Phases of Canonical Wnt Signaling During the Development of Mouse Intestinal Epithelium

Background & Aims: Intestinal crypts constitute a niche in which epithelial progenitors respond to Wnt signals, replicate, and prepare to differentiate. Because mutations in Wnt pathway genes lead to intestinal cancer, the role of Wnt signaling in gut epithelial homeostasis is a subject of intense investigation. We studied how Wnt signaling is established during intestine development. Methods: We studied spatiotemporal features of Wnt signaling at formative stages in mouse embryos, when villous projections appear and crypt precursors occupy intervillus regions. We used TOP-GAL transgenic and Axin2LacZ mice, which report faithfully on canonical Wnt activity, relevant molecular markers, and embryos with aberrant β-catenin activation. Results: Developing intestines first display evidence for Wnt signaling after appearance of villi. During villus morphogenesis, intervillus cells proliferate actively but lack signs of canonical Wnt signaling. Surprisingly, in late gestation and briefly thereafter, conspicuous Wnt activity is evident in differentiated, postmitotic villus epithelium. Neither Tcf4, a principal transcriptional effector of intestinal Wnt signals, nor candidate Wnt targets CD44 and cyclinD1 are expressed in late fetal villus cells that show high Wnt activity. Instead, those cells express the related factor Tcf3 and a different Wnt target, c-Myc. Premature and deregulated β-catenin activation causes severe villus dysmorphogenesis in transgenic mice. Conclusions: Relationships among Wnt signaling, epithelial proliferation, and tissue differentiation are reversed in the developing and adult gut. The canonical Wnt pathway has independent, albeit possibly overlapping, functions in early intestinal villi and adult crypts. These observations advance understanding of Wnt functions in intestinal development and disease. Background & Aims: Intestinal crypts constitute a niche in which epithelial progenitors respond to Wnt signals, replicate, and prepare to differentiate. Because mutations in Wnt pathway genes lead to intestinal cancer, the role of Wnt signaling in gut epithelial homeostasis is a subject of intense investigation. We studied how Wnt signaling is established during intestine development. Methods: We studied spatiotemporal features of Wnt signaling at formative stages in mouse embryos, when villous projections appear and crypt precursors occupy intervillus regions. We used TOP-GAL transgenic and Axin2LacZ mice, which report faithfully on canonical Wnt activity, relevant molecular markers, and embryos with aberrant β-catenin activation. Results: Developing intestines first display evidence for Wnt signaling after appearance of villi. During villus morphogenesis, intervillus cells proliferate actively but lack signs of canonical Wnt signaling. Surprisingly, in late gestation and briefly thereafter, conspicuous Wnt activity is evident in differentiated, postmitotic villus epithelium. Neither Tcf4, a principal transcriptional effector of intestinal Wnt signals, nor candidate Wnt targets CD44 and cyclinD1 are expressed in late fetal villus cells that show high Wnt activity. Instead, those cells express the related factor Tcf3 and a different Wnt target, c-Myc. Premature and deregulated β-catenin activation causes severe villus dysmorphogenesis in transgenic mice. Conclusions: Relationships among Wnt signaling, epithelial proliferation, and tissue differentiation are reversed in the developing and adult gut. The canonical Wnt pathway has independent, albeit possibly overlapping, functions in early intestinal villi and adult crypts. These observations advance understanding of Wnt functions in intestinal development and disease. See editorial on page 710. See editorial on page 710. In the adult small intestine, monoclonal populations of epithelial stem cells reside in crypts of Lieberkühn and give rise to 4 daughter cell lineages: Paneth cells, which remain at the crypt base, and enterocytes, goblet, and enteroendocrine cells, which populate the villi.1Cheng H. Leblond C.P. Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine V. Unitarian theory of the origin of the four epithelial cell types.Am J Anat. 1974; 141: 537-561Crossref PubMed Scopus (1173) Google Scholar Because gut mucosa is renewed every 3–5 days, intestinal stem cells or their immediate progeny replicate frequently,2Potten C.S. Booth C. Pritchard D.M. The intestinal epithelial stem cell: the mucosal governor.Int J Exp Pathol. 1997; 78: 219-243Crossref PubMed Scopus (412) Google Scholar, 3Stappenbeck T.S. Wong M.H. Saam J.R. Mysorekar I.U. Gordon J.I. Notes from some crypt watchers: regulation of renewal in the mouse intestinal epithelium.Curr Opin Cell Biol. 1998; 10: 702-709Crossref PubMed Scopus (130) Google Scholar and their behavior is regulated by canonical Wnt signals.4Sancho E. Batlle E. Clevers H. Signaling pathways in intestinal development and cancer.Annu Rev Cell Dev Biol. 2004; 20: 695-723Crossref PubMed Scopus (440) Google Scholar Crypt-villus junctions delimit a zone of active Wnt signaling and cell proliferation in crypts from that of postmitotic differentiation and absence of Wnt activity in villi. Epithelial progenitors in the human colon are frequent targets of cancer. Somatic mutations that initiate colon tumorigenesis occur in the adenomatous polyposis coli (APC) or β-catenin (CTNNB1) genes and cause constitutive activation of the Wnt pathway.4Sancho E. Batlle E. Clevers H. Signaling pathways in intestinal development and cancer.Annu Rev Cell Dev Biol. 2004; 20: 695-723Crossref PubMed Scopus (440) Google Scholar, 5Kinzler K.W. Vogelstein B. Lessons from hereditary colorectal cancer.Cell. 1996; 87: 159-170Abstract Full Text Full Text PDF PubMed Scopus (4326) Google Scholar β-catenin accumulates in affected cells and functions as a coactivator for transcription factors of the Tcf/LEF family, mainly Tcf4.6Korinek V. Barker N. Morin P.J. van Wichen D. de Weger R. Kinzler K.W. Vogelstein B. Clevers H. Constitutive transcriptional activation by a b-catenin-Tcf complex in APC−/− colon carcinoma.Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2977) Google Scholar, 7Morin P.J. Sparks A.B. Korinek V. Barker N. Clevers H. Vogelstein B. Kinzler K.W. Activation of b-catenin-Tcf signaling in colon cancer by mutations in b-catenin or APC.Science. 1997; 275: 1787-1790Crossref PubMed Scopus (3556) Google Scholar The resulting molecular derangements impose properties of crypt epithelial progenitors, including replication capacity, on transformed cells.8van 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 β-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells.Cell. 2002; 111: 241-250Abstract Full Text Full Text PDF PubMed Scopus (1764) Google Scholar Acute APC loss in the adult gut rapidly expands the crypt compartment and impairs differentiation of secretory cell lineages,9Andreu P. Colnot S. Godard C. Gad S. Chafey P. Niwa-Kawakita M. Laurent-Puig P. Kahn A. Robine S. Perret C. Romagnolo B. Crypt-restricted proliferation and commitment to the Paneth cell lineage following Apc loss in the mouse intestine.Development. 2005; 132: 1443-1451Crossref PubMed Scopus (249) Google Scholar, 10Sansom O.J. Reed K.R. Hayes A.J. Ireland H. Brinkmann H. Newton I.P. Batlle E. Simon-Assmann P. Clevers H. Nathke I.S. Clarke A.R. Winton D.J. Loss of Apc in vivo immediately perturbs Wnt signaling, differentiation, and migration.Genes Dev. 2004; 18: 1385-1390Crossref PubMed Scopus (674) Google Scholar whereas inhibition of Wnt signaling in adult mouse intestine reduces epithelial proliferation, coincident with loss of nuclear β-catenin and of crypt structures.11Pinto D. Gregorieff A. Begthel H. Clevers H. Canonical Wnt signals are essential for homeostasis of the intestinal epithelium.Genes Dev. 2003; 17: 1709-1713Crossref PubMed Scopus (827) Google Scholar Less is known about the timing and manner in which a local niche for Wnt signaling is established in the developing gut. Intestinal villus morphogenesis begins when mesenchymal aggregates impinge on the basal aspect of the epithelium to produce primitive folds. The pseudostratified squamous epithelium then converts into a single layer of columnar cells that line mesenchymal stalks or the lamina propria (Figure 1A); this occurs around embryonic