A Possible Role for the High Mobility Group Box Transcription Factor Tcf-4 in Vertebrate Gut Epithelial Cell Differentiation
1999; Elsevier BV; Volume: 274; Issue: 3 Linguagem: Inglês
10.1074/jbc.274.3.1566
ISSN1083-351X
AutoresYoung‐Joo Lee, Bethany Swencki, Sarah A. Shoichet, Ramesh A. Shivdasani,
Tópico(s)Epigenetics and DNA Methylation
ResumoThe Wingless (Wg)/Wnt signaling pathway activates High Mobility Group (HMG)-box transcription factors of the T-cell Factor (Tcf)/Lymphoid Enhancer Factor (LEF) subfamily and mediates diverse functions in development, possibly including endoderm and gut differentiation. Determinants of tissue specificity in the response to Wg/Wnt signaling remain unknown. We have identified Tcf-4 as the predominant Tcf/LEF factor in the developing mouse gut. During fetal development, Tcf-4 mRNA expression is restricted to gut epithelium and specific regions of the brain, the thalamus and roof of the midbrain. In adults, expression is widespread, with highest levels observed in the liver, an endodermally derived organ, and persists in the gastrointestinal tract. Murine Tcf-4 has multiple RNA splice variants with consequently significant heterogeneity in sequences 3′ to the HMG box. Microinjection of mRNA or plasmid DNA encoding Tcf-4 into Xenopus embryos results in ectopic expression of molecular markers of endoderm and differentiated gut epithelium in isolated animal cap explants. Taken together, these findings point to a potentially important function for Tcf-4 in development of the vertebrate gastrointestinal tract. The Wingless (Wg)/Wnt signaling pathway activates High Mobility Group (HMG)-box transcription factors of the T-cell Factor (Tcf)/Lymphoid Enhancer Factor (LEF) subfamily and mediates diverse functions in development, possibly including endoderm and gut differentiation. Determinants of tissue specificity in the response to Wg/Wnt signaling remain unknown. We have identified Tcf-4 as the predominant Tcf/LEF factor in the developing mouse gut. During fetal development, Tcf-4 mRNA expression is restricted to gut epithelium and specific regions of the brain, the thalamus and roof of the midbrain. In adults, expression is widespread, with highest levels observed in the liver, an endodermally derived organ, and persists in the gastrointestinal tract. Murine Tcf-4 has multiple RNA splice variants with consequently significant heterogeneity in sequences 3′ to the HMG box. Microinjection of mRNA or plasmid DNA encoding Tcf-4 into Xenopus embryos results in ectopic expression of molecular markers of endoderm and differentiated gut epithelium in isolated animal cap explants. Taken together, these findings point to a potentially important function for Tcf-4 in development of the vertebrate gastrointestinal tract. The Wingless (Wg) 1The abbreviations used are: Wg, Wingless; HMG, High Mobility Group; Tcf, T-cell factor; LEF, Lymphoid Enhancer Factor; IFABP, intestinal fatty acid binding protein; PCR, polymerase chain reaction; ED, embryonic day; UTR, untranslated region; BCIP, 5-bromo-4-chloro-3-indolyl phosphate. 1The abbreviations used are: Wg, Wingless; HMG, High Mobility Group; Tcf, T-cell factor; LEF, Lymphoid Enhancer Factor; IFABP, intestinal fatty acid binding protein; PCR, polymerase chain reaction; ED, embryonic day; UTR, untranslated region; BCIP, 5-bromo-4-chloro-3-indolyl phosphate. /Wnt signaling pathway mediates essential aspects of early development and has been elucidated through a combination of genetic and biochemical studies in several species (1Miller J.R. Moon R.T. Genes Dev. 1996; 10: 2527-2539Crossref PubMed Scopus (606) Google Scholar). One critical component of this signaling pathway is the cytoplasmic protein Armadillo/β-catenin, which is maintained at a low concentration in the free form in the cytoplasm. Wg/Wnt signaling raises the free β-catenin concentration to permit association with High Mobility Group (HMG)-box proteins of the T-cell Factor (Tcf)/Lymphoid Enhancer Factor (LEF) sub-family and translocation to the nucleus (2Behrens J. von Kries J.P. Kuhl M. Bruhn L. Wedlich D. Grosschedl R. Birchmeier W. Nature. 