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• W4255177710 abstract "Background & Aims: In the intestine, the canonical Wnt signaling cascade plays a crucial role in driving the proliferation of epithelial cells. Furthermore, aberrant activation of Wnt signaling is strongly associated with the development of colorectal cancer. Despite this evidence, little is known about the precise identity and localization of Wnts and their downstream effectors in the adult intestine. To address this issue, we examined the expression pattern of all Wnts, Frizzleds (Fzs), low-density lipoprotein receptor-related proteins, Wnt antagonists, and T-cell factors in the murine small intestine and colon and adenomas. Methods: Embryonic, postnatal, and adult intestinal samples were subjected to in situ hybridization by using specific RNA probes for the various genes tested. Results: Our analysis showed high expression of several signaling components (including Wnt-3, Wnt-6, Wnt-9b, Frizzled 4, Frizzled 6, Frizzled 7, low-density lipoprotein receptor-related protein 5, and secreted Frizzled-related protein 5) in crypt epithelial cells. We also detected Wnt-2b, Wnt-4, Wnt-5a, Wnt-5b, Frizzled 4, and Frizzled 6 in differentiated epithelial and mesenchymal cells of the small intestine and colon. Finally, several factors (Frizzled 4, T-cell factor 1, lymphoid enhancer factor, Dickkopf 2, Dickkopf 3, and Wnt-interacting factor) displayed differential expression in normal vs neoplastic tissue. Conclusions: Our study predicts a much broader role for Wnt signaling in gut development and homeostasis than was previously anticipated from available genetic studies and identifies novel factors likely involved in promoting canonical and noncanonical Wnt signals in the intestine. Background & Aims: In the intestine, the canonical Wnt signaling cascade plays a crucial role in driving the proliferation of epithelial cells. Furthermore, aberrant activation of Wnt signaling is strongly associated with the development of colorectal cancer. Despite this evidence, little is known about the precise identity and localization of Wnts and their downstream effectors in the adult intestine. To address this issue, we examined the expression pattern of all Wnts, Frizzleds (Fzs), low-density lipoprotein receptor-related proteins, Wnt antagonists, and T-cell factors in the murine small intestine and colon and adenomas. Methods: Embryonic, postnatal, and adult intestinal samples were subjected to in situ hybridization by using specific RNA probes for the various genes tested. Results: Our analysis showed high expression of several signaling components (including Wnt-3, Wnt-6, Wnt-9b, Frizzled 4, Frizzled 6, Frizzled 7, low-density lipoprotein receptor-related protein 5, and secreted Frizzled-related protein 5) in crypt epithelial cells. We also detected Wnt-2b, Wnt-4, Wnt-5a, Wnt-5b, Frizzled 4, and Frizzled 6 in differentiated epithelial and mesenchymal cells of the small intestine and colon. Finally, several factors (Frizzled 4, T-cell factor 1, lymphoid enhancer factor, Dickkopf 2, Dickkopf 3, and Wnt-interacting factor) displayed differential expression in normal vs neoplastic tissue. Conclusions: Our study predicts a much broader role for Wnt signaling in gut development and homeostasis than was previously anticipated from available genetic studies and identifies novel factors likely involved in promoting canonical and noncanonical Wnt signals in the intestine. The intestine harbors a variety of cell types that are organized into highly regular structures known as villi and crypts of Lieberkühn. The absorptive and protective functions of the gut are ensured by enterocytes, goblet cells, Paneth cells, and enteroendocrine cells. These differentiated epithelial cells arise from transit-amplifying progenitors and, ultimately, stem cells, both of which reside in the crypts. The intestine also consists of a layer of loose connective tissue, or mesenchyme, which is thought to provide structural support and growth signals to the overlying epithelium. Finally, embedded within the various