day (E) 14 in the mouse, E18 in the rat, and between 9 and 10 weeks in human gestation.12Trier J.S. Moxey P.C. Morphogenesis of the small intestine during fetal development.Ciba Found Symp. 1979; 70: 3-29PubMed Google Scholar Cells within the multilayered precursor tissue are joined by tight junctions and desmosomes, which rearrange during villus morphogenesis.13Mathan M. Moxey P.C. Trier J.S. Morphogenesis of fetal rat duodenal villi.Am J Anat. 1976; 146: 73-92Crossref PubMed Scopus (161) Google Scholar, 14Madara J.L. Neutra M.R. Trier J.S. Junctional complexes in fetal rat small intestine during morphogenesis.Dev Biol. 1981; 86: 170-178Crossref PubMed Scopus (52) Google Scholar Rudimentary features of cell maturation, including microvilli and secretory granules, appear coincidentally, and cell numbers remain roughly constant, with attrition of a minority of superficial cells and virtually no dilution of bromodeoxyuridine (BrdU) or [3H]-thymidine labels introduced earlier.12Trier J.S. Moxey P.C. Morphogenesis of the small intestine during fetal development.Ciba Found Symp. 1979; 70: 3-29PubMed Google Scholar, 13Mathan M. Moxey P.C. Trier J.S. Morphogenesis of fetal rat duodenal villi.Am J Anat. 1976; 146: 73-92Crossref PubMed Scopus (161) Google Scholar, 14Madara J.L. Neutra M.R. Trier J.S. Junctional complexes in fetal rat small intestine during morphogenesis.Dev Biol. 1981; 86: 170-178Crossref PubMed Scopus (52) Google Scholar Thus, the earliest differentiated cells to populate intestinal villi are the same cells that recently were stacked in layers. By contrast, subsequent generations of villus epithelial cells originate in ordered differentiation and migration of intervillus or crypt progenitors.2Potten C.S. Booth C. Pritchard D.M. The intestinal epithelial stem cell: the mucosal governor.Int J Exp Pathol. 1997; 78: 219-243Crossref PubMed Scopus (412) Google Scholar, 3Stappenbeck T.S. Wong M.H. Saam J.R. Mysorekar I.U. Gordon J.I. Notes from some crypt watchers: regulation of renewal in the mouse intestinal epithelium.Curr Opin Cell Biol. 1998; 10: 702-709Crossref PubMed Scopus (130) Google Scholar To determine when the earliest Wnt-responsive stem/progenitor cells appear, we examined gene expression in relation to mouse gut epithelial development. TOP-GAL transgenic mice and strain-matched (CD1) controls were purchased from Jackson Laboratories (Bar Harbor, ME). Sonic hedgehog (Shh)+/Cre mice originated by targeted insertion of a GFP-Cre fusion complementary DNA (cDNA) into the Shh locus, allowing expression of Cre recombinase under control of Shh regulatory sequences.15Harfe B.D. Scherz P.J. Nissim S. Tian H. McMahon A.P. Tabin C.J. Evidence for an expansion-based temporal Shh gradient in specifying vertebrate digit identities.Cell. 2004; 118: 517-528Abstract Full Text Full Text PDF PubMed Scopus (775) Google Scholar Catnb+/lox(ex3) mice carry an allele with LoxP sites flanking exon 3 of the β-catenin gene.16Harada N. Tamai Y. Ishikawa T. Sauer B. Takaku K. Oshima M. Taketo M.M. Intestinal polyposis in mice with a dominant stable mutation of the β-catenin gene.EMBO J. 1999; 18: 5931-5942Crossref PubMed Scopus (993) Google Scholar Axin2LacZ mice have the β-galactosidase reporter cDNA embedded in the Axin2 locus25Yu H.M. Jerchow B. Sheu T.J. Liu B. Costantini F. Puzas J.E. Birchmeier W. Hsu W. The role of Axin2 in calvarial morphogenesis and craniosynostosis.Development. 2005; 132: 1995-2005Crossref PubMed Scopus (281) Google Scholar and were kindly provided by Max-Delbrück Center, Berlin, Germany. Animals were housed and handled according to protocols approved by institutional committees. The morning of identification of a copulation plug was designated as day 0.5 of gestation. Whole mouse embryos or organs were isolated in Ca2+- and Mg2+-free Hank’s balanced salt solution (Invitrogen, Carlsbad, CA), fixed for 15 minutes with 4% paraformaldehyde in phosphate-buffered saline (PBS), washed 3 times in PBS, and incubated in staining solution (PBS, pH 7.2 containing 1 mg/mL 5-bromo-4-chloro-3-indoyl-β-D-galactoside, 5 mmol/L K3Fe(CN)6, 5 mmol/L K4Fe(CN)63H2O, 1 mmol/L MgCl2, 0.01% sodium deoxycholate, 0.02 % NP-40) for 9–10 