1996; 382: 638-642Crossref PubMed Scopus (2579) Google Scholar, 3Molenaar M. van de Wetering M. Oosterwegel M. Peterson-Maduro J. Godsave S. Korinek V. Roose J. Destree O. Clevers H. Cell. 1996; 86: 391-399Abstract Full Text Full Text PDF PubMed Scopus (1601) Google Scholar), where the complex is presumed to effect a Wg/Wnt-responsive program of gene expression (4Cavallo R. Rubenstein D. Peifer M. Curr. Opin. Genet. Dev. 1997; 7: 459-466Crossref PubMed Scopus (77) Google Scholar). Transcriptional targets of this signaling pathway include the developmentally regulated genes engrailed,siamois, labial, and ultrabithorax(5Brannon M. Gomperts M. Sumoy L. Moon R.T. Kimelman D. Genes Dev. 1997; 11: 2359-2370Crossref PubMed Scopus (464) Google Scholar, 6Bienz M. Curr. Opin. Genet. Dev. 1997; 7: 683-688Crossref PubMed Scopus (52) Google Scholar, 7Riese J. Yu X. Munnerlyn A. Eresh S. Hsu S.-C. Grosschedl R. Bienz M. Cell. 1997; 88: 777-787Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar), although many other genes are undoubtedly controlled through Wg/Wnt signaling in diverse cell types.Besides the established role of Wg/Wnt signaling in vertebrate mesodermal differentiation and axis formation and in development of the larval cuticle in Drosophila, several lines of evidence point to a role for this pathway in the differentiation of endodermal derivatives. First, genetic and biochemical studies inDrosophila suggest that larval midgut development depends on the Wg signal (8Hoppler S. Bienz M. EMBO J. 1995; 14: 5016-5026Crossref PubMed Scopus (82) Google Scholar, 9van 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. Clevers H. Cell. 1997; 88: 789-799Abstract Full Text Full Text PDF PubMed Scopus (1056) Google Scholar). Second, recent genetic evidence implicates homologs of Wg/Wnt signaling pathway components in gut development inCaenorhabditis elegans (10Rocheleau C.E. Downs W.D. Lin R. Wittmann C. Bei Y. Cha Y. Ali M. Priess J.R. Mello C.C. Cell. 1997; 90: 707-716Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar, 11Thorpe C.J. Schlesinger A. Carter J.C. Bowerman B. Cell. 1997; 90: 695-705Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar). Finally, latency of cytoplasmic β-catenin may be maintained in part through the function of the product of the adenomatous polyposis coli(APC) gene (12Rubinfeld B. Souza B. Albert I. Muller O. Chamberlain S.H. Masiarz F.R. Munemitsu S. Polakis P. Science. 1993; 262: 1731-1734Crossref PubMed Scopus (1173) Google Scholar, 13Su L.-K. Vogelstein B. Kinzler K.W. Science. 1993; 262: 1734-1737Crossref PubMed Scopus (1111) Google Scholar), a frequent target of mutation in human colorectal and other gastrointestinal epithelial malignancies (14Powell S.M. Zilz N. Beazer-Barclay Y. Bryan T.M. Hamilton S.R. Thibodeau S.N. Vogelstein B. Kinzler K.W. Nature. 1992; 359: 235-237Crossref PubMed Scopus (1659) Google Scholar). This potential role of APC in the Wg/Wnt signaling cascade likely reflects a critical function in maintaining gastrointestinal epithelial cell homeostasis. Indeed, a fraction of colorectal tumors with intact APC harbor activating mutations in the β-catenin gene (15Morin P.J. Sparks A.B. Korinek V. Barker N. Clevers H. Vogelstein B. Kinzler K.W. Science. 