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Wnt signaling in adult intestinal stem cells and cancer - an insight from transgenic mice Vladimir Korinek Department of Cell and Developmental Biology Institute of Molecular Genetics, v.v.i., Prague, CZ Hierarchical model of adult tissue - all tissues contain three basic cell compartments stem cells (101-104 divisions/life) selfrenewal via asymetric cell division progenitors (Transit Amplifying cells; TA cells) differentiated cells Two fundamental questions of stem cell biology How to find stem cells? How to prove that you have them? Find unique marker(s) and perform functionality tests: • • • transplantation to recipient = reconstitution of given tissue in vivo reconstitution of given tissue in vitro lineage tracing experiment track stem cell and its progeny in mouse The gastrointestinal epithelia: the most rapidly renewing tissue of the human body Stem cells reside in the crypts Small intestine villi: non-dividing cells life span – 5 days Colon colon lumen: non-dividing cells life span – 5 days crypts: dividing cells crypts: dividing cells stem cells stem cells Ki67 staining in the small intestine villi: non-dividing cells The crypts contain both dividing and non-dividing cells crypts: dividing cells Stem cell in the epithelium differentiates in four main cell types © Crosnier et al., 2006; Cheng & Leblond, 1974 Assymetric division of stem cell: one stem cell and its progeny is shown in blue Signaling pathways governing the architecture of the intestinal epithelium © Krausova & Korinek, 2012 The Wnt signaling pathway (a simplified schema) The Wnt gene family mammals: 19 members (i.e. Wnts) • all metazoans contain elements of Wnt signaling • genes activated by Wnt signaling: c-Myc, Cyclin D1, Twist, Axin2 • Wnt signaling regulates various cellular processes during development and in adult tissues • aberrant activation can lead to cancer For details visit the Wnt signaling homepage: http://www.stanford.edu/group/nusselab/cgibin/wnt/ adopted from Reya and Clevers, 2006 b-catenin, a protein with a dual function: adhesion and signaling • • b-catenin binds cadherins the protein is highly conserved during evolution Adherens junctions: strong mechanical attachments between adjacent cells anti-b-catenin staining P P P P protein stability cadherin transactivation domain Nuclear b-catenin can be detected in the crypts of the large (and small) intestine © van de Wetering et al., 2002 In the mouse the Wnt-signaling deficient gut displays dramatically reduced numbers of epithelial cells Existence of gene families complicates gene knockout studies (problems with redundancy) wild type 19 Wnt genes Tcf4 KO 4 Lef/Tcf genes © Korinek et al. 1998 The APC gene, the main tumor suppressor gene in the intestine • • • • polyp, a small clump of cells that forms on the lining of the intestine 1-10% of polyps progress to malignancy (carcinoma) the majority of colorectal cancer is sporadic (no familial history) 10-15% of colorectal cancers occur in dominantly inherited patterns (Lynch syndrome, FAP syndrome) Polyp observed during colonoscopy Resected carcinoma © www.wikipedia.org • inactivation of APC is the rate-limiting step in tumor initiation • APC mutations are found in >40% sporadic colorectal tumors Rizk & Barker, 2012 Aberrant Wnt signaling in colorectal tumors • APC binds b-catenin and mediates b-catenin degradation • APC-deficient colorectal tumors display constitutively active Wnt signaling Tumor histology (b-catenin + hematoxyline stainings) Situation in tumor cell (no Wnt added but the APC gene is mutated) anti-b-catenin staining in tumor; detail 1900-2007: search for intestinal stem cells = search for markers of these cells • • • Cheng and Leblond (1974): description of CBC cells = Crypt Base Columnar cells → prediction that CBC cells are the intestinal stem cells van de Wetering et al. (2002): high throughput screen for Wnt signaling target genes in the intestinal cells Barker et al. (2007): Lgr5 is the marker of CBC stem cells Lgr5=leucine-rich-repeat-containing G-protein coupled receptor 5 Transmission Electron Microscopy (bottom of the crypt) © Porter et al. 1997 Paneth cell CBC cells Lgr5 mRNA in situ hybridization in the small intestine © Barker et al. 2007 Labeling of Lgr5-positive cells using knock-in technique © Barker et al., 2007 Lgr5-EGFP-IRES-CreERT knock-in mouse Lgr5+ cells (green fluorescence) in the small intestine CBC stem cells (Lgr5) Paneth cells (lysosyme) © Snipert et al. 2010 Growing organoid derived from one Lgr5+ cell Epithelial organoids (or „miniguts“) – an in vitro system to study the gut epithelium freshly isolated crypt 2 days Organoids culturing conditions: EGF noggin (BMP signaling inhibitor) R-spondin (Wnt signaling agonist) 7 days 9 days