layers of the intestine, one additionally finds blood vessels, smooth muscle, and neuronal cells, as well as lymphocytes.Cellular proliferation and differentiation in the intestine depends on a large array of signaling molecules.1de Santa B.P. van den Brink G.R. Roberts D.J. Development and differentiation of the intestinal epithelium.Cell Mol Life Sci. 2003; 60: 1322-1332Crossref PubMed Scopus (256) Google Scholar, 2Sancho E. Batlle E. Clevers H. Signaling pathways in intestinal development and cancer.Annu Rev Cell Dev Biol. 2004; 20: 695-723Crossref PubMed Scopus (431) Google Scholar One such example includes the Wnt family of secreted growth factors.3Logan C.Y. Nusse R. The Wnt signaling pathway in development and disease.Annu Rev Cell Dev Biol. 2004; 20: 781-810Crossref PubMed Scopus (4177) Google Scholar, 4Cadigan K.M. Nusse R. Wnt signaling a common theme in animal development.Genes Dev. 1997; 11: 3286-3305Crossref PubMed Scopus (2210) Google Scholar, 5Wodarz A. Nusse R. Mechanisms of Wnt signaling in development.Annu Rev Cell Dev Biol. 1998; 14: 59-88Crossref PubMed Scopus (1728) Google Scholar Wnts are evolutionarily conserved, cysteine-rich glycoproteins capable of signaling in both a paracrine and autocrine fashion. Wnts induce a wide range of biological effects by binding to the 7-span transmembrane protein Frizzled (Fz)6Bhanot P. Brink M. Samos C.H. Hsieh J.C. Wang Y. Macke J.P. Andrew D. Nathans J. Nusse R. A new member of the frizzled family from Drosophila functions as a Wingless receptor.Nature. 1996; 382: 225-230Crossref PubMed Scopus (1213) Google Scholar and the single-span low-density lipoprotein receptor-related protein (LRP).7Pinson K.I. Brennan J. Monkley S. Avery B.J. Skarnes W.C. An LDL-receptor-related protein mediates Wnt signalling in mice.Nature. 2000; 407: 535-538Crossref PubMed Scopus (876) Google Scholar, 8Tamai K. Semenov M. Kato Y. Spokony R. Liu C. Katsuyama Y. Hess F. Saint-Jeannet J.P. He X. LDL-receptor-related proteins in Wnt signal transduction.Nature. 2000; 407: 530-535Crossref PubMed Scopus (1078) Google Scholar, 9Wehrli M. Dougan S.T. Caldwell K. O’Keefe L. Schwartz S. Vaizel-Ohayon D. Schejter E. Tomlinson A. DiNardo S. Arrow encodes an LDL-receptor-related protein essential for Wingless signalling.Nature. 2000; 407: 527-530Crossref PubMed Scopus (715) Google Scholar In a complex with Fz/LRP, Wnts trigger the release of β-catenin from a so-called destruction complex, which in the absence of Wnts promotes ubiquitin-mediated degradation of β-catenin.3Logan C.Y. Nusse R. The Wnt signaling pathway in development and disease.Annu Rev Cell Dev Biol. 2004; 20: 781-810Crossref PubMed Scopus (4177) Google Scholar Subsequently, free β-catenin shuttles to the nucleus, where it associates with DNA-binding factors of the T-cell factor (TCF) family to activate the transcription of target genes.10Behrens J. von Kries J.P. Kuhl M. Bruhn L. Wedlich D. Grosschedl R. Birchmeier W. Functional interaction of beta-catenin with the transcription factor LEF-1.Nature. 1996; 382: 638-642Crossref PubMed Scopus (2577) Google Scholar, 11Molenaar M. van de Wetering M. Oosterwegel M. Peterson-Maduro J. Godsave S. Korinek V. Roose J. Destree O. Clevers H. XTcf-3 transcription factor mediates beta-catenin-induced axis formation in Xenopus embryos.Cell. 1996; 86: 391-399Abstract Full Text Full Text PDF PubMed Scopus (1600) Google Scholar More recent evidence has shown that Wnts may also stimulate cellular responses independently of β-catenin and TCF.12Strutt D. Frizzled signalling and cell polarisation in Drosophila and vertebrates.Development. 