1997; 275: 1787-1790Crossref PubMed Scopus (3480) Google Scholar), and at least one Tcf/LEF protein, human (h) Tcf-4, is commonly expressed in colon cancer cell lines and mediates transcriptional activation therein (16Korinek V. Barker N. Morin P.J. van Wichen D. de Weger R. Kinzler K.W. Vogelstein B. Clevers H. Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2911) Google Scholar). The sum of these observations strongly implicates β-catenin and Tcf/LEF family proteins in normal gut development and in the pathogenesis of gastrointestinal tumors.The important question of how Wg/Wnt signaling achieves lineage-specific outcomes in diverse cell types remains unresolved and relies in part on a better understanding of the transcriptional effectors of the signaling pathway. In Drosophila, mutations in dTCF (also known as pangolin) result in phenotypes that are identical to those seen in wg mutants (9van 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. Clevers H. Cell. 1997; 88: 789-799Abstract Full Text Full Text PDF PubMed Scopus (1056) Google Scholar, 17Brunner E. Peter O. Schweizer L. Basler K. Nature. 1997; 385: 829-833Crossref PubMed Scopus (444) Google Scholar), implying that Pangolin functions exclusively within this pathway. The correspondence may, however, be more complicated in vertebrates, which have multiple Tcf/LEF-related proteins with varying patterns of expression in embryos and adults. Both Tcf-1 and LEF-1 were originally identified through studies in lymphocytes, where their expression is restricted in adult mice (18Travis A. Amsterdam A. Belanger C. Grosschedl R. Genes Dev. 1991; 5: 880-894Crossref PubMed Scopus (492) Google Scholar, 19van de Wetering M. Oosterwegel M. Dooijes D. Clevers H. EMBO J. 1991; 10: 123-132Crossref PubMed Scopus (447) Google Scholar, 20Waterman M.L. Fischer W.H. Jones K.A. Genes Dev. 1991; 5: 656-669Crossref PubMed Scopus (289) Google Scholar); during fetal development, their expression is wide and largely overlapping (21Oosterwegel M. van de Wetering M. Dooijes D. Klomp L. Winoto A. Georgopoulos K. Meijlink F. Clevers H. Development. 1993; 118: 439-448PubMed Google Scholar, 22van Genderen C. Okamura R.M. Farinas I. Quo R.G. Parslow T.G. Bruhn L. Grosschedl R. Genes Dev. 1994; 8: 2691-2703Crossref PubMed Scopus (814) Google Scholar). Mice lacking Tcf-1 develop normally (23Verbeek S. Izon D. Hofhuis F. Robanus-Maandag E. te Riele H. van de Wetering M. Oosterwegel M. Wilson A. MacDonald H.R. Clevers H. Nature. 1995; 374: 70-74Crossref PubMed Scopus (428) Google Scholar), whereas LEF-1−/− mice manifest developmental abnormalities consistent with a role for LEF-1 in inductive interactions between mesenchymal and epithelial cells (22van Genderen C. Okamura R.M. Farinas I. Quo R.G. Parslow T.G. Bruhn L. Grosschedl R. Genes Dev. 1994; 8: 2691-2703Crossref PubMed Scopus (814) Google Scholar). Development of the gut has long been recognized to depend upon such inductive interactions but Tcf-1 and LEF-1 are not expressed in this organ and absence of either gene does not lead to obvious gut anomalies. Characterization of other members of this HMG-box protein subfamily has been less detailed, and the full extent of the subfamily is unknown.We sought to identify Tcf/LEF proteins that are expressed in the developing vertebrate gut and to examine their function in differentiation of the epithelium. Using degenerate polymerase chain reaction (PCR) cloning, we isolated a single Tcf/LEF family member as the dominant protein of this class in the developing mouse gut. The mRNA and predicted amino acid sequence of this clone are most closely related to those of hTcf-4, previously identified through near uniform expression in colon cancer cell lines (16Korinek V. Barker N. Morin P.J. van Wichen D. de Weger R. Kinzler K.W. Vogelstein B. Clevers H. Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2911) Google Scholar); during preparation of this report, Korinek et al. also reported the cloning of murine (m) Tcf-4 (24Korinek V. Barker N. Willert K. Molenaar M. Roose J. Wagenaar G. Markman M. Lamers W. Destree O. Clevers H. Mol. Cell. Biol. 1998; 18: 1248-1256Crossref PubMed Scopus (292) Google Scholar). In mouse embryos, expression of Tcf-4 mRNA is restricted to the gut epithelium and specific regions of the developing brain; in adults, expression is widespread, with highest levels observed in the liver, an embryonic midgut derivative. mTcf-4 mRNA possesses multiple alternative splice forms, the significance of which is presently unclear. Ectopic expression of one of these mTcf-4 mRNA isoforms in Xenopus embryos induces expression of gastrointestinal epithelial markers in isolated animal cap explants. These observations point to a possibly important function for Tcf-4 in differentiation of the gastrointestinal epithelium and vertebrate gut development.DISCUSSIONThe molecular mechanisms by which the endoderm-derived epithelial cells lining the aerodigestive tract differentiate are largely unknown. Several lines of evidence point to a role for Wg/Wnt signaling in differentiation of the gastrointestinal epithelium, including genetic studies in invertebrates (6Bienz M. Curr. Opin. Genet. Dev. 1997; 7: 683-688Crossref PubMed Scopus (52) Google Scholar, 10Rocheleau C.E. Downs W.D. Lin R. Wittmann C. Bei Y. Cha Y. Ali M. Priess J.R. Mello C.C. Cell. 1997; 90: 707-716Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar) and biochemical analysis of colon carcinomas (16Korinek V. Barker N. Morin P.J. van Wichen D. de Weger R. Kinzler K.W. Vogelstein B. Clevers H. Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2911) Google Scholar). This raises the intriguing possibility that there are gut-specific components or modifiers of the Wnt signaling pathway that mediate tissue-specific responses. However, many of the components of this signaling pathway in gut epithelial cells are largely presumed on the basis of biochemical studies in other developmental systems. We have, therefore, focused on characterizing gut epithelium-specific aspects of Wnt signaling. Here we report that the predominant Tcf/LEF subfamily member present in the developing vertebrate gut is Tcf-4, which is only expressed here and in selected regions of the central nervous system during fetal development.mTcf-4 Structure and ExpressionMouse and human Tcf-4 are virtually identical at the amino acid level, at least in sequences N-terminal to the HMG box. The relationship of Tcf-4 to Tcf-1 extends to a remarkable similarity in splice variants and of a second reading frame in one of the terminal exons that encodes the isoform designated Tcf-4B. This potentially leads to considerable heterogeneity in the C termini of both proteins (32van de Wetering M. Castrop J. Korinek V. Clevers H. Mol. Cell. Biol. 1996; 16: 745-752Crossref PubMed Scopus (178) Google Scholar), and its evolutionary conservation suggests that this may be of functional importance. Although the various C termini of Tcf-1 do not reveal detectable differences in transactivation properties in transient transfection assays (32van de Wetering M. Castrop J. Korinek V. Clevers H. Mol. Cell. Biol. 1996; 16: 745-752Crossref PubMed Scopus (178) Google Scholar), the Tcf-4 isoforms may well harbor functional heterogeneity that is relevant in vivo. Tcf-1 is also heterogeneous at the N terminus, in part reflecting use of alternate promoters (32van de Wetering M. Castrop J. Korinek V. Clevers H. Mol. Cell. Biol. 1996; 16: 745-752Crossref PubMed Scopus (178) Google Scholar). Although we