Tcf as Wnt effector

In 1991, we reported the cloning of a T cell specific transcription factor that we termed TCF1 (1). Related genes exist in genomes throughout the animal kingdom. We have shown in frogs (4), flies (7) and worms (11) that the TCF proteins constitute the effectors of the canonical Wnt pathway. Upon Wnt signaling, ß-catenin binds and activates nuclear TCFs by providing a trans-activation domain. For these studies, we designed the widely used pTOPFLASH Wnt reporters. In the absence of Wnt signaling, we found that Tcf factors associate with proteins of the Groucho family of transcriptional repressors to repress target gene transcription (9).

Wnt signaling in cancer

The tumor suppressor protein APC forms the core of a cytoplasmic complex which binds ß-catenin and targets it for degradation in the proteasome. In APC-deficient colon carcinoma cells, we demonstrated that ß-catenin accumulates and is constitutively complexed with the TCF family member TCF4, providing a molecular explanation for the initiation of colon cancer (5).

Wnt signaling in adult stem cells

In mammals, physiological Wnt signaling is intimately involved with the biology of adult stem cells and self-renewing tissues (18,19). We were the first to link Wnt signaling with adult stem cell biology, when we showed that TCF4 gene disruption leads to the abolition of crypts of the small intestine (8), and that TCF1 gene knockout severely disables the stem cell compartment of the thymus (2). The Tcf4-driven target gene program in colorectal cancer cells is the malignant counterpart of a physiological gene program in selfrenewing crypts (13, 14, 21).

Lgr5 as adult stem cell marker

Amongst the Wnt target genes, we found the Lgr5 gene to be unique in that it marks small cycling cells at crypt bottoms. These cells represent the epithelial stem cells of the small intestine and colon (23), the hair follicle (24), the stomach (28) and many other tissue stem cell types.They also represent the cells-of-origin of adenomas in the gut (25) and within adenomas Lgr5 stem cells act as adenoma stem cells (36). Lgr6 marks multipotent skin stem cells (29).

Lgr5 stem cell biology

Lgr5 crypt stem cells behave in unanticipated ways: Against common belief, they divide constantly and in a symmetric fashion. Stem cells numbers remain fixed because stem cells compete ‘neutrally’  for niche space. Thus, they do not divide asymmetrically (31), a phenomenon that was confirmed by in vivo imaging (43). Daughters of the small intestinal stem cells, the Paneth cells, serve as crypt niche cells by providing Wnt, Notch and EGF signals (30).

The Wnt target gene encoding the transcription factor Achaete scute-like 2 controls the fate of the intestinal stem cell (26).

Lgr5 is the R-spondin receptor

Lgr5 resides in Wnt receptor complexes and mediates signaling of the R-spondin Wnt agonists (32), explaining the unique dependence of Lgr5 stem cells on R-spondins in vivo and in vitro. Two other Wnt target genes, RNF43 and ZNRF3, encode stem cell-specific E3 ligases that downregulate Wnt receptors. They serve in a negative feedback loop to control the size of the stem cell zone (34). Independent work by the Feng Cong lab has first shown that R-spondin, when bound to Lgr5, captures and inactivates RNF43/ZNRF3.

Long-term clonal culturing of organoids from Lgr5 stem cells

Wnt signaling intimately interacts with the BMP and Notch cascades to drive proliferation and inhibit differentiation in intestinal crypts and adenomas (17, 20). Based on these combined insights, we have established Lgr5/R-spondin-based culture systems that allow the outgrowth of single mouse or human Lgr5 stem cells into ever-expanding mini-guts (27), mini-stomachs (28), liver organoids (38, 45), prostate organoids (44) and organoids representing other adult tissues. These epithelial organoid cultures are genetically and phenotypically extremely stable, allowing transplantation of the cultured offspring of a single stem cell, as well as disease modeling by growing organoids directly from diseased patient tissues (45). 

As proof-of-concept, the CFTR locus was repaired in single gut stem cells from two Cystic Fibrosis patients, using CRISPR/Cas9 technology in conjunction with homologous recombination. Repaired stem cells were clonally expanded into mini-guts and shown to contain a functional CFTR channel (42).

