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• W2104490911 abstract "Article9 December 2004free access FGF-20 and DKK1 are transcriptional targets of β-catenin and FGF-20 is implicated in cancer and development Mario N Chamorro Mario N Chamorro Cancer Biology and Genetics Program, Sloan-Kettering Institute, Varmus Laboratory, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Cell Biology Program, Cornell University, Weill Graduate School of Medical Sciences, New York, NY, USA Search for more papers by this author Donald R Schwartz Donald R Schwartz Department of Pathology, The University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Alin Vonica Alin Vonica The Laboratory of Vertebrate Embryology, The Rockefeller University, New York, NY, USA Search for more papers by this author Ali H Brivanlou Ali H Brivanlou The Laboratory of Vertebrate Embryology, The Rockefeller University, New York, NY, USA Search for more papers by this author Kathleen R Cho Kathleen R Cho Department of Pathology, The University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Harold E Varmus Corresponding Author Harold E Varmus Cancer Biology and Genetics Program, Sloan-Kettering Institute, Varmus Laboratory, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Search for more papers by this author Mario N Chamorro Mario N Chamorro Cancer Biology and Genetics Program, Sloan-Kettering Institute, Varmus Laboratory, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Cell Biology Program, Cornell University, Weill Graduate School of Medical Sciences, New York, NY, USA Search for more papers by this author Donald R Schwartz Donald R Schwartz Department of Pathology, The University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Alin Vonica Alin Vonica The Laboratory of Vertebrate Embryology, The Rockefeller University, New York, NY, USA Search for more papers by this author Ali H Brivanlou Ali H Brivanlou The Laboratory of Vertebrate Embryology, The Rockefeller University, New York, NY, USA Search for more papers by this author Kathleen R Cho Kathleen R Cho Department of Pathology, The University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Harold E Varmus Corresponding Author Harold E Varmus Cancer Biology and Genetics Program, Sloan-Kettering Institute, Varmus Laboratory, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Search for more papers by this author Author Information Mario N Chamorro1,4, Donald R Schwartz2,‡, Alin Vonica3,‡, Ali H Brivanlou3, Kathleen R Cho2 and Harold E Varmus 1 1Cancer Biology and Genetics Program, Sloan-Kettering Institute, Varmus Laboratory, Memorial Sloan-Kettering Cancer Center, New York, NY, USA 2Department of Pathology, The University of Michigan Medical School, Ann Arbor, MI, USA 3The Laboratory of Vertebrate Embryology, The Rockefeller University, New York, NY, USA 4Cell Biology Program, Cornell University, Weill Graduate School of Medical Sciences, New York, NY, USA ‡These authors contributed equally to this work *Corresponding author. Cancer Biology and Genetics Program, Sloan-Kettering Institute, Varmus Laboratory-RRL717, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, Box 62, New York, NY 10021, USA. Tel.: +1 212 639 6561; Fax: +1 212 717 3125; E-mail: [email protected] The EMBO Journal (2005)24:73-84https://doi.org/10.1038/sj.emboj.7600460 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info β-catenin is the major effector of the canonical Wnt signaling pathway. Mutations in components of the pathway that stabilize β-catenin result in augmented gene transcription and play a major role in many human cancers. We employed microarrays to identify transcriptional targets of deregulated β-catenin in a human epithelial cell line (293) engineered to produce mutant β-catenin and in ovarian endometrioid adenocarcinomas characterized with respect to mutations affecting the Wnt/β-catenin pathway. Two genes strongly induced in both systems—FGF20 and DKK1—were studied in detail. Elevated levels of FGF20 RNA were also observed in adenomas from mice carrying the ApcMinallele. Both XFGF20 and Xdkk-1 are expressed early in Xenopus embryogenesis under the control of the Wnt signaling pathway. Furthermore, FGF20 and DKK1 appear to be direct targets for β-catenin/TCF