have not observed the same structure in mTcf-4 cDNAs isolated from the fetal gut, the major mRNA isoform present in the brain is distinct from that in other tissues (Fig. 5 A) and probably reflects 5′ heterogeneity; the precedent with Tcf-1 suggests that this also may represent dual promoter usage. The roughly equal frequency with which we isolated alternatively spliced clones suggests, but does not prove, that these splice variants are expressed in low but equal proportions in the fetal gut; our data do not address the existence or relative abundance of mTcf-4 splice variants in the brain.Recently, Korinek et al. (24Korinek V. Barker N. Willert K. Molenaar M. Roose J. Wagenaar G. Markman M. Lamers W. Destree O. Clevers H. Mol. Cell. Biol. 1998; 18: 1248-1256Crossref PubMed Scopus (292) Google Scholar) reported cloning the mouse Tcf-4 gene, and our in situ hybridization studies confirm localization of fetal expression to the gut epithelium and di/mesencephalon. Whereas these investigators failed to detect appreciable mTcf-4 RNA levels in most adult tissues, however, we note that the gene is widely expressed postnatally, with highest levels in the liver, an endoderm-derived organ, and brain. The same pattern is seen in Northern analysis with either a full-length cDNA probe or one corresponding to a fragment of the 3′-UTR, which argues strongly in favor of specificity over cross-reactivity with related species. This apparent discrepancy is best explained by our use of high specific activity probes against Northern blots of poly(A)+ rather than total RNA. Indeed, Korinek et al. (24Korinek V. Barker N. Willert K. Molenaar M. Roose J. Wagenaar G. Markman M. Lamers W. Destree O. Clevers H. Mol. Cell. Biol. 1998; 18: 1248-1256Crossref PubMed Scopus (292) Google Scholar) readily detected mTcf-4 transcripts in poly(A)+ RNA isolated from various segments of the adult intestine, and we were also repeatedly frustrated in efforts to demonstrate expression outside the brain using total RNA. Thus, the expression pattern of Tcf-4 departs significantly from that of either Tcf-1 or LEF-1, both of which are expressed broadly during fetal development but restricted to lymphocytes in adult mice.The restricted fetal expression of Tcf-4 might suggest that it mediates essential aspects of signaling by Wnts or related molecules during development of the gut epithelium and, especially, of the diencephalon (thalamus), where expression levels are highest. Notably, at least 7 of the 16 known mammalian Wntgenes are expressed in various regions of the central nervous system and at least one of these, Wnt-1, is required for mid- and hind-brain development (33McMahon A.P. Bradley A. Cell. 1990; 62: 1073-1085Abstract Full Text PDF PubMed Scopus (1233) Google Scholar, 34Thomas K.R. Capecchi M.R. Nature. 1990; 346: 847-850Crossref PubMed Scopus (725) Google Scholar). The lack of brain abnormalities in mouse embryos lacking Tcf-1 or LEF-1 further hints at a possible requirement for Tcf-4 in central nervous system development or function.Tcf-4 FunctionOur most important finding pertains to the potential role of Tcf-4 in differentiation of endodermally derived tissues. Injection of mTcf-4 in the early Xenopus embryo leads to ectopic expression of endodermal and gut markers in animal cap explants; at sibling tadpole stages beyond 35–36, endodermin specifically marks endodermal derivatives in the gut (29Sasai Y. Lu B. Piccolo S. De Robertis E.M. EMBO J. 1996; 15: 4547-4555Crossref PubMed Scopus (214) Google Scholar), whereas Xlhbox8 and IFABP are specific markers of the duodenum and pancreas (30Gamer L.W. Wright C.V.E. Dev. Biol. 1995; 171: 240-251Crossref PubMed Scopus (113) Google Scholar) and small intestine (31Shi