Redundant sources of Wnt regulate intestinal stem cells and promote formation of Paneth cells.

BACKGROUND & AIMS Wnt signaling regulates multiple aspects of intestinal physiology, including stem cell maintenance. Paneth cells support stem cells by secreting Wnt, but little is known about the exact sources and primary functions of individual Wnt family members. METHODS We analyzed intestinal tissues and cultured epithelial cells from adult mice with conditional deletion of Wnt3 (Vil-CreERT2;Wnt3fl/fl mice). We also analyzed intestinal tissues and cells from Atoh1 mutant mice, which lack secretory cells. RESULTS Unexpectedly, Wnt3 was dispensable for maintenance of intestinal stem cells in mice, indicating a redundancy of Wnt signals. By contrast, cultured crypt organoids required Paneth cell-derived Wnt3. Addition of exogenous Wnt, or coculture with mesenchymal cells, restored growth of Vil-CreERT2;Wnt3fl/fl crypt organoids. Intestinal organoids from Atoh1 mutant mice did not grow or form Paneth cells; addition of Wnt3 allowed growth in the absence of Paneth cells. Wnt signaling had a synergistic effect with the Lgr4/5 ligand R-spondin to induce formation of Paneth cells. Mosaic expression of Wnt3 in organoids using a retroviral vector promoted differentiation of Paneth cells in a cell-autonomous manner. CONCLUSIONS Wnt is part of a signaling loop that affects homeostasis of intestinal stem and Paneth cells in mice. Wnt3 signaling is required for growth and development of organoid cultures, whereas nonepithelial Wnt signals could provide a secondary physiological source of Wnt.

[1]  H. Clevers,et al.  Tumour suppressor RNF43 is a stem-cell E3 ligase that induces endocytosis of Wnt receptors , 2012, Nature.

[2]  Alexander van Oudenaarden,et al.  The Lgr 5 intestinal stem cell signature : robust expression of proposed quiescent ‘ þ 4 ’ cell markers , 2012 .

[3]  Matthias Stelzner,et al.  A nomenclature for intestinal in vitro cultures. , 2012, American journal of physiology. Gastrointestinal and liver physiology.

[4]  K. Venken,et al.  A Mouse Model of Acrodermatitis Enteropathica: Loss of Intestine Zinc Transporter ZIP4 (Slc39a4) Disrupts the Stem Cell Niche and Intestine Integrity , 2012, PLoS genetics.

[5]  Franck Letourneur,et al.  Functional intestinal stem cells after Paneth cell ablation induced by the loss of transcription factor Math1 (Atoh1) , 2012, Proceedings of the National Academy of Sciences.

[6]  S. Quake,et al.  Identification of a cKit(+) colonic crypt base secretory cell that supports Lgr5(+) stem cells in mice. , 2012, Gastroenterology.

[7]  Bruce J. Aronow,et al.  The Pan-ErbB Negative Regulator Lrig1 Is an Intestinal Stem Cell Marker that Functions as a Tumor Suppressor , 2012, Cell.

[8]  R. Shivdasani,et al.  Intact function of Lgr5 receptor-expressing intestinal stem cells in the absence of Paneth cells , 2012, Proceedings of the National Academy of Sciences.

[9]  O. Klein,et al.  A reserve stem cell population in small intestine renders Lgr5-positive cells dispensable , 2012, Nature.

[10]  H. Clevers,et al.  Controlled gene expression in primary Lgr5 organoid cultures , 2011, Nature Methods.

[11]  J. Epstein,et al.  Interconversion Between Intestinal Stem Cell Populations in Distinct Niches , 2011, Science.

[12]  Matthias Stelzner,et al.  Intestinal Subepithelial Myofibroblasts Support in vitro and in vivo Growth of Human Small Intestinal Epithelium , 2011, PloS one.

[13]  Hans Clevers,et al.  Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. , 2011, Gastroenterology.

[14]  B. Spencer‐Dene,et al.  Delta1 Expression, Cell Cycle Exit, and Commitment to a Specific Secretory Fate Coincide within a Few Hours in the Mouse Intestinal Stem Cell System , 2011, PloS one.

[15]  Hans Clevers,et al.  Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling , 2011, Nature.

[16]  T. Van Loy,et al.  Lgr4 is required for Paneth cell differentiation and maintenance of intestinal stem cells ex vivo , 2011, EMBO reports.

[17]  N. Salzman,et al.  Paneth cells, antimicrobial peptides and maintenance of intestinal homeostasis , 2011, Nature Reviews Microbiology.

[18]  K. Kaestner,et al.  Dll1- and dll4-mediated notch signaling are required for homeostasis of intestinal stem cells. , 2011, Gastroenterology.

[19]  Hans Clevers,et al.  Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts , 2011, Nature.

[20]  Camilla A. Richmond,et al.  Mouse telomerase reverse transcriptase (mTert) expression marks slowly cycling intestinal stem cells , 2010, Proceedings of the National Academy of Sciences.

[21]  Nobutoshi Ito,et al.  Development of a novel selective inhibitor of the Down syndrome-related kinase Dyrk1A. , 2010, Nature communications.

[22]  N. Shroyer,et al.  Atonal homolog 1 is required for growth and differentiation effects of notch/gamma-secretase inhibitors on normal and cancerous intestinal epithelial cells. , 2010, Gastroenterology.