Selected publications

1) van de Wetering, M., Oosterwegel, M., Dooijes, D., and Clevers, H.C. Identification and cloning of TCF-1, a T cell-specific transcription factor containing a sequence-specific HMG box.
EMBO J ., 10:123-132 (1991)

2) Verbeek, J.S., Ison, D., Hofhuis, F., Robanus-Maandag, E., te Riele, H., van de Wetering, M., Oosterwegel, M., Wilson, A., MacDonald, H.R. and Clevers, H.C. An HMG box containing T-cell factor required for thymocyte differentiation.
Nature 374: 70-74 (1995)

3) Schilham, M., Oosterwegel, M., Moerer, P., Jing Ya, de Boer, P., van de Wetering, M., Verbeek, S., S., Lamers, W., Kruisbeek, A., Cumano, A., and Clevers, H .Sox-4 gene is required for cardiac outflow tract formation and pro-B lymphocyte expansion.
Nature , 380: 711-714 (1996)

4) Molenaar, M., Van de Wetering, M., Oosterwegel, M., Peterson-Maduro, J., Godsave, S., Korinek, V., Roose, J., Destrée, O. And Clevers, H. Xtcf-3 Transcription factor mediates beta-catenin-induced axis formation in xenopus embryos.
Cell , 86, 391-399 (1996)

5) Korinek, V, Barker, N., Morin, P.J., van Wichen, D., de Weger, R., Kinzler, K.W., Vogelstein, B., and Clevers, H. Constitutive Transcriptional Activation by a beta-catenin-Tcf complex in APC -/- Colon Carcinoma.
Science , 275: 1784-1787 (1997)

6) Morin, P.J., Sparks, A., Korinek, V., Barker, N., Clevers, H., Vogelstein, B., and Kinzler, K. Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC.
Science 275: 1787-1790 (1997)

7) Van de Wetering, M., Cavallo, R., Dooijes, D., Van Beest, M., Van Es, J., Loureiro, J., Ypma, A., Hursh, D., Jones, T., Bejsovec, A., Peifer, M., Mortin, M., and Clevers, H. Armadillo co-activates transcription driven by the product of the Drosophila segment polarity gene dTCF .
Cell , 88, 789-799 (1997)

8) Korinek, V., Barker, N., Moerer, P., van Donselaar, E., Huls, G., Peters, P.J. and Clevers, H. Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4.
Nat Genet 19(4): 379-383 (1998)

9) Roose, J., Molenaar, M., Peterson, J., Hurenkamp, J., Brantjes, H., Moerer, P., van de Wetering, M., Destree, O., and Clevers, H. The Xenopus Wnt effector XTcf-3 interacts with Groucho-related transcriptional repressors.
Nature 395(6702): 608-612 (1998)

10) Roose, J., Huls, G., van Beest, M., Moerer, P., van der Horn, K., Goldschmeding, R., Logtenberg, T., and Clevers, H. Synergie between tumor suppressor APC and the beta-catenin/Tcf4 target gene Tcf1. 
Science 
285: 1923-1926 (1999)

11) Korswagen, R., Herman, M. and Clevers, H. Separate beta-catenins mediate Wnt signaling and cadherin adhesion in C. elegans.
Nature 406: 527-532 (2000)

12) Bienz, M., and Clevers, H. Linking colorectal cancer to Wnt signaling. Review
Cell 103: 311-320 (2000)

13) Van de Wetering, M., Sancho, E., Verweij, C., de Lau, W., Oving, I., Hurlstone, A., Van der Horn, K., Batlle, E., Coudreuse, D., Haramis, A-P., Tjon-Pon-Fong, M., Moerer, P., Van den Born, M., Soete, G., Pals, S., Eilers, M., Medema, R., Clevers, H. The beta-catenin/TCF4 complex imposes a crypt progenitor phenotype on colorectal cancer cells.
Cell 111: 241-250 (2002)

14) Battle, E., Henderson, J.T., Beghtel, H., van den Born, M., Sancho, E., Huls, G., Meeldijk, J., Robertson, J., van de Wetering, M., Pawson, T., Clevers, H. Beta- catenin and TCF mediate cell positioning in the intestinal epithelium by controlling the expression of EphB/ephrinB.
Cell 111: 251-263 (2002)