transcriptional regulation via LEF/TCF-binding sites. Finally, by using small inhibitory RNAs specific for FGF20, we show that continued expression of FGF20 is necessary for maintenance of the anchorage-independent growth state in RK3E cells transformed by β-catenin, implying that FGF-20 may be a critical element in oncogenesis induced by the Wnt signaling pathway. Introduction Wnt proteins are secreted glycoproteins that bind and activate two classes of co-receptors, LDL-related proteins (LRPs) and members of the Frizzled protein family. Signaling initiated by Wnts and their receptors controls a wide variety of cell processes, including cell fate specification, differentiation, migration, and polarity (reviewed in Peifer and Polakis, 2000). β-Catenin is the major effector of the canonical Wnt signaling pathway. In the absence of Wnt, cytosolic β-catenin forms a complex with Axin and adenomatous polyposis coli (APC) proteins, and is rapidly degraded by the ubiquitination–proteosome system. Wnt signaling inactivates the β-catenin destruction complex, so that β-catenin is stabilized, accumulates in the cytoplasm and nucleus, and forms heterodimers with the DNA-binding factors belonging to the LEF/TCF family (reviewed in Huelsken and Behrens, 2002). As a result, significant changes occur in the gene expression program (reviewed in Hecht and Kemler, 2000; Miller et al, 2001). In addition, Wnt signaling can be antagonized extracellularly by secreted factors such as Wnt inhibitory factor-1 (WIF-1), Cerberus, members of the Dickkopf (DKK) family, and soluble Frizzled-related proteins (sFRP) (Kawano and Kypta, 2003). Genetic alterations that stabilize β-catenin are found in tumors in mice and humans. The most commonly observed genetic alterations involve either the loss of APC or Axin or mutations that affect the amino-terminus of β-catenin, all occurring in a mutually exclusive manner, and the most commonly affected organs are the colon, liver, skin, stomach, ovaries, pancreas, and prostate. For example, loss of APC occurs in 70–80% of human colorectal cancers (reviewed in Bienz and Clevers, 2000) and mutations affecting β-catenin are found in about half of the remaining tumors, implying that stabilization of β-catenin is a major early event in colonic carcinogenesis. Similarly, in ApcMin/+ mice, the loss of the wild-type allele initiates adenoma formation (Moser et al, 1993; Oshima et al, 1995), and expression of an oncogenic form of β-catenin produces adenomas in the mouse intestine (Romagnolo et al, 1999). Identification of the transcriptional targets of the Wnt/β-catenin signaling pathway is a potentially important means to understand the role of the canonical pathway in oncogenesis and development. A number of candidate target genes have been identified in human cell lines and tumors. (For more information on Wnt pathway targets, consult the Wnt webpage at http://www.stanford.edu/~rnusse/wntwindow.html.) Our objective in the study reported here has been to identify novel β-catenin target genes that are regulated directly by DNA binding of β-catenin/TCF heterodimers and are potentially relevant to carcinogenesis. To this end, we employed microarray technology to identify genes with significantly altered levels of expression in human epithelial (293) cells expressing mutant (stabilized) β-catenin in which serine 37 has been replaced with alanine; we then compared a list of these genes to a similar list obtained using the same methods to measure the abundance of RNAs in a well-characterized set of primary human ovarian endometrioid adenocarcinomas (OEAs) with and without Wnt pathway defects (Wu et al, 2001). Merging of microarray data from cell culture and OEA tumors revealed at least 17 genes that are regulated in common when similar criteria were applied. Two such genes, FGF20, a putative proto-oncogene (Jeffers et al, 2001), and DKK1, a Wnt pathway antagonist (Glinka et al, 1998), were studied in greater detail in mouse and human tumors, in frog development, in tests of the direct action of β-catenin-TCF heterodimers on gene expression, and in tests for a role of FGF20 during maintenance of the β-catenin-induced transformed state. We present evidence supporting the conclusions that FGF20 and