hours at 37°C. Samples were photographed under a binocular microscope, postfixed in 4% paraformaldehyde for 1 hour at 4°C, dehydrated in ethanol, and embedded in paraffin. Ten-micrometer sections, counterstained with eosin or nuclear fast red, were examined by light microscopy, and images were captured with a CCD camera using QCapture and Adobe Photoshop 7.0 software. Intestines were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, and sections of 5- to 6-μm thickness were prepared. For antigen retrieval, slides were incubated in a pressure cooker for 3 minutes in 10 mmol/L sodium citrate, pH 6.0. Samples were incubated for 24 hours at 4°C with one of the following mouse monoclonal antibodies (Ab): Tcf4 (1:500, Upstate Biotechnology, Charlottesville, VA), Tcf3/4 (1:250, Upstate), activated β-catenin (1:500, Upstate), CD44 (1:100, Becton Dickinson, San Diego, CA), BrdU (1:100, Developmental Studies Hybridoma Bank, University of Iowa), proliferating cell nuclear antigen (1:150, Zymed, San Francisco, CA), Cdx2 (1:20, Biogenex, San Ramon, CA; MU392A-UC) or with rabbit antisera against c-Myc (1:200, Santa Cruz Biotechnology, Santa Cruz, CA), cyclin D1 (1:500, Santa Cruz), Lef-1 (1:1000, gift of Rudi Grosschedl), or Ki67 (1:2000, Vector Labs, Burlingame, CA). Samples were washed, incubated with biotinylated goat anti-mouse or anti-rabbit IgG, and treated with avidin-biotin-peroxidase complex (Vector Laboratories). The reaction was visualized with diaminobenzidine (DAB) hydrochloride (Sigma Chemical Co., St. Louis, MO). RNA in situ hybridization was performed as described previously15Harfe B.D. Scherz P.J. Nissim S. Tian H. McMahon A.P. Tabin C.J. Evidence for an expansion-based temporal Shh gradient in specifying vertebrate digit identities.Cell. 2004; 118: 517-528Abstract Full Text Full Text PDF PubMed Scopus (775) Google Scholar using digoxigenin-labeled Apoa1, Ybx1, or sense control riboprobes (1 μg/mL), alkaline phosphatase-conjugated anti-digoxigenin Ab (Roche, Indianapolis, IN), and nitroblue-tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. Transcriptional profiles of the developing mouse gut reveal widespread activation of molecular markers of the mature epithelium between E13 and E15, coincident with early villus morphogenesis.17Lepourcelet M. Tou L. Cai L. Sawada J. Lazar A.J. Glickman J.N. Williamson J.A. Everett A.D. Redston M. Fox E.A. Nakatani Y. Shivdasani R.A. Insights into developmental mechanisms and cancers in the mammalian intestine derived from serial analysis of gene expression and study of the hepatoma-derived growth factor (HDGF).Development. 2005; 132: 415-427Crossref PubMed Scopus (77) Google Scholar Even in the earliest villi, in situ hybridization localized many messenger RNA (mRNA) in patterns characteristic of adult intestine (Figure 1B). Transcripts that appear in differentiated cells in adults are confined to villi in E15 mouse embryos, whereas those restricted to adult crypts localize in intervillus cells. Thus, the earliest villus-lining cells, which converted from undifferentiated squamous epithelium, are readily distinguished from intervening crypt precursors at the level of gene expression; even in the absence of structured crypts, boundaries of gene expression between villi and intervillus regions appear at the same time that the epithelium is remodeled. This demarcation of gene expression domains precedes zonation of epithelial cell replication. Ki67 (Figure 1C) and proliferating cell nuclear antigen (data not shown) immunostaining of E15.5 gut identifies proliferation not only in intervillus cells but also in many cells that lie along the newly formed villi and express differentiation-related transcripts. Thus, gene expression in bowel mucosa is regionalized in conjunction with early villus morphogenesis. Mucosal Wnt signaling in adult intestine is confined to the crypt, including progenitor and Paneth cells.4Sancho E. Batlle E. Clevers H. Signaling pathways in intestinal development and cancer.Annu Rev Cell Dev Biol. 2004; 20: 695-723Crossref PubMed Scopus (440) Google Scholar, 18van Es J.H. Jay P. Gregorieff A. van Gijn M.E. Jonkheer S. Hatzis P. Thiele A. van den Born M. Begthel H. Brabletz T. Taketo M.M. Clevers H. Wnt signalling induces maturation of Paneth cells in intestinal crypts.Nat Cell Biol. 2005; 7: 381-386Crossref PubMed Scopus (525) Google Scholar To determine when prospective crypt cells first acquire this distinguishing feature, we assessed LacZ staining in TOP-GAL transgenic embryos, which carry the Echerichia coli β-galactosidase gene under the control of Wnt/Tcf-responsive cis-elements.19DasGupta R. Fuchs E. Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and differentiation.Development. 1999; 126: 4557-4568Crossref PubMed Google Scholar These animals report faithfully on canonical Wnt activity, as we reported previously20Kim B.M. Buchner G. Miletich I. Sharpe P.T. Shivdasani R.A. The stomach mesenchymal transcription factor Barx1 specifies gastric epithelial identity through inhibition of transient Wnt signaling.Dev Cell. 2005; 8: 611-622Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar and verified in the vibrissae, brain, and tracheobronchial tree (Figure 2A and 2B), known sites of active Wnt signaling in midgestation embryos.19DasGupta R. Fuchs E. Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and differentiation.Development. 1999; 126: 4557-4568Crossref PubMed Google Scholar, 21Joyner A.L. Engrailed, Wnt and Pax genes regulate midbrain-hindbrain development.Trends Genet. 1996; 12: 15-20Abstract Full Text PDF PubMed Scopus (385) Google Scholar, 22Okubo T. Hogan B.L. Hyperactive Wnt signaling changes the developmental potential of embryonic lung endoderm.J Biol. 2004; 3: 11.1-11.17Crossref Google Scholar In adult gut epithelium, LacZ signal is confined to the crypt base (Figure 2C), corresponding to the location of Paneth cells, known recipients of intestinal Wnt signals.18van Es J.H. Jay P. Gregorieff A. van Gijn M.E. Jonkheer S. Hatzis P. Thiele A. van den Born M. Begthel H. Brabletz T. Taketo M.M. Clevers H. Wnt signalling induces maturation of Paneth cells in intestinal crypts.Nat Cell Biol. 2005; 7: 381-386Crossref PubMed Scopus (525) Google Scholar Surprisingly, LacZ staining in intervillus regions, the functional precursors of intestinal crypts, is prominent only on the third postnatal day (Figure 2N). Rather, it is the earliest intestinal villi that display strong β-galactosidase activity in late gestation and immediately after birth. On the CD1 genetic background, LacZ activity reproducibly appears first between E16.25 and E16.5 (Figure 2G and 2H). Prior to this period, β-galactosidase activity is detected with radial asymmetry in stomach endoderm (Figure 2F), as we reported previously,20Kim B.M. Buchner G. Miletich I. Sharpe P.T. Shivdasani R.A. The stomach mesenchymal transcription factor Barx1 specifies gastric epithelial identity through inhibition of transient Wnt signaling.Dev Cell. 2005; 8: 611-622Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar but not at all in intestine (Figure 2D and 2E), including the pseudostratified squamous lining at E13 or E14 (data not shown). Villus LacZ staining increases steadily in intensity until E18.5 (Figure 2J and 2M) and persists until approximately 2 days after birth. Intervillus cells, which subsequently show strong β-galactosidase activity (Figure 2N), lack a signal for most of the duration that one is detected in villus surface cells. Brünner’s glands, a submucosal network of branching secretory ducts23Grossman M.L. The glands of Brunner.Physiol Rev. 1958; 38: 675-690PubMed Google Scholar that is confined to the first portion of the duodenum in mice, also show strong LacZ staining in weanling TOP-GAL mice (Figure 2P). The embryos for our study were derived from crosses between wild-type and hemizygote transgenic mice so that only half the progeny carried the TOP-GAL reporter; we observed villus β-galactosidase activity in all transgenic embryos and never in nontransgenic littermates (Figure 2I). β-galactosidase activity in any tissue likely correlates imperfectly with the duration of active Wnt signaling: the enzyme must first accumulate, and, because it is highly stable, its activity may outlast brief periods of Wnt signaling. Moreover, although differences in LacZ signal intensity likely reflect true variation