Organoid transplantation assay The lineage tracing experiments: Cre recombinase is used to mark irreversibly one cell and its progeny • Cre is type I topoisomerase of phage P1 • catalyzes recombination between loxP sites (34 bps) • sequence of the loxP site is not present in mammalian genome • Cre recombinase fused to the estrogen receptor (=CreERT) is used Deletion of „floxed“ sequences “Reporter” mouse • • • • Rosa26 gene is active in all cell types lacZ encodes b-galactosidase enzyme (=reporter gene) genome of the reporter mouse contains floxed transcription blocker lacZ is produced only when the blocker is recombined out by Cre intercrossing CreERT-expressing mouse Lineage tracing using CreERT-expressing and ROSA26 „reporter“ mouse Cre recombinase fused to the estrogen receptor (CreERT) can be regulated by tamoxifen Situation in genomic DNA of cells expressing CreERT upon addition of tamoxifen Progeny of the breeding + tamoxifen The CreERT/lacZ induction in the adult Lgr5-EGFP-IRES-CreERT mice by tamoxifen Situation in genomic DNA of Lgr5+ cells + tamoxifen Days after tamoxifen injection © Barker et al. 2007 Our aim: to find genes important for physiology of healthy and diseased gut tissue 2007: Chromatin Immunoprecipitation (ChIP) with DNA microarray analysis (ChIP-on-chips) of chromatin isolated from colorectal cancer cells displaying aberrant Wnt signaling APC-deficient cells: Colo320, DLD1, SW480 Cells harboring oncogenic mutations in b-catenin: LS174T Antibodies: anti-TCF; anti-b-catenin Promoter regions (bound by TCF4) of 18 genes were precipitated in all (four) tested cell lines Two genes – Troy and Nkd1 – were selected for further study tumor (APC-Min mouse) Troy mRNA in situ hybridization crypts Generation of Troy reporter mouse using BAC transgenesis Cre expression/activity = Troy expression Situation in genomic DNA of Troy+ cells + Tamoxifen TROY-CreERT x Rosa26 reporter mouse Troy-CreERT2 transgene expression phenocopies the expression pattern of endogenous Troy Long-term labeling of cells (originally) expressing Troy LacZ staining in the small intestine of Troy-CreERT x Rosa26 reporter mice (indicated days after tamoxifen injection) 1 day 3 days 1 day 60 days 60 days Conclusions of the study • • • • COLON Troy is Wnt signaling target gene Troy is a marker of CBC stem cells Troy interacts with Lgr5 Troy inhibits (negatively modulates) Wnt signaling The additional aim: Analysis of the Nkd1 gene All details can be found in the recently published article: The Crew Bobeš Fafílek Nikol Baloghová Kateřina Gajdůšková Adéla Hlavatá/Monika Horázná Dušan Hrčkulák Jitka Stančíková Collaborators Zbynek Kozmik & his lab different mouse strains and ingredients Radek Sedlacek, Inken Beck (Transgenic Unit) transgenic mice Antonio Pombinho & Petr Bartunek HTS David Stanek & his lab microscopy Hynek Strnad & Jan Paces & Michal Kolar (Vlcek Lab) Chips and Bioinformatics Michaela Krausová Vít Kříž Eva Šloncová Jiří Švec Lucie Tůmová Martina Vojtěchová To produce conditional knockouts you need two mouse strains (1) Mouse expressing Cre in a specific tissue (the mouse is generated by transgenesis) Conditional (= tissue-specific) deletion of gene of interest (2) Mouse harboring „floxed“ alleles of gene of interest (gene targeting in ES cells; floxed is mostly smaller but essential part of the gene) Testing Cre-expressing mice (question: in which tissue is Cre produced?) “Reporter” mouse (Rosa26 gene/locus is active in all cell types) Cre-expressing mouse X Progeny of the reporter and Cre mouse Situation in cells expressing Cre Example: Cre is driven by a promoter active in tracheal endoderm of the lung + X-gal lacZ = b-galactosidase Inactivation of the floxed-APC gene in Lgr5(Troy)-positive CBC stem cells without Tamoxifen + Tamoxifen Non-functional APC gene/protein in Lgr5+ cells APC is deleted in CBC stem cells Tumor formation in Lgr5-CreERT mouse harboring a conditional allele of the APC gene b-catenin-positive tumors in the large and small intestine 14 days after tamoxifen injection (floxed-APC x Lgr5-CreERT2 mice) 0.5 mg of tamoxifen per animal 2 mg of tamoxifen per animal small intestine small intestine colon The Yin and Yang of the Wnt pathway in the intestine Nuclear b-catenin can be detected in the crypts of the healthy large (and small) intestine polyp: aberrant Wnt signaling Adherens junctions © van de Wetering et al., 2002 Reminder: nucleus of the cell stimulated with Wnt the crypt: physiological Wnt signaling APC-Min mice – a mouse model of the intestinal cancer • produced using chemical mutagenesis of laboratory mice in the lab (Moser et al., 1990) • APC-Min mice are APC+/- (Su et al., 1992) Min=Multiple intestinal neoplasia Reminder: wild-type APC (2842 AA) truncated APC protein (produced in APC-Min mice)