DKK1 are directly regulated by β-catenin during development and tumorigenesis, and that continued expression of FGF20 is required to maintain the anchorage-independent growth state established by Wnt/β-catenin signaling. Results Genes regulated by mutant β-catenin in a human epithelial cell line In an initial effort to identify genes regulated by mutant β-catenin, whether directly or indirectly, we used an efficient virus-based gene delivery system to introduce mutant β-catenin into virtually all cells in a cultured epithelial cell line, the human embryonic kidney cell line 293. This approach obviated a need to select individual clones of β-catenin-expressing cells and thus minimized variations in gene expression that might have been attributed to clonal variation. 293 cells were engineered to produce Tva, the avian leukosis subgroup A virus receptor, allowing efficient infection by the avian retroviral vector RCAS (Fisher et al, 1999). In addition, the β-catenin/TCF reporter construct, pOT, was inserted in the genome of this cell line to monitor β-catenin/TCF activity (Rubinfeld et al, 1993). The resulting cell line, 293Top, was infected with RCAS vectors encoding either GFP, to serve as a control, or HA-tagged β-cateninS37A, a stable mutant protein (Wu et al, 2001). Typically, about 80% of 293 cells were infected with either virus, as judged by anti-HA immunofluorescence or GFP fluorescence (data not shown). In the cells infected with RCAS-β-cateninS37A (293Top-S37A), the pOT reporter was typically induced 100–300-fold compared to cells infected with RCAS-GFP (293Top-GFP; data not shown). RNA for microarray analysis was isolated 7 days after viral infection from four independently infected cultures, and chromophore-tagged cDNAs were hybridized to human Affymetrix U133A oligonucleotide microarrays to compare the messenger RNA (mRNA) expression profile of 293Top-S37A cells with that of 293Top-GFP cells, as described in greater detail in Materials and Methods. The criteria for identifying genes as up- or downregulated by β-cateninS37A included differential expression of at least two-fold, with a P-value of less than 0.05 using a parametric test. In all, 62 genes were represented by higher levels of RNA in cells expressing β-cateninS37A than in GFP-expressing cells, and 15 genes were represented by lower RNA levels. Genes previously reported to be regulated by β-catenin-mediated Wnt signaling, such as CCND1 (CyclinD1), ENC1, ABCB1 (MDR1), LEF1, ENPP2 (Autotaxin), MSX1, and MSX2, were among the genes upregulated in the 293 cells expressing β-cateninS37A, as compared to the 293 cells expressing GFP (Supplementary Table 1S). Among the top 20 upregulated genes, we identified several known or suspected proto-oncogenes, such as FGF20 (Jeffers et al, 2001), ETV5 (Ets-5) (Dhulipal, 1997), LMO2 (Rabbitts et al, 1997; Hacein-Bey-Abina et al, 2003), and some genes implicated in Wnt signaling, such as DKK1 (Glinka et al, 1998) and WNT11 (Table I). Reverse transcription followed by semiquantitative polymerase chain reaction-mediated amplification (RT–PCR assay) was used to confirm the findings, with some of the most dramatically upregulated genes by microarray tests (Figure 1A). Figure 1.Validation of microarray data by semiquantitative RT–PCR. (A) To verify oligonucleotide microarray results, semiquantitative RT–PCR was used to estimate the amount of RNA from five of the upregulated genes in 293Top cells (Table I). The same total RNA sample was used to prepare probes for microarray hybridization. (B) Expression of FGF20 RNA as assessed by RT–PCR with RNA from OEA cell line TOV112D, ovarian clear cell carcinoma-derived line TOV21G, and colon cancer cell lines SW480 and LS123. The letters D and N refer to β-catenin status (Deregulated and Normal). (C) Measurement of FGF20 RNA from RK3E cells transformed by β-cateninS33Y (clones A and D) and N-terminal-deleted β-cateninΔN132 (ΔN132B) by RT–PCR. GAPDH mRNA was reverse transcribed and amplified to control for the amount of RNA loaded. Download figure Download PowerPoint Table 1. List of the twenty most strongly upregulated genes in 293Top cells by mutant β-cateninS37A Gene symbol Gene name Fold change P-value Function FGF20 Fibroblast growth factor 20 17.7 0.003 