15) Hurlstone A.F., Haramis A.P., Wienholds E., Begthel H., Korving J., Van Eeden F., Cuppen E., Zivkovic D., Plasterk R.H., Clevers H. , The Wnt/beta-catenin pathway regulates cardiac valve formation.
Nature 425:633-7 (2003)

16) Baas A.F., Kuipers J., van der Wel N.N., Batlle E., Koerten H.K., Peters P.J., Clevers H.C. , Complete polarization of single intestinal epithelial cells upon activation of LKB1 by STRAD.
Cell. 116:457-66 (2004)

17) Haramis A.P., Begthel H., van den Born M., van Es J., Jonkheer S., Offerhaus G.J., Clevers H. , De novo crypt formation and Juvenile Polyposis upon BMP inhibition
Science. 303:1684-6 (2004)

18) Radtke, F and Clevers, H., Self-renewal and cancer of the gut: Two sides of a coin. Review
Science. 307:1904-1909 (2005)

19) Reya T., Clevers H., Wnt signalling in stem cells and cancer. Review
Nature 434:843-850 (2005)

20) Van Es J.H., Van Gijn M.E., Riccio O., Van den Born M., Vooijs M., Begthel H., Cozijnsen M., Robine S., Winton D.J., Radtke F., Clevers H. Notch pathway/gamma-secretase inhibition turns proliferative cells in intestinal crypts and neoplasia into Goblet cells.
Nature 435:959-963 (2005)

21) Batlle E., Bacani J., Begthel H., Jonkheer S., Gregorieff A., Van de Born M., Malats N., Sancho E., Boon E., Pawson T., Gallinger S., Pals S., Clevers H., EphB activity suppresses colorectal cancer progression
Nature 435:1126-1130 (2005)

22) Clevers, H. Wnt/ß-catenin signaling in development and disease.
Cell 127: 469-480 (2006)

23) Barker, N, van Es, J.H., Kuipers, J., Kujala P., van den Born, M., Cozijnsen, M., Korving, J., Begthel, H., Peters, P.C., and Clevers, H. Identification of Stem Cells in Small Intestine and Colon by a Marker Gene LGR5 
Nature, 449:1003-1007 (2007)

24) Jaks V., Barker N., Kasper M., van Es J.H., Snippert H.J., Clevers H., Toftgård R. Lgr5 marks cycling, yet long-lived, hair follicle stem cells.
Nat Genet. 40 : 1291-1299 (2008)

25) Barker N., Ridgway R.A., van Es J.H.,van de Wetering M., Begthel H., van den Born M., Danenberg E., Clarke A.R., Sansom O.J., Clevers H. Crypt Stem Cells as the Cells-of-Origin of Intestinal Cancer
Nature 457:608-611(2009)

26) van der Flier, L.G., van Gijn, M.E., .., and Clevers H. Transcription factor Achaete scute-like 2 (Ascl2) controls intestinal stem cell fate
Cell 136: 903-12 (2009)

27) Sato, T., Vries, R., Snippert, H., van de Wetering, M., Barker, N., Stange, D., van Es, J., Abo, A., Kujala, P., Peters, P., and Clevers, H. Single lgr5 gut stem cells build crypt-villus structures in vitro without a stromal niche 459(7244):262-5
Nature 459 :262-5 (2009)

28) Barker, N, Huch, M., …, and Hans Clevers.  Lgr5+ve stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro
Cell Stem Cell, 6: 25-36 (2010)

29) Snippert, H.J., Haegebarth, A., Kasper, M., Jaks, V., van Es, J.H., Barker, N., van de Wetering,
M., van den Born, M., Begthel, H., Vries, R.G., Stange, D.E., Toftgård, R., Clevers H.  Lgr6 marks stem cells in the hair follicle that generate all cell lineages of the skin.
Science 327: 1385-1389 (2010)

30) Sato, T., van Es, J.H., Snippert, H.J., Stange, D.E., Vries, R.G., van den Born, M., Barker, N., Shroyer, N.F., van de Wetering, M., Clevers, H. Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts.
Nature 469: 415-418 (2010)

