regeneration through Cell-Autonomous Regulation of mTORC1 in Reserve Stem Cells

Summary: Aging is a complex process associated with a decline in functionality of adult stem cells affecting tissue homeostasis and regeneration. Calorie restriction (CR) is the only experimental manipulation known to extend lifespan and reduce the incidence of age-related disorders across numerous species. These benefits are likely mediated, at least in part, through the preservation of stem cell function. Here, we show that CR enhances the regenerative capacity of the intestinal epithelium through preservation of an injury-resistant reserve intestinal stem cell (ISC) pool. Cell-autonomous activity of mechanistic target of rapamycin complex 1 (mTORC1) governs the sensitivity of reserve ISCs to injury. CR inhibits mTORC1 in these cells, protecting them against DNA damage, while mTORC1 stimulation, either genetically or through nutrient sensing, sensitizes reserve ISCs to injury, thus compromising regeneration of the epithelium. These data delineate a critical role for mTORC1 in epithelial regeneration and inform clinical strategies based on nutrient modulation. : In this study, Yousefi et al. identify reserve intestinal stem cell-autonomous suppression of mTORC1 activity in response to calorie restriction as the basis for enhanced regeneration of the intestinal epithelium after DNA-damaging injury. Conversely, the authors demonstrate that acute nutrient-based stimulation of mTORC1 prior to injury results in reserve stem cell apoptosis and intestinal regenerative failure. Keywords: calorie restriction, radiation injury, reserve intestinal stem cells, intestine, stem cells, mTORC1 signaling, radiosensitivity, regeneratio

Multiphasic and Dynamic Changes in Alternative Splicing during Induction of Pluripotency Are Coordinated by Numerous RNA-Binding Proteins

Alternative splicing (AS) plays a critical role in cell fate transitions, development, and disease. Recent studies have shown that AS also influences pluripotency and somatic cell reprogramming. We profiled transcriptome-wide AS changes that occur during reprogramming of fibroblasts to pluripotency. This analysis revealed distinct phases of AS, including a splicing program that is unique to transgene-independent induced pluripotent stem cells (iPSCs). Changes in the expression of AS factors Zcchc24, Esrp1, Mbnl1/2, and Rbm47 were demonstrated to contribute to phase-specific AS. RNA-binding motif enrichment analysis near alternatively spliced exons provided further insight into the combinatorial regulation of AS during reprogramming by different RNA-binding proteins. Ectopic expression of Esrp1 enhanced reprogramming, in part by modulating the AS of the epithelial specific transcription factor Grhl1. These data represent a comprehensive temporal analysis of the dynamic regulation of AS during the acquisition of pluripotency

The histone variant macroH2A confers functional robustness to the intestinal stem cell compartment

<div><p>A stem cell’s epigenome directs cell fate during development, homeostasis, and regeneration. Epigenetic dysregulation can lead to inappropriate cell fate decisions, aberrant cell function, and even cancer. The histone variant macroH2A has been shown to influence gene expression, guide cell fate, and safeguard against genotoxic stress. Interestingly, mice lacking functional macroH2A histones (hereafter referred to as macroH2A DKO) are viable and fertile; yet suffer from increased perinatal death and reduced weight and size compared to wildtype (WT). Here, we ask whether the ostensible reduced vigor of macroH2A DKO mice extends to intestinal stem cell (ISC) function during homeostasis, regeneration, and oncogenesis. <i>Lgr5-eGFP-IRES-CreERT2</i> or <i>Hopx-CreERT2</i>::<i>Rosa26-LSL-tdTomato</i> ISC reporter mice or the <i>C57BL/6J-Apc</i>^<i>min</i><i>/J</i> murine intestinal adenoma model were bred into a macroH2A DKO or strain-matched WT background and assessed for ISC functionality, regeneration and tumorigenesis. High-dose (12Gy) whole-body γ-irradiation was used as an injury model. We show that macroH2A is dispensable for intestinal homeostasis and macroH2A DKO mice have similar numbers of active crypt-base columnar ISCs (CBCs). MacroH2A DKO intestine exhibits impaired regeneration following injury, despite having significantly more putative reserve ISCs. DKO reserve ISCs disproportionately undergo apoptosis compared to WT after DNA damage infliction. Interestingly, a macroH2A DKO background does not significantly increase tumorigenesis in the <i>Apc</i>^<i>min</i> model of intestinal adenoma. We conclude that macroH2A influences reserve ISC number and function during homeostasis and regeneration. These data suggest macroH2A enhances reserve ISC survival after DNA damage and thus confers functional robustness to the intestinal epithelium.</p></div

Regeneration and DNA damage response in macroH2A DKO intestine.