Signal transduction; cell–cell signaling DKK1 Dickkopf (Xenopus laevis) homolog 1 15.5 <0.001 Extracellular Wnt signaling antagonist MEGT1 Megakaryocyte-enhanced gene transcript 1 9.4 0.012 Unknown BIK BCL2-interacting killer 9.2 0.002 Apoptotic program; induction of apoptosis EST GenBank acc. # AK022120 9.1 0.014 Unknown ETV5 Ets variant gene 5 6.2 0.007 Transcription factor WNT11 Wingless-type MMTV integration site, member 11 5.3 0.006 Signal transduction; cell–cell signaling; embryogenesis; and morphogenesis SLC2A3 Solute carrier family 2, member 3 5.2 0.026 Glucose transport; carbohydrate metabolism SERPIND1 Serine proteinase inhibitor, clade D, member 1 5 0.049 Plasma glycoprotein; proteinase inhibitor SLC7A8 Solute carrier family 7, member 8 4.9 0.007 Cationic amino-acid transporter ENPP2 Ectonucleotide pyrophosphatase/phosphodiesterase 2 (autotaxin) 4.7 0.001 Cell motility; G-protein-linked receptor signaling pathway; transcription factor binding DUSP6 Dual-specificity phosphatase 6 4.6 0.024 Apoptosis; MAPKKK cascade; cell cycle control; inactivation of MAPK LMO2 LIM domain only 2 (rhombotin-like 1) 4.2 <0.001 Oncogenesis; developmental processes SLC2A14 Solute carrier family 2, member 14 4.0 0.001 Carbohydrate transport ARHGAP26 GTPase regulator associated with FAK 3.8 0.028 Neurogenesis; cell growth/maintenance GAD1 Glutamate decarboxylase 1 (brain, 67 kDa) 3.6 0.003 Synaptic transmission; glutamate decarboxylation FUT1 Fucosyltransferase 1 3.5 0.036 Carbohydrate metabolism QPTC Glutaminyl-peptide cyclotransferase (glutaminyl cyclase) 3.5 <0.001 Protein modification RBP1 Cellular retinol-binding protein 1 3.3 0.024 Retinoid binding; vitamin A metabolism ABCB1 MDR/TAP member 1 3.1 0.002 Drug resistance; small-molecule transport RNA for microarray analysis was isolated from 293Top cells 7 days after viral infection from four cultures independently infected with either RCAS-β-cateninS37A or RCAS-GFP. The top 20 upregulated genes are shown in descending order with respect to fold increase in RNA levels. P-values were obtained using a parametric test. Known or suspected proto-oncogenes and genes implicated in the Wnt pathway are shown in bold-faced letters. FGF20 mRNA is undetectable in 293Top cells that express GFP, although readily observed in cells expressing activated β-catenin. In a survey of normal human tissues with RT–PCR assays, FGF20 RNA was found exclusively in the adult central nervous system, suggesting that expression of FGF20 is tightly controlled in normal development. The gene is, however, expressed in human cancers; for example, FGF20 RNA was detected in five of 15 human colon cancer cell lines (Jeffers et al, 2001). One of those lines, the SW480 line, is known to have deregulated β-catenin due to loss of APC (Munemitsu et al, 1995). We have corroborated this finding with SW480 cells, and we have also found FGF20 RNA in the ovarian endometrioid cell line TOV112D, which contains mutant form of β-catenin (Figure 1B). In contrast, FGF20 RNA is not detectable in TOV21G or LS123, ovarian and colorectal carcinoma lines respectively, harboring wild-type β-catenin (Rutzky et al, 1983; Wu et al, 2001; MN Chamorro, unpublished data, 2004; Figure 1B). Prior studies have shown that activated mutants of β-catenin promote neoplastic transformation of RK3E cells, a rat epithelial cell line (Kolligs et al, 1999). In agreement with our expression profile of FGF20 in 293Top cells, FGF20 RNA is readily detectable in the β-catenin-transformed RK3E lines by RT–PCR, but not in the parental line (Figure 1C). Increases in FGF-20 and DKK1 RNA are associated with deregulated β-catenin in human ovarian endometrioid adenocarcinomas We next extended our findings with 293Top cells by examining the gene expression profiles of our large collection of OEAs. Approximately 40% of these tumors have mutations in CTNNB1 (β-catenin), APC, AXIN1 or AXIN2 (Wu et al, 2001), providing a test of the proposed correlation between Wnt/β-catenin pathway defects and induction of candidate target genes for regulation by β-catenin, as illustrated previously for the β-catenin-regulated genes, ITF-2 and AXIN2 (Kolligs et al, 2002; Leung et al, 2002). Affymetrix U133A oligo microarrays were again used to profile gene expression