2003; 130: 4501-4513Crossref PubMed Scopus (212) Google Scholar, 13Veeman M.T. Axelrod J.D. Moon R.T. A second canon. Functions and mechanisms of beta-catenin-independent Wnt signaling.Dev Cell. 2003; 5: 367-377Abstract Full Text Full Text PDF PubMed Scopus (1145) Google Scholar Examples of these so-called noncanonical pathways involve either the intracellular release of calcium ions and activation of Ca2+-dependent kinases or morphogenic changes dependent on RhoA and Jun kinase stimulation, also termed the planar cell polarity pathway.Genetic studies have shown an essential role for canonical Wnt signaling in regulating intestinal epithelial cell proliferation. One of the earliest indications of this came from the analysis of mutations in core Wnt signaling components such as adenomatous polyposis coli (APC) and β-catenin.14Giles R.H. van Es J.H. Clevers H. Caught up in a Wnt storm Wnt signaling in cancer.Biochim Biophys Acta. 2003; 1653: 1-24Crossref PubMed Scopus (1323) Google Scholar, 15Kolligs F.T. Bommer G. Goke B. Wnt/beta-catenin/tcf signaling a critical pathway in gastrointestinal tumorigenesis.Digestion. 2002; 66: 131-144Crossref PubMed Scopus (254) Google Scholar, 16Tejpar S. Cassiman J.J. Van Cutsem E. The molecular basis of colorectal cancer.Acta Gastroenterol Belg. 2001; 64: 249-254PubMed Google Scholar These genetic alterations lead to the formation of intestinal adenomas as a result of deregulated nuclear accumulation of β-catenin and constitutive activation of target genes associated with proliferation of epithelial cells.17Sansom 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 (652) Google Scholar, 18van 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/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells.Cell. 2002; 111: 241-250Abstract Full Text Full Text PDF PubMed Scopus (1132) Google Scholar, 19Korinek V. Barker N. Morin P.J. van Wichen D. de Weger R. Kinzler K.W. Vogelstein B. Clevers H. Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC−/− colon carcinoma.Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2910) Google Scholar, 20Morin P.J. Sparks A.B. Korinek V. Barker N. Clevers H. Vogelstein B. Kinzler K.W. Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC.Science. 1997; 275: 1787-1790Crossref PubMed Scopus (3480) Google Scholar Conversely, blockage of canonical Wnt signals in the intestine, either through deletion of TCF-4 or overexpression of the Wnt antagonist Dickkopf (Dkk)-1, results in arrested epithelial cell proliferation.21Korinek 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 (1310) Google Scholar, 22Kuhnert F. Davis C.R. Wang H.T. Chu P. Lee M. Yuan J. Nusse R. Kuo C.J. Essential requirement for Wnt signaling in proliferation of adult small intestine and colon revealed by adenoviral expression of Dickkopf-1.Proc Natl Acad Sci U S A. 2004; 101: 266-271Crossref PubMed Scopus (498) Google Scholar, 23Pinto 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 (790) Google Scholar Another function ascribed to Wnt signaling in epithelial cells was recently uncovered through DNA microarray analysis. EphB2 and B3 are TCF-4-responsive genes that regulate upward movement of epithelial cells and the positioning of Paneth cells within the crypt-villus axis.24Batlle E. Henderson J.T. Beghtel H. van den Born M.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. 2002; 111: 251-263Abstract Full Text Full Text PDF PubMed Scopus (927) Google Scholar Together these data provide clear evidence that proliferation and sorting of crypt epithelial cells depend on the correct dosage of canonical Wnt signals.Despite the functional evidence described previously, relatively little is known about the localization of Wnt signaling components within the intestine. Expression studies in mice and chicks have shown that during embryonic development, Wnt gene products are broadly expressed throughout the intestinal tract.25Lickert H. Kispert A. Kutsch S. Kemler R. Expression patterns of Wnt genes in mouse gut development.Mech Dev. 2001; 105: 181-184Crossref PubMed Scopus (89) Google Scholar, 26McBride H.J. Fatke B. Fraser S.E. Wnt signaling components in the chicken intestinal tract.Dev Biol. 2003; 256: 18-33Crossref PubMed Scopus (30) Google Scholar, 27Theodosiou N.A. Tabin C.J. Wnt signaling during development of the gastrointestinal tract.Dev Biol. 2003; 259: 258-271Crossref PubMed Scopus (100) Google Scholar In the adult intestine, Wnt expression is maintained,28Holcombe R.F. Marsh J.L. Waterman M.L. Lin F. Milovanovic T. Truong T. Expression of Wnt ligands and Frizzled receptors in colonic mucosa and in colon carcinoma.Mol Pathol. 