<p>(A) Left: representative images of Ki-67 immmunohistochemistry within macroH2A WT and DKO proximal jejunum 3 days after exposure of mice to 12 Gy whole body γ-irradiation. 10x objective. Right: quantitation of Ki67⁺ nascent crypt foci per mm. N = 3 mice per condition, mean ± SD. (B) Top: post-IR lineage tracing scheme: macroH2A WT or DKO <i>Hopx-CreERT2</i>::<i>Rosa26-LSL-tdTomato</i> mice were injected with 2mg tamoxifen 48h and 24h prior to treatment with 12 Gy whole-body gamma irradiation, and 72h later sacrificed for analysis. Bottom: representative immunofluorescence of tdTomato lineage tracing (red) counterstained with DAPI (blue) within macroH2A WT and DKO crypts 72 hours after γ-irradiation. 30x objective (C) Left: quantitation of tdTomato tracing events per 500μm, N = 3 mice per condition, mean ± SD. Right: quantitation of tdTomato tracing events per 500μm normalized to percentage of <i>Hopx-tdTomato</i>^<i>+</i> ISCs during homeostasis (values in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185196#pone.0185196.g004" target="_blank">Fig 4A</a>), N = 3 mice per condition, mean ± SD. (D) Experimental scheme highlighting the timing of DNA damage and apoptosis analysis (24h post IR) and regeneration and lineage tracing analysis (72h post IR) (E) Left: flow cytometry plots of SSC-A vs. cleaved caspase-3 content within total crypt epithelium or <i>Hopx-tdTomato</i>^<i>+</i> subpopulations of macroH2A WT and DKO proximal jejunal crypt cells 24 hours after γ-irradiation. Right: quantitation of total crypt epithelium CC3 positivity and <i>Hopx-tdTomato</i>⁺/CC3 double positivity as defined by boxed subpopulation on left. N = 3 mice per condition, mean ± SD. *p<0.05, ***p<0.0005, ns = not significant, Student’s <i>t</i>-test. Scale bars = 100μm.</p

CBC frequency and activity in macroH2A DKO intestine.

<p>(A) Representative phase contrast images of macroH2A WT and DKO crypt-derived organoids, 7 days into culture. Left: 4x objective. Right: 10x objective. (B) Average resulting organoids per well (24-well tissue culture plate) from 100 crypts from macroH2A WT or DKO proximal jejunum from 2-month or 2-year old mice. N = 6 mice per condition, medians, quartiles and ranges of values shown. (C) Aged organoid formation capacity as defined by the average number of organoids that formed as a percent of the number of corresponding organoids that formed from 2-month old crypts per genotype. 10x objective. (D) Left: representative anti-eGFP immunofluorescence of macroH2A WT and DKO jejunum counterstained with DAPI (blue). Right: average Lgr5-eGFP⁺ cells per crypt. N = 6 mice per condition, medians, quartiles and ranges of values shown. (E) Left: representative flow cytometry plots of EdU content vs. DAPI of within <i>Lgr5-eGFP</i>^<i>+</i> subpopulations of macroH2A WT and DKO proximal jejunal crypt cells. Right: quantitation of <i>Lgr5-eGFP</i>/EdU double positivity as defined by boxed subpopulation on left. N = 4 mice per condition, medians, quartiles and ranges of values shown. *p<0.05, ns = not significant, Student’s <i>t</i>-test. Scale bars = 100μm.</p

Reserve ISC frequency and activity in macroH2A DKO intestine.

<p>(A) Left: representative flow cytometry plots of SSC-A vs. <i>Hopx-tdTomato</i>^<i>+</i> signal in proximal small intestine crypt cells from macroH2A WT or DKO mice. Right: quantitation of <i>Hopx-tdTomato</i>^<i>+</i> population as a percentage of crypt epithelial cells. N = 5 mice per condition, mean ± SD. (B) Top: homeostatic lineage-tracing scheme: macroH2A WT and DKO <i>Hopx-CreERT2</i>::<i>Rosa26-LSL-tdTomato</i> mice were injected with 2mg tamoxifen for 2 consecutive days followed by a 2-week chase. Bottom: representative anti-tdTomato immunofluorescence (red) counterstained with DAPI (blue) of macroH2A WT and DKO proximal jejunum 2-weeks after induction of <i>Hopx-tdTomato</i> lineage tracing. 4x objective. (C) Left: quantitation of percentage of villi with tracing events after 2 week chase, N = 3 mice per condition, mean ± SD. Right: percentage of villi with tracing events normalized to percentage of <i>Hopx-tdTomato</i>^<i>+</i> ISCs during homeostasis (values in Fig 4A). N = 3 mice per condition, mean ± SD. (D) Left: representative flow cytometry plots of EdU content vs. DAPI of within <i>Hopx-tdTomato</i>^<i>+</i> subpopulations of macroH2A WT and DKO proximal jejunal crypt cells. Right: quantitation of <i>Hopx-tdTomato</i>/EdU double positivity as defined by boxed subpopulation on left. N = 7 mice per condition, medians, quartiles and ranges of values shown. *p<0.05, ns = not significant, Student’s <i>t</i>-test. Scale bar = 100μm.</p