in 18 OEAs with an intact β-catenin pathway and 12 OEAs with deregulated β-catenin, in a fashion similar to recently published work using lower density HuGeneFL arrays (Schwartz et al, 2003). By comparing the profiles from tumors with normal versus mutant Wnt/β-catenin pathways, we identified a list of 563 genes differentially regulated by at least 1.75-fold with a P-value of less than 0.05 (Schwartz et al, 2003). To develop a shorter list of genes that are commonly regulated by Wnt/β-catenin signaling in different cell types, we compared our list of regulated genes using modestly different criteria from 293Top cells (two-fold regulation, P<0.05) and OEAs (1.75-fold regulation, P<0.05). By these criteria, at least 17 genes appeared to be regulated in common; 16 of these genes were upregulated and one was downregulated (Table II). It is noteworthy that eight of the 20 most dramatically upregulated genes in 293Top cells are present in the combined list of 16 upregulated genes, including three proto-oncogenes and two genes involved in Wnt signaling (Tables I and II). Table 2. List of genes regulated by β-catenin in both 293Top cells and in OEAs Gene symbol Gene name 293Top fold change P-values OEAs fold change P-values FGF-20 Fibroblast growth factor 20 17.70 0.003 7.39 <0.001 DKK1 Dickkopf homolog 1 (Xenopus laevis) 15.55 <0.001 12.95 <0.001 ETV5 Ets variant gene 5 (ets-related molecule) 6.23 0.007 1.82 0.037 WNT11 Wingless-type MMTV integration site family, member 11 5.32 0.006 2.20 0.011 LMO2 LIM domain only 2 (rhombotin-like 1) 4.27 <0.001 4.05 <0.001 GAD1 Glutamate decarboxylase 1 3.66 0.003 6.89 <0.001 QPCT Glutaminyl-peptide cyclotransferase (glutaminyl cyclase) 3.57 <0.001 3.90 <0.001 ABCB1 ATP-binding cassette, subfamily B (MDR/TAP), member 1 3.15 0.002 1.75 <0.001 SNK Serum-inducible kinase 2.75 0.006 3.49 <0.001 IRS1 Insulin receptor substrate 1 2.62 0.002 2.68 0.001 TBX3 T-box 3 (ulnar mammary syndrome) 2.49 <0.001 2.17 0.024 MSX2 Msh homeo box homolog 2 (Drosophila) 2.47 <0.001 6.03 0.000 MSX1 Msh homeo box homolog 1 (Drosophila) 2.46 0.001 3.31 0.047 DNCI1 Dynein, intermediate polypeptide 1 2.20 <0.001 3.70 <0.001 CCND1 Cyclin D1 (PRAD1: parathyroid adenomatosis 1) 2.05 <0.001 2.90 0.001 NMA Putative transmembrane protein 2.01 0.003 7.28 <0.001 ISYNA1 Myo-inositol 1-phosphate synthase A1 −2.1 <0.001 −2.1 0.009 Regulated genes are shown in descending order with respect to fold change in upregulation in 293Top cells. The two genes that appear to be most highly induced by stabilized β-catenin in both the 293Top cell system and in OEA primary tumors are FGF20 and DKK1 (Table II). Figure 2A demonstrates that both of these genes are expressed at markedly elevated levels in eight of 12 OEAs harboring mutations that deregulate β-catenin, but neither was induced in the 19 tumors lacking such mutations. Figure 2.FGF20 and DKK1 gene expression in individual human and mouse tumors. (A) FGF20 (upper panel) and DKK1 (lower panel) gene expression in OEAs. Relative RNA levels were determined by Affymetrix microarray data analysis. White boxes represent the relative gene expression in tumors with an intact Wnt pathway and black boxes represent tumors with a deregulated Wnt pathway. (B) FGF20 RT–PCR products using 200 ng of RNA from adenomas from ApcMin/+ mice and from normal intestinal mucosa samples from 15-day-old and adult ApcMin/+ mice. Download figure Download PowerPoint FGF-20 is expressed in adenomas from ApcMin/+ mice but not in normal intestinal mucosa Of the suspected proto-oncogenes on our combined list of candidate targets for regulation by β-catenin, FGF20 displayed the strongest correlation between RNA levels and the status of the Wnt/β-catenin pathway in OEA tumors. We therefore extended our studies of FGF20 expression to other tumor types. In mice heterozygous for loss-of-function mutations at the Apc locus, such as ApcMin/+ mice and other heterozygous Apc knockout mice, loss of the wild-type allele initiates adenoma formation in the small intestine (Oshima et al, 1995). Using RT–PCR to detect expression of RNA, we found little or no Fgf20 RNA in non-neoplastic intestinal tissues from 15-day-old and adult ApcMin/+ mice, but readily detected Fgf20 RNA in all the adenomas analyzed (Figure 2B). Stabilization of β-catenin in colonic epithelial cells by loss of APC is an early event in the majority of human colon cancers (Polakis, 2000). In preliminary experiments, we used the RT–PCR assay to measure FGF20 RNA in six primary human colon adenocarcinomas and in three samples of normal colon mucosa. We found FGF20 RNA in half of the tumors, but in none of the normal mucosas (Supplementary Figure IS). XFGF20 and Xdkk-1 are downstream of the Wnt pathway in Xenopus laevis embryos Much of our knowledge about the Wnt pathway and its role in vertebrate development comes from studies in Xenopus laevis (reviewed in Harland and Gerhart, 1997; Huelsken and Birchmeier, 2001). We therefore sought to extend the evidence for regulation of some of our candidate genes by the Wnt/β-catenin pathway by testing for transcripts in Xenopus embryos at relevant times in development. Both FGF20 and DKK1 were first identified in X. laevis (Glinka et al, 1998; Koga et al, 1999) and are closely related to their human counterparts (79% amino-acid identity between Xenopus and human FGF-20; 56% identity between Xdkk-1 and human DKK-1). Xdkk-1 is expressed at the beginning of gastrulation (stage 10) in the future anterior endomesoderm, on the dorsal side of the embryo (Glinka et al, 1998). The localization of XFGF20 RNA was previously reported for the neurula stage, but the RNA could be detected from late blastula onwards by RT–PCR (Koga et al, 1999). As in Xenopus early developmental stages are the most accessible experimentally, we confirmed zygotic expression from stage 10 onwards (Figure 3B, left panel), and localized XFGF20 RNA by RT–PCR exclusively to the equatorial region (the marginal zone) of stage 10 embryos, which is fated to produce mesoderm, (Figure 3B, right panel). The pattern of XFGF20 expression is similar to that described for other FGF genes like eFGF and XFGF3 (Isaacs et al, 1994; Schohl and Fagotto, 2003), and for a group of genes expressed in the marginal zone, which require a zygotic Wnt pathway (Figure 3A), such as XmyoD and Xbra (Hoppler et al, 1996; Vonica and Gumbiner, 2002). Figure 3.Wnt signaling regulates XFGF20 and Xdkk-1 expression in Xenopus embryos. (A) Schematic description of a pregastrula Xenopus embryo (left panel) and the localizations and timing of the maternal and zygotic Wnt pathways (right panel). (B) XFGF20 is expressed zygotically and localized exclusively in the marginal zone. RT–PCR for XFGF20 in various developmental stages (left panel) and various locations of stage 10 (early gastrula) embryos (right panel). ODC RNA and Xwnt-8 RNA serve as controls for amounts loaded and localization (ODC is normally expressed weakly in vegetal cells). (C) Modulation of the Wnt signaling pathway alters XFGF20 and Xdkk-1 expression. Embryos were injected marginally in each cell at the four-cell stage with 100 pg DN-Xtcf-3 RNA or 20 pg VP16-Xtcf-3 RNA, and collected at stage 9.5 for RT–PCR analysis. XFGF20, Xdkk-1, siamois, chordin, and Xbra all required early activation of the Wnt pathway, while Xwnt-8 is repressed under the same conditions. (D) Synergistic effect of the Wnt pathway and the endomesoderm inducer VegT on ectopic expression of XFGF20 and Xdkk-1 in animal caps. Two-cell stage embryos were injected in the animal pole of each cell with 400 pg VegT RNA or 10 pg VP16 Xtcf-3 RNA, as indicated, and animal caps were cut and collected at stage 9.5 for RT–PCR analysis. Download figure Download PowerPoint Activating or inhibiting the Wnt pathway had a dramatic effect on FGF20 and DKK1 expression. We injected RNA encoding an activated form of the downstream component of the pathway (VP16Xtcf-3) or a dominant-negative mutant (DNXtcf-3) (Vonica et al, 2000) into the equatorial zone of each blastomere at the four-cell stage (Figure 3C). XFGF20 and Xdkk-1 RNAs were increased by VP16Xtcf-3, and undetectable when Wnt signaling was blocked with DNXtcf-3RNA. Known direct (siamois) (Carnac et al, 1996; Brannon et al, 1997) and indirect (chordin) (Kessler, 1997) target genes of the maternal dorsal Wnt pathway (Figure 3A), as well as the zygotic Wnt target and mesodermal marker Xbra (Smith et al, 1991; Vonica and Gumbiner, 2002), showed a similar response. In contrast, Xwnt-8, a marker