2002; 55: 220-226Crossref PubMed Scopus (153) Google Scholar but their precise localization remains unclear. To address this issue in detail, we screened the expression patterns of all known Wnts, Fz/LRP and Wnt antagonists, and TCF family members in the adult intestine by in situ hybridization (ISH).Materials and MethodsProbesThe following probes used in this study were described elsewhere: Wnt-124Batlle E. Henderson J.T. Beghtel H. van den Born M.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. 2002; 111: 251-263Abstract Full Text Full Text PDF PubMed Scopus (927) Google Scholar, 29McMahon A.P. Bradley A. The Wnt-1 (int-1) proto-oncogene is required for development of a large region of the mouse brain.Cell. 1990; 62: 1073-1085Abstract Full Text PDF PubMed Scopus (1233) Google Scholar; Wnt-2, Wnt-4, Wnt-5b, Wnt-6, Wnt-7a, and Wnt-7b30Gavin B.J. McMahon J.A. McMahon A.P. Expression of multiple novel Wnt-1/int-1-related genes during fetal and adult mouse development.Genes Dev. 1990; 4: 2319-2332Crossref PubMed Scopus (402) Google Scholar; Wnt-3 and Wnt-3a31Roelink H. Nusse R. Expression of two members of the Wnt family during mouse development—restricted temporal and spatial patterns in the developing neural tube.Genes Dev. 1991; 5: 381-388Crossref PubMed Scopus (238) Google Scholar; Wnt-8a32Bouillet P. Oulad-Abdelghani M. Ward S.J. Bronner S. Chambon P. Dolle P. A new mouse member of the Wnt gene family, mWnt-8, is expressed during early embryogenesis and is ectopically induced by retinoic acid.Mech Dev. 1996; 58: 141-152Crossref PubMed Scopus (88) Google Scholar; Wnt-8b32Bouillet P. Oulad-Abdelghani M. Ward S.J. Bronner S. Chambon P. Dolle P. A new mouse member of the Wnt gene family, mWnt-8, is expressed during early embryogenesis and is ectopically induced by retinoic acid.Mech Dev. 1996; 58: 141-152Crossref PubMed Scopus (88) Google Scholar, 33Richardson M. Redmond D. Watson C.J. Mason J.O. Mouse Wnt8B is expressed in the developing forebrain and maps to chromosome 19.Mamm Genome. 1999; 10: 923-925Crossref PubMed Scopus (25) Google Scholar; Wnt-9a/14 and Wnt-9b/14b33Richardson M. Redmond D. Watson C.J. Mason J.O. Mouse Wnt8B is expressed in the developing forebrain and maps to chromosome 19.Mamm Genome. 1999; 10: 923-925Crossref PubMed Scopus (25) Google Scholar, 34Katoh M. Molecular cloning and expression of mouse Wnt14, and structural comparison between mouse Wnt14-Wnt3a gene cluster and human WNT14-WNT3A gene cluster.Int J Mol Med. 2002; 9: 221-227PubMed Google Scholar; Wnt-10a and Wnt-10b35Wang J. Shackleford G.M. Murine Wnt10a and Wnt10b cloning and expression in developing limbs, face and skin of embryos and in adults.Oncogene. 1996; 13: 1537-1544PubMed Google Scholar; Wnt-1136Kispert A. Vainio S. Shen L. Rowitch D.H. McMahon A.P. Proteoglycans are required for maintenance of Wnt-11 expression in the ureter tips.Development. 1996; 122: 3627-3637Crossref PubMed Google Scholar; Fz-4 and Fz-737Wang Y. Macke J.P. Abella B.S. Andreasson K. Worley P. Gilbert D.J. Copeland N.G. Jenkins N.A. Nathans J. A large family of putative transmembrane receptors homologous to the product of the Drosophila tissue polarity gene frizzled.J Biol Chem. 1996; 271: 4468-4476Crossref PubMed Scopus (314) Google Scholar; Dkk-138Monaghan A.P. Kioschis P. Wu W. Zuniga A. Bock D. Poustka A. Delius H. Niehrs C. Dickkopf genes are co-ordinately expressed in mesodermal lineages.Mech Dev. 1999; 87: 45-56Crossref PubMed Scopus (173) Google Scholar; and lymphoid enhancer factor (Lef)39Oosterwegel M. van de Wetering M. Timmerman J. Kruisbeek A. Destree O. Meijlink F. Clevers H. Differential expression of the HMG box factors TCF-1 and LEF-1 during murine embryogenesis.Development. 