[23]  Bassem A. Hassan,et al.  Intestinal stem cells lacking the Math1 tumour suppressor are refractory to Notch inhibitors , 2010, Nature communications.

[24]  Hans Clevers,et al.  The ets-domain transcription factor Spdef promotes maturation of goblet and paneth cells in the intestinal epithelium. , 2009, Gastroenterology.

[25]  R. Nusse,et al.  Towards an integrated view of Wnt signaling in development , 2009, Development.

[26]  M. Lu,et al.  Wnt2/2b and beta-catenin signaling are necessary and sufficient to specify lung progenitors in the foregut. , 2009, Developmental cell.

[27]  M. Trivett,et al.  Colony stimulating factor-1 dependence of paneth cell development in the mouse small intestine. , 2009, Gastroenterology.

[28]  R. Odze,et al.  Paneth cell differentiation in colonic epithelial neoplasms: evidence for the role of the Apc/beta-catenin/Tcf pathway. , 2009, Human pathology.

[29]  H. Clevers,et al.  Single Lgr5 stem cells build crypt–villus structures in vitro without a mesenchymal niche , 2009, Nature.

[30]  Hans Clevers,et al.  Transcription Factor Achaete Scute-Like 2 Controls Intestinal Stem Cell Fate , 2009, Cell.

[31]  H. Clevers,et al.  Stem cells, self-renewal, and differentiation in the intestinal epithelium. , 2009, Annual review of physiology.

[32]  P. Laurent-Puig,et al.  A genetic study of the role of the Wnt/beta-catenin signalling in Paneth cell differentiation. , 2008, Developmental biology.

[33]  Ujunwa C. Okoye,et al.  Wnt and Frizzled RNA expression in human mesenchymal and embryonic (H7) stem cells , 2008, Journal of molecular signaling.

[34]  P. Sharp,et al.  A system for Cre-regulated RNA interference in vivo , 2008, Proceedings of the National Academy of Sciences.

[35]  M. Capecchi,et al.  Bmi1 is expressed in vivo in intestinal stem cells , 2008, Nature Genetics.

[36]  A. Abo,et al.  R-Spondin family members regulate the Wnt pathway by a common mechanism. , 2008, Molecular biology of the cell.

[37]  H. Clevers,et al.  Identification of stem cells in small intestine and colon by marker gene Lgr5 , 2007, Nature.

[38]  Philippe Blache,et al.  Sox9 regulates cell proliferation and is required for Paneth cell differentiation in the intestinal epithelium , 2007, The Journal of cell biology.

[39]  H. Zoghbi,et al.  Intestine-specific ablation of mouse atonal homolog 1 (Math1) reveals a role in cellular homeostasis. , 2007, Gastroenterology.

[40]  Hans Clevers,et al.  SOX9 is required for the differentiation of paneth cells in the intestinal epithelium. , 2006, Gastroenterology.

[41]  H. Zoghbi,et al.  Gfi1 functions downstream of Math1 to control intestinal secretory cell subtype allocation and differentiation. , 2005, Genes & development.

[42]  Hans Clevers,et al.  Expression pattern of Wnt signaling components in the adult intestine. , 2005, Gastroenterology.

[43]  Louise Howard,et al.  Cellular inheritance of a Cre‐activated reporter gene to determine paneth cell longevity in the murine small intestine , 2005, Developmental dynamics : an official publication of the American Association of Anatomists.

[44]  H. Clevers,et al.  Wnt signalling induces maturation of Paneth cells in intestinal crypts , 2005, Nature Cell Biology.

[45]  Pierre Laurent-Puig,et al.  Crypt-restricted proliferation and commitment to the Paneth cell lineage following Apc loss in the mouse intestine , 2005, Development.

[46]  Ossama Tawfik,et al.  BMP signaling inhibits intestinal stem cell self-renewal through suppression of Wnt–β-catenin signaling , 2004, Nature Genetics.

[47]  Daniel Metzger,et al.  Tissue‐specific and inducible Cre‐mediated recombination in the gut epithelium , 2004, Genesis.

[48]  Hans Clevers,et al.  De Novo Crypt Formation and Juvenile Polyposis on BMP Inhibition in Mouse Intestine , 2004, Science.

[49]  Hans Clevers,et al.  Canonical Wnt signals are essential for homeostasis of the intestinal epithelium. , 2003, Genes & development.

[50]  Andrew P McMahon,et al.  Ectodermal Wnt3/beta-catenin signaling is required for the establishment and maintenance of the apical ectodermal ridge. , 2003, Genes & development.

[51]  Tony Pawson,et al.  β-Catenin and TCF Mediate Cell Positioning in the Intestinal Epithelium by Controlling the Expression of EphB/EphrinB , 2002, Cell.

[52]  Hans Clevers,et al.  The β-Catenin/TCF-4 Complex Imposes a Crypt Progenitor Phenotype on Colorectal Cancer Cells , 2002, Cell.

[53]  Allan Bradley,et al.  Requirement for Wnt3 in vertebrate axis formation , 1999, Nature Genetics.

[54]  Philippe Soriano.  Generalized lacZ expression with the ROSA26 Cre reporter strain , 1999, Nature Genetics.

[55]  Hans Clevers,et al.  Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4 , 1998, Nature Genetics.

[56]  J. Gordon,et al.  Examining the Role of Paneth Cells in the Small Intestine by Lineage Ablation in Transgenic Mice* , 1997, The Journal of Biological Chemistry.