The Lgr5 intestinal stem cell signature: robust expression of proposed quiescent ‘+4’ cell markers

Muñoz, Javier; Stange, Daniel E.; Schepers, Arnout; Wetering, Marc van de; Koo, Bon–Kyoung; Itzkovitz, Shalev; Volckmann, Richard; Kung, Kevin S.; Koster, Jan; Radulescu, Sorina; Myant, Kevin; Versteeg, Rogier; Sansom, Owen J.; Es, Johan H. van; Barker, Nick; Oudenaarden, Alexander van; Mohammed, Shabaz; Heck, Albert J. R.; Clevers, Hans

doi:10.1038/emboj.2012.166

Cited by 675 publications

(778 citation statements)

References 48 publications

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“…In control mice, ISCs and secretory Paneth cells were both restricted to the crypt base, as assessed by RNA in situ hybridization for Olfm4 23 and Lysozyme IHC, respectively (Figure 3a and Supplementary Figure 4). Olfm4-positive ISCs had characteristic wedge-like shapes and were mostly proliferative, as shown by Ki67 staining.…”

Section: Resultsmentioning

confidence: 99%

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Transgenic expression of oncogenic BRAF induces loss of stem cells in the mouse intestine, which is antagonized by β-catenin activity

et al. 2014

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Colon cancer cells frequently carry mutations that activate the β-catenin and mitogen-activated protein kinase (MAPK) signaling cascades. Yet how oncogenic alterations interact to control cellular hierarchies during tumor initiation and progression is largely unknown. We found that oncogenic BRAF modulates gene expression associated with cell differentiation in colon cancer cells. We therefore engineered a mouse with an inducible oncogenic BRAF transgene, and analyzed BRAF effects on cellular hierarchies in the intestinal epithelium in vivo and in primary organotypic culture. We demonstrate that transgenic expression of oncogenic BRAF in the mouse strongly activated MAPK signal transduction, resulted in the rapid development of generalized serrated dysplasia, but unexpectedly also induced depletion of the intestinal stem cell (ISC) pool. Histological and gene expression analyses indicate that ISCs collectively converted to short-lived progenitor cells after BRAF activation. As Wnt/β-catenin signals encourage ISC identity, we asked whether β-catenin activity could counteract oncogenic BRAF. Indeed, we found that intestinal organoids could be partially protected from deleterious oncogenic BRAF effects by Wnt3a or by small-molecule inhibition of GSK3β. Similarly, transgenic expression of stabilized β-catenin in addition to oncogenic BRAF partially prevented loss of stem cells in the mouse intestine. We also used BRAF V637E knock-in mice to follow changes in the stem cell pool during serrated tumor progression and found ISC marker expression reduced in serrated hyperplasia forming after BRAF activation, but intensified in progressive dysplastic foci characterized by additional mutations that activate the Wnt/β-catenin pathway. Our study suggests that oncogenic alterations activating the MAPK and Wnt/β-catenin pathways must be consecutively and coordinately selected to assure stem cell maintenance during colon cancer initiation and progression. Notably, loss of stem cell identity upon induction of BRAF/MAPK activity may represent a novel fail-safe mechanism protecting intestinal tissue from oncogene activation.

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Section: Resultsmentioning

confidence: 99%

“…To ascertain the loss of ISCs in crypts upon transgenic BRAF V600K induction, we analyzed the expression of several ISC marker genes 23 by qRT-PCR and found all markers strongly reduced in the induced intestines ( Figure 3b).…”

Section: Above)mentioning

confidence: 99%

Transgenic expression of oncogenic BRAF induces loss of stem cells in the mouse intestine, which is antagonized by β-catenin activity

et al. 2014

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“…We plan subsequently to micro-dissect these LRCs to see if FOXF1 or indeed Bmi1, Tert, Hopx and Lrig1 are factors dictating fate 46 .…”