1993; 118: 439-448PubMed Google Scholar. Other probes were derived from reverse-transcription polymerase chain reaction products and correspond to the following nucleotides: 781–1640 for Wnt-16, 1488–1815 for Fz-1, 1245–1784 for Fz-2, 1371–1733 for Fz-3, 1500–2500 for Fz-5, 418–940 for Fz-8, 1296–1864 for Fz-9, and 994–1413 for Fz-10. The remaining probes correspond to expressed sequence tags obtained from the IMAGE consortium (MRC geneservice, Babraham, United Kingdom). The GenBank accession numbers for these probes are as follows: Wnt-5a, 4317623 ; Fz-6, 4973661 ; secreted Frizzled-related protein 1 (sFRP-1), BG975485 ; sFRP-2, BQ958218 ; sFRP-3, AA399912 ; sFRP-4, BC034853 ; sFRP-5, BC032921 ; Dkk-2, BE310466 ; Dkk-3, BQ933260 ; Dkk-4, BC018400 ; Wnt-interacting factor (WIF), BG246236 ; cerberus, AA120122 ; TCF-4, BG921346 ; TCF-1, BF141520 ; and TCF-3, AA015280 . To ensure the specificity of the probes, we generated both sense and antisense probes for Wnt-3, Wnt-9b, Wnt-4, Wnt-5b, Wnt-6, Fz-4, Fz-6, sFRP-1, sFRP-5, WIF, Dkk-2, Dkk-3, TCF-4, TCF-1, and TCF-3 and tested these in parallel in our ISH protocol. Representative examples are shown in Supplementary Figure 1 (see supplementary material online at http://www.niob.knaw.nl/researchpages/clevers/files/Figure%201%20supplementary.jpg; see supplementary Figure legend online at http://www.niob.knaw.nl/researchpages/clevers/files/Figure%20Legends%20Supplementary%20Figure%201%20and%202.pdf).In Situ HybridizationIntestines from normal or APCmin mice (C57BL/6, 3 months old) were flushed and fixed overnight in formalin. Samples were then dehydrated and embedded in paraffin, sectioned at 8 micrometers, and processed for hybridization as described below. Sections were dewaxed, rehydrated, treated with 0.2N HCl, digested in proteinase K solution, postfixed, treated in acetic anhydride solution, and hybridized overnight for 24–48 hours at 68°C with various probes in 5× standard saline citrate (SSC; pH 4.5), 50% formamide, 2% blocking powder (Roche, Almere, The Netherlands), 5 mmol/L ethylenediaminetetraacetic acid, 50 μg/mL yeast transfer RNA, 0.1% Tween 20, 0.05% 3[3-cholaminopropyl diethylammonio]-1-propane sulfonate, and 50 μg/mL heparin. Sections were then rinsed in 2× SSC and washed for 3 × 20 minutes at either 60°C or 65°C in 2× SSC/50% formamide. After several rinses in tris-buffered saline/Tween, sections were then blocked for 30 minutes in tris-buffered saline/Tween containing 0.5% blocking powder (Roche). Next, sections were incubated in blocking solution overnight at 4°C with alkaline phosphatase-conjugated anti-digoxigenin (1:2000 dilution; Roche). After washing several times in tris-buffered saline/Tween, the color reaction was performed with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate solution. For image analysis, sections were temporally mounted in glycerol or permanently mounted after dehydration in Pertex (Klinipath, The Netherlands). A complete protocol will be provided upon request. In vitro transcription reactions to generate labeled probe was performed as follows: 1 μg of linearized DNA was incubated at 37°C for more than 2 hours with 4 μL of transcription buffer (Promega, Leiden, The Netherlands), 2 μL of dithiothreitol 0.1 mol/L (Promega), 2 μL of Dig RNA labeling mix (Roche), 1 μL of ribonuclease inhibitor (Promega), and 1.5 μL of T7, T3, SP6 (Promega) in a total volume of 20 μL.Antibody StainingThe immunohistochemistry procedure is described elsewhere.24Batlle E. Henderson J.T. Beghtel H. van den Born M.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. 2002; 111: 251-263Abstract Full Text Full Text PDF PubMed Scopus (927) Google Scholar Briefly, sections were pretreated with peroxidase blocking buffer (120 mmol/L Na2HPO4, 43 mmol/L citric acid, 30 mmol/L NaN3, and 0.2% H2O2; pH 5.8) for 20 minutes at room temperature. Antigen retrieval was performed by boiling samples in sodium citrate buffer (10 mmol/L; pH 6.0). After 20 minutes, the boiling pan was allowed to slowly cool down to room temperature. Incubation of antibodies was performed in 1% bovine serum albumin in phosphate-buffered saline overnight at 4°C. The primary antibodies used in this study were rabbit anti-lysozyme (1:500; Dako, Glostrup, Denmark) and goat anti-TCF-4 (1:500; Santa Cruz, Santa Cruz, CA). Rabbit anti-goat (Dako) and rabbit EnVision+ (Dako) were used as secondary antibodies.ResultsExpression of Wnt Genes