31) Snippert, .J., van der Flier, L.G., Sato, T., van Es, J.H., van den Born, M., Kroon-Veenboer, C., Barker, N.,Klein, A.M., van Rheenen, J. Benjamin D. Simons, B.D. and Clevers, H. Intestinal Crypt Homeostasis results from Neutral Competition between Symmetrically Dividing Lgr5 Stem Cells.
Cell 143:134-44 (2010)

32) de Lau, W., Barker, N., … and Clevers, H. Lgr5 homologues associate with Wnt receptors and mediate R-spondin signaling
Nature 476: 293-297 (2011)

33) Li, V.S., Ng, S.S., Boersema, P.J., Low, T.Y., Karthaus, W.R., Gerlach, J.P., Mohammed, S., Heck, A.J., Maurice, M.M., Mahmoudi, T. and Clevers H. Wnt signaling inhibits proteasomal β-catenin degradation within a compositionally intact Axin1 complex.
Cell  149: 1245-1256 (2012)

34) Koo, B-K., Spit, M. Jordens, I., Low, T.Y., Stange, D.E., van de Wetering, M., van Es, J.H., Mohammed, S., Heck, A.J.R., Maurice, M.M. and Hans Clevers. Tumour suppressor RNF43 is a stem-cell E3 ligase that induces endocytosis of Wnt receptors.
Nature 488: 665-669 (2012)

35) Schepers, A.G., Snippert, H.J., Stange, D.E., van den Born, M., van Es, J.H., van de Wetering, M., Clevers, H. Lineage Tracing Reveals Lgr5+ Stem Cell Activity in Mouse Intestinal Adenomas. Science337: 730-735 (2012)

36) van Es, J.H., Sato, T., van de Wetering, M., Lyubimova, A., Yee Nee, A.N., Gregorieff, A., Sasaki, N., Zeinstra, L., van de Born, M., Korving, J., Martens, A.C., Barker, N., van Oudenaarden, A., Clevers, H. DII (+) secretory progenitor cells revert to stem cells upon crypt damage.
Nat Cell Biol 14: 1099-1104 (2012)

37) Boj, S,F., van Es, J.H.,Huch. M., Li, V.S., Jose, A., Hatzis, P., Mokry, M., Haegebarth, A., van den Born, M., Chambon, P., Voshol, P., Dor, Y., Cuppenm E., Fillat, C., Clevers, H. Diabetes risk gene and Wnt effector Tcf7l2/TCF4 controls hepatic response to perinatal and adult metabolic demand.
Cell 151: 1595-1607 (2012)

38) Huch M., Dorell, C., Boj, S.F., van Es, J.H., van de Wetering, M., Li, V.S.W., Hamer, K., Sasaki, N., Finegold, M.J., Haft, A., Grompe, M., Clevers, H. In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration.
Nature 494: 247-250 (2013)

39) Sato, T., Clevers, H., Growing self-organizing mini-guts from a single intestinal stem cells: mechanism and applications. Review
Science 340: 1190-1194 (2013)

40) Clevers, H. The intestinal crypt, a prototype stem cell compartment.
Cell 154: 274-284 (2013)

41) Stange, D.E., Koo, B.K., Huch, M., Sibbel, G., Basak, O., Lyubimova, A.,Kujalla, P., Bartfeld, S., Koster, J., Geahlen, J.H., Peters, P.J., van Es, J., van de Wetering, M., Mills, J.C., Clevers, H. Differentiated Troy+ chief cells act as ‘reserve’ stem cells to generate all lineages of stomach epithelium.
Cell 155: 357-368 (2013)

42) Schwank, G., Koo, B.K., Sasselli, V., Dekkers, J.F., Heo, I., Demircan, T., Sasaki, N., Boymans, S., Cuppen, E., van der Ent, C.K., Nieuwenhuis, E.E., Beekman, J.M. and Clevers, H. Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients.
Cell Stem Cell 13: 653-658 (2013)

43) Ritsma, L., Ellenbroek, S.I., Zomer, A., Snippert, H.J., de Sauvage, F.J., Simons, B.D., Clevers, H., van Rheenen, J.. Intestinal crypt homeostasis revealed at single-stem-cell level by in vivo imaging.
Nature 507: 362-5 (2014)

44) Karthaus, W.R., Iaquinta, P.J., Drost, J., Gracanin, A.., van Boxtel, R., Wongvipat, J., Dowling, C.M., Gao, D., Begthel, H., Sachs, N., Vries, R.G., Cuppen, E., Chen, Y., Sawyers, C.L., Clevers, H.C. Identification of multipotent luminal progenitor cells in human prostate organoid cultures.
Cell 159: 163-75 (2014).