Y.B. Hayes W.P. Dev. Biol. 1994; 161: 48-58Crossref PubMed Scopus (111) Google Scholar), respectively. This implicates Tcf-4 as functioning within a biochemical pathway that promotes gastrointestinal epithelial cell differentiation.Several aspects of this finding merit further discussion. First, the assay does not directly address the extent of cytodifferentiation promoted by Tcf-4; indeed, complete differentiation of endodermal derivatives in vivo is highly dependent on inductive interactions with mesenchyme (35Birchmeier C. Birchmeier W. Annu. Rev. Cell Biol. 1993; 9: 511-540Crossref PubMed Scopus (210) Google Scholar) and probably does not occur in isolated animal caps (26Jones E.A. Abel M.H. Woodland H.R. Roux Arch. Dev. Biol. 1993; 202: 233-239Crossref PubMed Scopus (45) Google Scholar). However, induction of Xlhbox8 mRNA, a specific marker of differentiated foregut derivatives (30Gamer L.W. Wright C.V.E. Dev. Biol. 1995; 171: 240-251Crossref PubMed Scopus (113) Google Scholar), suggests that Tcf-4 might promote relatively advanced differentiation and may be particularly relevant to development of the pancreas. Second, the mechanism of cellular changes induced by Tcf-4 in the animal cap remains uncertain. Untreated animal caps differentiate into atypical, ciliated epidermis in isolation but retain considerable developmental plasticity early on. Ectopic expression of gastrointestinal markers in this tissue may reflect selective expansion of rare progenitor cells with intrinsic endodermal potential, realization of such potential in naive embryonic cells, or perhaps some combination of these possibilities; this consideration applies to all experiments withXenopus animal caps. Finally, loss-of-function studies, including gene targeting in mice, are necessary to establish a functional requirement for Tcf-4 in development of the endoderm and its derivatives in vivo. During the review of this manuscript, Korinek et al. reported that Tcf-4 knockout mice die at birth and their only detectable histopathologic abnormality is a reduced number of cells in small intestinal crypts, suggestive of a depleted stem cell compartment (36Korinek V. Barker N. Moerer P. van Donselaar E. Huls G. Peters P.J. Clevers H. Nat. Genet. 1998; 19: 379-383Crossref PubMed Scopus (1310) Google Scholar). Together with the restricted fetal expression in mice, this observation and our gain-of-function studies in Xenopus implicate Tcf-4 as a regulator of the vertebrate gut epithelium. The similar results with either mRNA or DNA injection in Xenopus embryos further suggest that Tcf-4 can function in this capacity relatively late, i.e. after the mid-blastula transition, in embryonic development.Although genetic experiments in Drosophila suggest that dTcf/Pangolin exclusively subserves Wg signaling (9van 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. Clevers H. Cell. 1997; 88: 789-799Abstract Full Text Full Text PDF PubMed Scopus (1056) Google Scholar, 17Brunner E. Peter O. Schweizer L. Basler K. Nature. 1997; 385: 829-833Crossref PubMed Scopus (444) Google Scholar), it is entirely possible that in the vertebrate gut ligands other than Wnt signal through Tcf-4 to influence epithelial cell differentiation. The full complement of Wnt-related proteins expressed in the developing gut remains unknown, and several proteins without a known connection to Wnt signaling have also been implicated in endodermal and gut epithelial differentiation (37Bronner G. Chu-Lagraff Q. Doe C.Q. Cohen B. Weigel D. Taubert H. Jackle H. Nature. 