in the Adult IntestineTo provide a comprehensive expression profile of Wnt signaling components in the intestinal mucosa, we first collected and generated RNA probes for all murine Wnts (19 genes), Fzs (10 genes), LRPs (2 genes), TCFs (4 genes), sFRPs (5 genes), Dkks (4 genes), WIF, and cerberus (see Materials and Methods). By ISH on sections, we initially examined the expression of Wnt genes in both embryonic and adult stages. Of the 19 Wnt probes tested, 7 Wnts were readily detected in the intestine (results are recapitulated in Figure 1 and Supplementary Table 1, see supplementary material online at http://www.niob.knaw.nl/researchpages/clevers/files/Supplementary%20Table%201.pdf). As shown in Figure 2, Figure 3, Wnt ligands are expressed in both the epithelial and mesenchymal layers of the intestine. In the epithelium, Wnt-3 was associated with the very bottom of the crypts of Lieberkühn, at the sites where Paneth cells are located (Figure 2A). Similarly, in the distal portion of the small intestine, Wnt-9b (also termed Wnt-14b) was found predominately at the bottom of the crypts (Figure 2B). To confirm the identity of these cells, we performed immunostainings on consecutive 4-μm sections for the classic Paneth cell marker lysozyme. As shown on corresponding sections (Figure 2C and D) of the same intestinal crypt, Wnt-9b-expressing cells also produced lysozyme and thus represent Paneth cells. It is interesting to note that in more proximal areas of the small intestine, Wnt-9b was also detected in epithelial progenitor cells above the Paneth cell compartment (Figure 2E). Contrary to the small intestine, in the colon, Wnt-9b was localized throughout the colonic epithelium (Figure 2F). Moreover, both Wnt-3 and 9b displayed weak expression in adenomas of APCmin mice (data not shown). Note that a recent report has additionally observed Wnt-9b expression in the fetal gut.40Qian J. Jiang Z. Li M. Heaphy P. Liu Y.H. Shackleford G.M. Mouse Wnt9b transforming activity, tissue-specific expression, and evolution.Genomics. 2003; 81: 34-46Crossref PubMed Scopus (33) Google Scholar Finally, we found Wnt-6 expressed throughout the crypts of the small intestine and colon (Figure 2G and H). Similarly, Wnt-6 was strongly expressed in adenomas (Figure 2I).Figure 2ISH analysis of Wnts expressed in intestinal epithelial cells. (A) shows Wnt-3 expressed in Paneth cells (arrowheads). (B and C) show Wnt-9b strongly and predominately expressed in Paneth cells in the distal portion of the intestine (ileum). (D) represents a consecutive 4-μm section of the same intestinal crypt as shown in (C) that was immunostained with an antibody recognizing the Paneth cell marker lysozyme. (E) represents a section of the duodenum and shows Wnt-9b expression in Paneth cells and crypt progenitor cells. (F) shows Wnt-9b expressed throughout the colonic epithelium. (G, H, and I) show Wnt-6 in crypt epithelial cells of the small intestine, colon, and adenomas, respectively.View Large Image Figure ViewerDownload (PPT)Figure 3ISH analysis of Wnts expressed in the mesenchyme of the small intestine and large intestine. (A) shows at E18.5 Wnt-5a accumulation in the mesenchyme of the villi tips (red arrowheads), in mesenchymal cells adjacent to proliferative epithelial cells (black arrowheads), and in the smooth muscle layer (green arrowhead). The border between the epithelium and the mesenchyme is indicated with dashed lines. In the mature intestine (B), Wnt-5a is strongly expressed in mesenchymal cells at the tips of villi (red arrowheads and inset), whereas levels are reduced along the villi toward the crypt-villus border (black arrowheads). (D) and a higher magnification in (E) show Wnt-5a up-regulation in stromal cells of intestinal polyps (black arrowheads). (C, F, and H) show Wnt-5a, Wnt-5b, and Wnt-4, respectively, just beneath the surface epithelium of the colon. Note that Wnt-5a is also detected lower in the crypts (red arrowheads). Inset in (F) shows a cross section of colonic crypts with Wnt-5b staining in the surrounding mesenchyme. (G) shows uniform Wnt-4 expression