of ventral mesoderm (Christian et al, 1991) inhibited by the maternal Wnt pathway, retained expression in embryos injected with DNXtcf-3 RNA, but was absent when VP16Xtcf-3 RNA was injected. We also asked whether ectopic activation of the Wnt pathway in animal cap explants, which are normally fated to produce only ectoderm, could induce expression of XFGF20 and Xdkk-1 (Figure 3D). On its own, VP16Xtcf-3 can induce both genes only slightly, but induction was significantly enhanced by co-injection of the endomesoderm inducer VegT (Kofron et al, 1999). The indirect target chordin was similarly activated, and the direct Wnt target twin, a homologue of siamois, was strongly activated with VP16Xtcf-3 alone. We conclude that expression of XFGF20 and Xdkk-1 in animal caps is augmented by Wnt signaling. The differences in embryonic expression patterns between the two genes could be a consequence of different requirements for cooperative signaling pathways, responses to different Wnt ligands, or both. FGF-20 and DKK1 are direct targets of the canonical Wnt signaling pathway To differentiate between the direct and indirect effects of β-catenin stabilization on the control of FGF20 and DKK1, we took advantage of a dexamethasone-inducible form of VP16 Xtcf-3 (TVGR) (Darken and Wilson, 2001). TVGR RNA was injected alone or in combination with VegT RNA in the animal pole of two cell stage Xenopus embryos (Figure 4). Animal caps were dissected at stage 8.5 and dexamethasone was added in the presence or absence of cycloheximide, an inhibitor of translation. Under these conditions, the transcription of only those genes directly activated by VP16 Xtcf-3 will increase upon addition of the inducer. Cycloheximide treatment decreased the levels of XFGF20 and Xdkk-1 RNA in VegT and TVGR-injected embryos, but addition of dexamethasone significantly raised them. siamois, a direct Tcf target, and, less dramatically, Xbra, showed similar variation. On the contrary, levels of chordin RNA, an indirect Wnt target, did not respond to dexamethasone when translation was inhibited. The low level of siamois induction and the absence of chordin stimulation upon addition of dexamethasone are due to the late timing of Wnt activation, at the limit of competence for activation of dorsal genes (Darken and Wilson, 2001). In conclusion, the injection experiments in frog embryos suggest that expression of XFGF20 and Xdkk-1 is subject to direct regulation by the Wnt/β-catenin signaling pathway. Figure 4.Xenopus XFGF20 and Xdkk-1 are direct targets of the Wnt pathway. Embryos were injected at the two-cell stage in both blastomeres with 400 pg VegT RNA and 20 pg TVGR RNA, and treated with cycloheximide (CHX) and dexamethasone (DEX) as indicated. XFGF20 and Xdkk-1 were induced when DEX was added to CHX-treated caps (compare lanes 4 and 5). Siamois is a control for direct induction by the Wnt pathway, and chordin for indirect induction. Download figure Download PowerPoint To address the implied direct relationship between the Wnt/β-catenin pathway and the regulation of FGF20 and DKK1 more rigorously, we have performed chromatin immunoprecipitation (ChIP) assays with an anti-β-catenin antibody in the 293Top cells (for ChIP methodology, see Orlando, 2000; Weinmann et al, 2001). Stabilized β-catenin accumulates in the nucleus in a complex with TCF, and the heterodimer influences transcription by binding to TCF recognition sites in DNA (Huelsken and Behrens, 2002). Regulatory domains of direct β-catenin/TCF targets are therefore expected to be enriched in anti-β-catenin immunoprecipitates, as compared to immunoprecipitates obtained with a control antibody. Formaldehyde-fixed chromatin from 293 cells expressing either β-cateninS37A or GFP" @default.
• W2104490911 created "2016-06-24" @default.
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• W2104490911 date "2004-12-09" @default.
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• W2104490911 title "FGF-20 and DKK1 are transcriptional targets of β-catenin and FGF-20 is implicated in cancer and development" @default.
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• W2104490911 doi "https://doi.org/10.1038/sj.emboj.7600460" @default.
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