45) Huch, M., Gehart, H., van Boxtel, R., Hamer, K., Blokzijl, F., Verstegen, M.A., Ellis, E., van Wenum, M., Fuchs, S., de Ligt, S., van de Wetering, M., Sasaki, N., Boers, S.J., Kemperman, H., de Jonge, J., Ijzermans, J.N.M., Niewenhuis, E.E.S., Hoekstra, R., Strom, S., Vries, R.G.J., van der Laan, L.J.W., Cuppen, E., Clevers, H. Long-term culture of genome-stable bipotent stem cells from adult human liver
Cell 160: 299-312 (2015)

46) Boj, S.F., Hwang, C.I., Baker, L.A., Chio, I.I., Engle, D.D., Corbo, V., Jager, M., Ponz-Sarvise, M., Tiriac, H., Spector, M.S., Gracanin, A., Oni, T., Yu, K.H., van Boxtel, R., Huch, M., Rivera, K.D., Wilson, J.P., Feigin, M.E., Öhlund, D., Handly-Santana, A., Ardito-Abraham, C.M., Ludwig, M., Elyada, E., Alagesan, B., Biffi, G., Yordanov, G.N., Delcuze, B., Creighton, B., Wright, K., Park, Y., Morsink, F.H., Molenaar, I.Q., Borel Rinkes, I.H., Cuppen, E., Hao, Y., Jin, Y., Nijman, I.J., Iacobuzio-Donahue, C., Leach, S.D., Pappin, D.J., Hammell, M., Klimstra, D.S., Basturk, O., Hruban RH, Offerhaus GJ, Vries RG, Clevers H, Tuveson DA. Organoid models of human and mouse ductal pancreatic cancer.
Cell 160: 324-338 (2015)

47) van de Wetering, M., Francies, H.E., Francis, J.M., Bounova, G., Iorio, F., Pronk, A., van Houdt, W., van Gorp, J., Taylor-Weiner, A., Kester, L., McLaren-Douglas, A., Blokker, J., Jaksani, S., Bartfeld, S., Volckman, R., van Sluis, P., Li, V.S.W., Seepo, S., Sekhar Pedamallu, C., Cibulskis, C., Carter, S.L., McKenna, A., Lawrence, M.S., Lichtenstein, L., Stewart, C., Koster, J., Versteeg, R., van Oudenaarden, A., Saez-Rodriguez, J., Vries, R.G.J., Getz, G., Wessels, L., Stratton, M.R., McDermott, U., Meyerson, M., Garnett, M.J., Clevers, H. Prospective derivation of a ‘Living Organoid Biobank’ of colorectal cancer patients.
Cell 161: 933-945 (2015)

48) Drost, J, van Jaarsveld, R.H., Ponsioen, B., Zimberlin, C., van Boxtel, R., Buijs, A.,Sachs, N., Overmeer, R.M., Offerhaus, G.J., Begthel, H. Korving, J., van de Wetering, M., Schwank, G. Logtenberg, M., Cuppen, E., Snippert, H.J., Medema, J.P., Kops, G. J. P. L., Clevers, H. Sequential cancer mutations in cultured human intestinal stem cells.
Nature 521: 43-47 (2015)

49) Farin, H.F., Jordens, I., Mosa, M.H., Basak, O., Korving, J., Tauriello, D.V.F., de Punder, K., Angers, S., Peters, P.J. Maurice, M.M. and Clevers, H. Visualization of the short-range Wnt gradient in the intestinal stem cell niche. Nature 530: 340-343 (2016)

50) Clevers, H. Modeling development and disease with organoids Cell 165:1586-1597 (2016)