1994; 369: 664-668Crossref PubMed Scopus (111) Google Scholar, 38Suh E. Traber P.G. Mol. Cell. Biol. 1996; 16: 619-625Crossref PubMed Scopus (450) Google Scholar, 39Kaestner K.H. Silberg D.G. Traber P.G. Schutz G. Genes Dev. 1997; 11: 1583-1595Crossref PubMed Scopus (188) Google Scholar). The required inductive effect of adjacent mesenchyme for complete maturation of the gut epithelium (35Birchmeier C. Birchmeier W. Annu. Rev. Cell Biol. 1993; 9: 511-540Crossref PubMed Scopus (210) Google Scholar) raises the further possibility that Tcf-4 is an effector for signals delivered by cell surface ligands. The interactions between the biochemical pathways involved in these processes and in gastrointestinal tumorigenesis remain to be determined. Identification of Tcf-4 as the predominant Tcf/LEF protein expressed in the developing and adult gut epithelium and its potential role in cytodifferentiation should facilitate molecular approaches to these questions. The Wingless (Wg) 1The abbreviations used are: Wg, Wingless; HMG, High Mobility Group; Tcf, T-cell factor; LEF, Lymphoid Enhancer Factor; IFABP, intestinal fatty acid binding protein; PCR, polymerase chain reaction; ED, embryonic day; UTR, untranslated region; BCIP, 5-bromo-4-chloro-3-indolyl phosphate. 1The abbreviations used are: Wg, Wingless; HMG, High Mobility Group; Tcf, T-cell factor; LEF, Lymphoid Enhancer Factor; IFABP, intestinal fatty acid binding protein; PCR, polymerase chain reaction; ED, embryonic day; UTR, untranslated region; BCIP, 5-bromo-4-chloro-3-indolyl phosphate. /Wnt signaling pathway mediates essential aspects of early development and has been elucidated through a combination of genetic and biochemical studies in several species (1Miller J.R. Moon R.T. Genes Dev. 1996; 10: 2527-2539Crossref PubMed Scopus (606) Google Scholar). One critical component of this signaling pathway is the cytoplasmic protein Armadillo/β-catenin, which is maintained at a low concentration in the free form in the cytoplasm. Wg/Wnt signaling raises the free β-catenin concentration to permit association with High Mobility Group (HMG)-box proteins of the T-cell Factor (Tcf)/Lymphoid Enhancer Factor (LEF) sub-family and translocation to the nucleus (2Behrens J. von Kries J.P. Kuhl M. Bruhn L. Wedlich D. Grosschedl R. Birchmeier W. Nature. 1996; 382: 638-642Crossref PubMed Scopus (2579) Google Scholar, 3Molenaar M. van de Wetering M. Oosterwegel M. Peterson-Maduro J. Godsave S. Korinek V. Roose J. Destree O. Clevers H. Cell. 1996; 86: 391-399Abstract Full Text Full Text PDF PubMed Scopus (1601) Google Scholar), where the complex is presumed to effect a Wg/Wnt-responsive program of gene expression (4Cavallo R. Rubenstein D. Peifer M. Curr. Opin. Genet. Dev. 1997; 7: 459-466Crossref PubMed Scopus (77) Google Scholar). Transcriptional targets of this signaling pathway include the developmentally regulated genes engrailed,siamois, labial, and ultrabithorax(5Brannon M. Gomperts M. Sumoy L. Moon R.T. Kimelman D. Genes Dev. 1997; 11: 2359-2370Crossref PubMed Scopus (464) Google Scholar, 6Bienz M. Curr. Opin. Genet. Dev. 1997; 7: 683-688Crossref PubMed Scopus (52) Google Scholar, 7Riese J. Yu X. Munnerlyn A. Eresh S. Hsu S.-C. Grosschedl R. Bienz M. Cell. 1997; 88: 777-787Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar), although many other genes are undoubtedly controlled through Wg/Wnt signaling in diverse cell types. Besides the established role of Wg/Wnt signaling in vertebrate mesodermal differentiation and axis formation and in development of the larval cuticle in Drosophila, several lines of evidence point to a role for this pathway in the differentiation of endodermal derivatives. First, genetic and biochemical studies inDrosophila suggest that larval midgut development depends on the Wg signal (8Hoppler S. Bienz M. EMBO J. 1995; 14: 5016-5026Crossref PubMed Scopus (82) Google Scholar, 9van 