Section: Discussionmentioning

confidence: 99%

Identification of Lineage-Uncommitted, Long-Lived, Label-Retaining Cells in Healthy Human Esophagus and Stomach, and in Metaplastic Esophagus

et al. 2013

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Background & Aims:The existence of slowly-cycling, adult stem cells has been challenged by the identification of actively cycling cells. We investigated the existence of uncommitted, slowly cycling cells by tracking 5-iodo-2'-deoxyuridine (IdU) label-retaining cells (LRCs) in normal esophagus, Barrett's esophagus (BE), esophageal dysplasia, adenocarcinoma, and healthy stomach tissues from patients.Methods: Four patients (3 undergoing esophagectomy, 1 undergoing esophageal endoscopic mucosal resection for dysplasia and an esophagectomy for esophageal adenocarcinoma) received intravenous infusion of IdU (200 mg per m 2 body surface area, maximum dose of 400 mg) over a 30-min period; the IdU had a circulation t 1/2 of 8hs. Tissues were collected at 7, 11, 29 and 67 days following infusion, from regions of healthy esophagus, BE, dysplasia, adenocarcinoma, and healthy stomach; they were analyzed by in situ hybridization, flow cytometry, and immunohistochemical analyses. Results:No LRCs were found in dysplasias or adenocarcinomas, but there were significant numbers of LRCs in the base of glands from BE tissue, in the papillae of the basal layer of the esophageal squamous epithelium, and in the neck/isthmus region of healthy stomach. These cells cycled slowly, because IdU was retained for at least 67 days and co-labeling with Ki-67 was infrequent. In glands from BE tissues, most cells did not express defensin-5, Muc-2, or chromogranin A, indicating that they were not lineage committed. Some cells labeled for endocrine markers and IdU at 67 days; these cells represented a small population (<0.1%) of epithelial cells at this timepoint. The epithelial turnover time of the healthy esophageal mucosa was approximately 11 days (twice that of the intestine). Conclusions:LRCs of human esophagus and stomach have many features of stem cells (long lived, slow cycling, uncommitted, and multipotent), and can be found in a recognized stem cell niche. Further analyses of these cells, in healthy and metaplastic epithelia is required.4

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“…We observed no differences in the intensity of Brg1 staining between CBC cells and other epithelial cells. Additionally, Brg1 was not found to be differentially expressed between Lgr5 high and Lgr5 low cell populations using various transcriptome and proteome platforms [28]. Therefore, its selective requirement for stem cell homeostasis is likely to be due to differential recruitment to the promoters of the genes involved in control of the stem cell fate.…”

Section: Discussion Brg1 Is Essential For the Small Intestinal Stem Cmentioning

confidence: 99%

“…Other small intestinal stem cell markers including Lrg5, Ascl2, and Bmi1 were not detected among the differentially expressed genes. In order to search for other genes potentially involved in Brg1-mediated stem cell maintenance, we compared the list of differentially expressed genes in Brg1 deficient epithelium to a comprehensive stem cell signature profile as defined by genes preferentially expressed in small intestinal Lgr5 positive cells [28]. Out of 86 differentially expressed genes, 9 genes were present in the intestinal stem cell signature (Supporting Information Table S1).…”

Section: Brg1 Loss Compromises Stem Cell Maintenance In the Murine Smmentioning

confidence: 99%

Brg1 is required for stem cell maintenance in the murine intestinal epithelium in a tissue-specific manner

et al. 2013

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Brg1 is a chromatin remodeling factor involved in mediation of a plethora of signaling pathways leading to its participation in various physiological processes both during development and in adult tissues. Among other signaling pathways, the Wnt pathway has been proposed to require Brg1 for transactivation of its target genes. Given the pivotal role of the Wnt pathway in the maintenance of normal intestinal homeostasis, we aimed to investigate the effects of Brg1 loss on the intestinal physiology. To this end, we deleted Brg1 in the murine small and large intestinal epithelia using a range of transgenic approaches. Pan-epithelial loss of Brg1 in the small intestine resulted in crypt ablation, while partial Brg1 deficiency led to gradual repopulation of the intestinal mucosa with wild-type cells. In contrast, Brg1 loss in the large intestinal epithelium was compensated by upregulation of Brm. We propose that while Brg1 is dispensable for the survival and function of the progenitor and differentiated cells in the murine intestinal epithelium, it is essential for the maintenance of the stem cell population in a tissuespecific manner.

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The Lgr5 intestinal stem cell signature: robust expression of proposed quiescent ‘+4’ cell markers

Cited by 675 publications

References 48 publications

Transgenic expression of oncogenic BRAF induces loss of stem cells in the mouse intestine, which is antagonized by β-catenin activity

Transgenic expression of oncogenic BRAF induces loss of stem cells in the mouse intestine, which is antagonized by β-catenin activity

Identification of Lineage-Uncommitted, Long-Lived, Label-Retaining Cells in Healthy Human Esophagus and Stomach, and in Metaplastic Esophagus

Brg1 is required for stem cell maintenance in the murine intestinal epithelium in a tissue-specific manner

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