in the degree of Wnt activity, there is probably a threshold below which Wnt signaling is undetected in TOP-GAL tissues. LacZ mRNA in situ hybridization confirmed exclusively villus expression in the fetal gut; we detected weak RNA expression at E16.0 (data not shown) and robust expression thereafter (Figure 3A). Our data hence reveal a perinatal window in which canonical Wnt signaling is active in newly formed villi but not in intervillus cells. Canonical Wnt signaling in the latter population first becomes evident on the day of birth in a fraction (∼20%) of villus profiles; at this stage, the signal remains considerably more prominent in villi, and the majority of intervillus regions still lack β-galactosidase activity (green arrowheads in Figure 3B). Wnt signaling shifts completely from villi to intervillus regions over the next 2 days (Figure 2N). These staining patterns were unchanged when transgenic TOP-GAL mice were crossed into mixed CD1-C57BL/6 and CD1-129/Sv genetic backgrounds. Because intestinal epithelium differentiates in a proximal to distal wave, we surveyed the length of the gut to determine the timing of villus activation of the Wnt pathway. TOP-GAL mice showed the predicted proximal-to-distal gradient of intestinal LacZ staining (see Supplemental Figure 1 online at www.gastrojournal.org). As an independent indicator of canonical Wnt signaling, we assessed fetal and postnatal mouse intestines for nuclear localization of β-catenin, a marker of Wnt pathway activation. As expected, immunohistologic detection of β-catenin is confined to the crypts of Lieberkühn in adult mice and especially to Paneth cells at the base (Figure 3C). In contrast, and in agreement with the β-galactosidase data shown in Figure 2, between E17 and 2 days after birth, nuclear β-catenin is found in cells lining each villus (Figure 3D) but not in intervillus cells (detail in Figure 3E); it appears consistently in the intervillus zone only after the second postnatal day (data not shown). We also examined expression of c-Myc, one of the best characterized targets of canonical Wnt signaling.8van 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 β-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells.Cell. 2002; 111: 241-250Abstract Full Text Full Text PDF PubMed Scopus (1764) Google Scholar, 11Pinto D. Gregorieff A. Begthel H. Clevers H. Canonical Wnt signals are essential for homeostasis of the intestinal epithelium.Genes Dev. 2003; 17: 1709-1713Crossref PubMed Scopus (827) Google Scholar, 24He T.C. Sparks A.B. Rago C. Hermeking H. Zawel L. da Costa L.T. Morin P.J. Vogelstein B. Kinzler K.W. Identification of c-MYC as a target of the APC pathway.Science. 1998; 281: 1509-1512Crossref PubMed Scopus (4120) Google Scholar c-Myc distribution is virtually identical to that of nuclear β-catenin, with highest expression in nuclei near villus tips, lower levels toward the villus base, and none in intervillus regions (Figure 3F, and detail in Figure 3G). Finally, we studied an independent murine reporter strain for Wnt activity, where LacZ cDNA replaces the coding sequence of the Axin2/conductin gene,25Yu H.M. Jerchow B. Sheu T.J. Liu B. Costantini F. Puzas J.E. Birchmeier W. Hsu W. The role of Axin2 in calvarial morphogenesis and craniosynostosis.Development. 2005; 132: 1995-2005Crossref PubMed Scopus (281) Google Scholar an established target of Wnt signaling in diverse tissues.26Jho E.H. Zhang T. Domon C. Joo C.K. Freund J.N. Costantini F. Wnt/beta-catenin/Tcf signaling induces the transcription of Axin2, a negative regulator of the signaling pathway.Mol Cell Biol. 2002; 22: 1172-1183Crossref PubMed Scopus (1366) Google Scholar Just as in TOP-GAL embryos, the intestine in E18.5 Axin2LacZ mice showed high β-galactosidase activity in villus epithelial cells and sparing of the intervillus regions (Figure 3H). Thus, 4 informative markers, reporter activity in TOP-GAL and Axin2LacZ mice, nuclear β-catenin, and c-Myc expression, reveal canonical Wnt signaling over the villus surface in mouse gestation and earlier than its restriction in prospective crypt precursors. Villus formation and early activation of intestinal differentiation genes both