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. Clevers H. Cell. 1997; 88: 789-799Abstract Full Text Full Text PDF PubMed Scopus (1056) Google Scholar). Second, recent genetic evidence implicates homologs of Wg/Wnt signaling pathway components in gut development inCaenorhabditis elegans (10Rocheleau C.E. Downs W.D. Lin R. Wittmann C. Bei Y. Cha Y. Ali M. Priess J.R. Mello C.C. Cell. 1997; 90: 707-716Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar, 11Thorpe C.J. Schlesinger A. Carter J.C. Bowerman B. Cell. 1997; 90: 695-705Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar). Finally, latency of cytoplasmic β-catenin may be maintained in part through the function of the product of the adenomatous polyposis coli(APC) gene (12Rubinfeld B. Souza B. Albert I. Muller O. Chamberlain S.H. Masiarz F.R. Munemitsu S. Polakis P. Science. 1993; 262: 1731-1734Crossref PubMed Scopus (1173) Google Scholar, 13Su L.-K. Vogelstein B. Kinzler K.W. Science. 1993; 262: 1734-1737Crossref PubMed Scopus (1111) Google Scholar), a frequent target of mutation in human colorectal and other gastrointestinal epithelial malignancies (14Powell S.M. Zilz N. Beazer-Barclay Y. Bryan T.M. Hamilton S.R. Thibodeau S.N. Vogelstein B. Kinzler K.W. Nature. 1992; 359: 235-237Crossref PubMed Scopus (1659) Google Scholar). This potential role of APC in the Wg/Wnt signaling cascade likely reflects a critical function in maintaining gastrointestinal epithelial cell homeostasis. Indeed, a fraction of colorectal tumors with intact APC harbor activating mutations in the β-catenin gene (15Morin P.J. Sparks A.B. Korinek V. Barker N. Clevers H. Vogelstein B. Kinzler K.W. Science. 1997; 275: 1787-1790Crossref PubMed Scopus (3480) Google Scholar), and at least one Tcf/LEF protein, human (h) Tcf-4, is commonly expressed in colon cancer cell lines and mediates transcriptional activation therein (16Korinek V. Barker N. Morin P.J. van Wichen D. de Weger R. Kinzler K.W. Vogelstein B. Clevers H. Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2911) Google Scholar). The sum of these observations strongly implicates β-catenin and Tcf/LEF family proteins in normal gut development and in the pathogenesis of gastrointestinal tumors. The important question of how Wg/Wnt signaling achieves lineage-specific outcomes in diverse cell types remains unresolved and relies in part on a better understanding of the transcriptional effectors of the signaling pathway. In Drosophila, mutations in dTCF (also known as pangolin) result in phenotypes that are identical to those seen in wg mutants (9van 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. Clevers H. Cell. 1997; 88: 789-799Abstract Full Text Full Text PDF PubMed Scopus (1056) Google Scholar, 17Brunner E. Peter O. Schweizer L. Basler K. Nature. 1997; 385: 829-833Crossref PubMed Scopus (444) Google Scholar), implying that Pangolin functions exclusively within this pathway. The correspondence may, however, be more complicated in vertebrates, which have multiple Tcf/LEF-related proteins with varying patterns of expression in embryos and adults. Both Tcf-1 and LEF-1 were originally identified through studies in lymphocytes, where their expression is restricted in adult mice (18Travis A. Amsterdam A. Belanger C. Grosschedl R. Genes Dev. 1991; 5: 880-894Crossref PubMed Scopus (492) Google Scholar, 19van de Wetering M. Oosterwegel M. Dooijes D. Clevers H. EMBO J. 1991; 10: 123-132Crossref PubMed Scopus (447) Google Scholar, 20Waterman M.L. Fischer W.H. Jones K.A. Genes Dev. 1991; 5: 656-669Crossref PubMed Scopus (289) Google Scholar); during fetal development, their expression is wide and largely overlapping (21Oosterwegel M. van de Weterin
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