throughout the villi. (I and J) show Wnt-2b in mesenchymal cells of the small intestine and endothelial or smooth muscle cells of the colon, respectively.View Large Image Figure ViewerDownload (PPT)Several Wnts were also found in specific compartments of the mesenchyme. In the fetal gut, Wnt-5a showed high expression at the tips of growing villi and in few discrete cells beneath the proliferative epithelium (Figure 3A). In the adult small intestine, Wnt-5a was similarly abundant in the villus tips, although weaker expression was also observed throughout the villi, as well as at the crypt-villus junction (Figure 3B). In the colon, Wnt-5a and the closely related Wnt-5b were restricted to the mesenchyme beneath the surface epithelium (Figure 3C and F). Finally, Wnt-5a and, to a lesser extent, Wnt-5b were both up-regulated in stromal cells of APCmin polyps (Figure 3D and E and data not shown). Unlike Wnt-5a, Wnt-4 was uniformly expressed along the villus mesenchyme and was absent from adenomas (Figure 3G and data not shown). However, in the colon, Wnt-4 expression closely resembled Wnt-5a and 5b stainings (Figure 3H). Finally, Wnt-2b was strongly expressed in the mesenchymal layer of the villi and more weakly in the crypts of the small intestine (Figure 3I). In the colon, Wnt-2b transcripts were less abundant and seemed to be markers of endothelial or smooth muscle cells (Figure 3J).Expression of Wnt Receptors in the Adult IntestineTo identify putative Wnt-responsive cells, we next examined the expression of Fz-related receptors 1–10. As indicated in Supplementary Table 1 (see supplementary material online at http://www.niob.knaw.nl/researchpages/clevers/files/Supplementary%20Table%201.pdf) and Figure 4, several Fz genes were located in both the fetal and adult intestine. As shown in Figure 4A and in van Es et al,41van 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 (495) Google Scholar Fz-5 was detected in epithelial cells of the intervillus pockets of fetal guts and in the crypts of adult intestines. In addition, Fz-7 and Fz-4 were dynamically expressed during intestinal development. Fz-7 was strongly expressed in the smooth muscle layer and in intervillus epithelial cells of neonates (P1; data not shown), although at later stages, Fz-7 was confined to the epithelium of the crypt bottom (Figure 4B and C). Fz-4 transcripts were also widely distributed in neonatal guts (Supplementary Table 1, see supplementary material online at http://www.niob.knaw.nl/researchpages/clevers/files/Supplementary%20Table%201.pdf), whereas expression in adults was comparatively weaker and restricted to differentiated epithelial cells of the villi (Figure 4D). One notable exception to this was the high levels of Fz-4 in adenomas, as shown in Figure 4E. Finally, Fz-6 was uniformly expressed throughout the epithelium of the small and large intestine (Figure 4F and G). We also tested the expression of the LRP coreceptors LRP-5 and LRP-6, given their absolute requirement in driving canonical Wnt signals. As expected, we found both LRP-5 and LRP-6 expressed in proliferative epithelial cells of the crypts (Figure 4H and data not shown).Figure 4ISH analysis of Wnt receptors in the intestine and colon. (A) Fz-5 in the epithelium of the crypts and crypt-villus border. (B and C) Fz-7 in the lower portions of adult intestinal and colonic crypts, respectively. (D) Fz-4 in differentiated epithelial cells of the villi. Note that expression of Fz-4 is strongest in the lower half of the villi. (E) Fz-4 up-regulated in intestinal polyps. (F and G) Fz-6 throughout the epithelium in the small and large intestine, respectively. (H) LRP-5 restricted to proliferative epithelial cells.View Large" @default.
• W4255177710 created "2022-05-12" @default.
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• W4255177710 date "2005-08-01" @default.
• W4255177710 modified "2023-10-18" @default.
• W4255177710 title "Expression Pattern of Wnt Signaling Components in the Adult Intestine" @default.
• W4255177710 cites W1504655784 @default.
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