occur before the first signs of intestinal Wnt signaling in TOP-GAL embryos (Figure 1, Figure 2); accordingly, villus Wnt activity in the perinatal period must be associated with processes other than these. In adult gut, canonical Wnt signaling promotes crypt-specific behaviors and distinguishes proliferating cells from their differentiated progeny.4Sancho E. Batlle E. Clevers H. Signaling pathways in intestinal development and cancer.Annu Rev Cell Dev Biol. 2004; 20: 695-723Crossref PubMed Scopus (440) Google Scholar We evaluated intestines after treating pregnant dams with a 2-hour pulse of BrdU. By E17.5, when Wnt-responsive TOP-GAL expression is readily detected in mouse intestine, only rare proliferating (BrdU+) cells are found in intestinal villi, the location of Wnt activity; epithelial proliferation is largely restricted to the intervillus regions, at which LacZ signals are absent (Figure 3I). Thus, Wnt signaling and epithelial cell proliferation are almost mutually exclusive in the late fetal gut. Wnt activity later localizes in the proliferative zone (Figure 2C and 2N, and data not shown), as expected. Canonical Wnt signals are transduced through Tcf/LEF family proteins. Tcf4 is especially abundant in, and virtually specific to, adult gut epithelium,6Korinek V. Barker N. Morin P.J. van Wichen D. de Weger R. Kinzler K.W. Vogelstein B. Clevers H. Constitutive transcriptional activation by a b-catenin-Tcf complex in APC−/− colon carcinoma.Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2977) Google Scholar, 27Barker N. Huls G. Korinek V. Clevers H. Restricted high level expression of Tcf-4 protein in intestinal and mammary gland epithelium.Am J Pathol. 1999; 154: 29-35Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar and Tcf4−/− mice die soon after birth from presumed complications of defects confined to the gut.28Korinek V. Barker N. Moerer P. van Donselaar E. Huls G. Peters P.J. Clevers H. Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4.Nat Genet. 1998; 19: 379-383Crossref PubMed Scopus (1344) Google Scholar Intervillus cells in the Tcf4-null small bowel mucosa fail to proliferate; the capacity to populate villi is consequently reduced, and the defect is evident before birth.28Korinek V. Barker N. Moerer P. van Donselaar E. Huls G. Peters P.J. Clevers H. Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4.Nat Genet. 1998; 19: 379-383Crossref PubMed Scopus (1344) Google Scholar Indeed, in E18.5 mouse embryos, near to term, epithelial Tcf4 protein expression is virtually restricted to cells in the intervillus space (Figure 4A). The findings in Tcf4-null mice helped establish the idea that gut epithelial cell proliferation requires Wnt signals. However, we show above that intervillus Wnt signaling in reporter mice is at best patchy at birth (Figure 3A). This implies that early intervillus cells may not respond to Wnt or depend on levels that are below the detection threshold for β-galactosidase activity in TOP-GAL or Axin2LacZ mice and of immunostaining for nuclear β-catenin in intervillus cells at E18.5 (Figure 3D–G) or 1 day after birth (data not shown). Later in life, Tcf4 expression occurs over the full surface of mouse intestinal villi (Figure 4B), similar to findings reported in the late human fetus,27Barker N. Huls G. Korinek V. Clevers H. Restricted high level expression of Tcf-4 protein in intestinal and mammary gland epithelium.Am J Pathol. 1999; 154: 29-35Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar although Wnt signaling is confined to crypts. In differentiated villus cells in adolescent mice, Tcf4 expression is highest in epithelial cells with basally displaced nuclei (Figure 4B, and data not shown), which seem to correspond to a subset of goblet cells. Tcf3 and Tcf4 are the dominant Tcf/LEF family proteins present in the gut.29Korinek V. Barker N. Willert K. Molenaar M. Roose J. Wagenaar G. Markman M. Lamers W. Destree O. Clevers H. Two members of the Tcf family implicated in Wnt/b-catenin signaling during mouse embryogenesis.Mol Cell Biol. 1998; 18: 1248-1256Crossref PubMed Scopus (296) Google Scholar To assess developmental expression of Tcf3, we used an Ab that recognizes b