A Novel Wilms Tumor 1 (WT1) Target Gene Negatively Regulates

A Novel Wilms Tumor 1 (WT1) Target Gene NegativelyRegulates the WNT Signaling Pathway*□S

Received for publication, December 14, 2009, and in revised form, February 22, 2010 Published, JBC Papers in Press, March 10, 2010, DOI 10.1074/jbc.M109.094334

Myoung Shin Kim‡, Seung Kew Yoon§, Frank Bollig¶, Jirouta Kitagaki�, Wonhee Hur§, Nathan J. Whye‡,Yun-Ping Wu‡, Miguel N. Rivera**, Jik Young Park‡, Ho-Shik Kim‡1, Karim Malik‡‡, Daphne W. Bell§§,Christoph Englert¶, Alan O. Perantoni�, and Sean Bong Lee‡2

From the ‡Genetics of Development and Disease Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, the§Department of Internal Medicine, World Health Organization Collaborating Center of Viral Hepatitis, Catholic Research Instituteof Medical Science, The Catholic University of Korea, Seoul 137-701, Korea, the ¶Leibniz Institute for Age Research, Fritz LipmannInstitute, Beutenbergstrasse 11, 07745 Jena, Germany, the �Cancer and Developmental Biology Laboratory, NCI-Frederick,National Institutes of Health, Frederick, Maryland 21702, the **Massachusetts General Hospital Cancer Center, Harvard MedicalSchool, Charlestown, Massachusetts 02129, the ‡‡Cancer and Leukaemia in Childhood Sargent Unit, Department of Cellular andMolecular Medicine, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom, and the §§Cancer Genetics Branch,National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892

Mammalian kidney development requires the functions ofthe Wilms tumor gene WT1 and the WNT/�-catenin signal-ing pathway. Recent studies have shown thatWT1 negativelyregulatesWNT/�-catenin signaling, but themolecularmech-anisms by which WT1 inhibits WNT/�-catenin signaling arenot completely understood. In this study, we identified a gene,CXXC5, which we have renamed WID (WT1-induced Inhibitorof Dishevelled), as a novelWT1 transcriptional target that neg-atively regulatesWNT/�-catenin signaling.WT1 activatesWIDtranscription through the upstream enhancer region. In thedeveloping kidney, Wid and Wt1 are coexpressed in podocytesof maturing nephrons. Structure-function analysis demon-strated that WID interacts with Dishevelled via its C-terminalCXXC zinc finger and Dishevelled binding domains andpotently inhibits WNT/�-catenin signaling in vitro and in vivo.WID is evolutionarily conserved, and ablation of wid inzebrafish embryos with antisense morpholino oligonucleotidesperturbs embryonic kidney development. Taken together, ourresults demonstrate that theWT1negatively regulatesWNT/�-catenin pathway via its target gene WID and further suggest arole forWID in nephrogenesis.

Wilms tumor is a childhood kidney cancer arising from cellsthat failed to differentiate during kidney development (1).About 10–15% of Wilms tumors have loss of function muta-tions in the Wilms tumor gene (WT1) encoding a zinc fingertranscription factor indispensable during multiple stages of

kidney development (2–4). More recently, mutations resultingin the activation of theWNT/�-catenin signaling pathway havebeen identified in Wilms tumors. These include the activatingmutations in CTNNB1 (5–7), which encodes �-catenin, andloss of function mutations in WTX (8–10), encoding an X-linked protein that promotes degradation of �-catenin (11).Interestingly, activating mutations in CTNNB1 have beenfoundmore frequently in the tumors harboringWT1mutations(6, 7). Recent studies have shown that WT1 inhibits WNT/�-catenin signaling (12–14), but direct evidence for WT1-medi-ated inhibition of �-catenin signaling pathway is still lacking.

Multiple isoforms of WT1 are produced as a result of differ-ent promoter usage, alternative splicing, internal translation,and alternative translational start (4). These isoforms can belargely classified into two major isoforms, WT1(�KTS) andWT1(�KTS), distinguished by the presence or absence of threeamino acids (lysine, threonine, and serine (KTS)) between zincfingers 3 and 4 (15). WT1(�KTS) functions as a transcriptionfactor, whereasWT1(�KTS) has been implicated in post-tran-scriptional regulation (1, 3). Mice nullizygous for Wt1 fail toinitiatemetanephric development, demonstrating a pivotal rolefor WT1 during the early steps of nephrogenesis (16). Addi-tional studies have also demonstrated clear roles for WT1 inlater stages of kidney development, such as the formation ofnephrons and differentiation of podocytes (17, 18), as well as inhomeostasis of adult kidneys (19, 20). Previously, we conductedgenome-wide analyses of WT1 target genes to delineate thepathways that WT1 might regulate (21). Here, we report theidentification of a novelWT1 target gene that encodes a nega-tive regulator of the WNT/�-catenin signaling pathway.

EXPERIMENTAL PROCEDURES

Cell Lines and Plasmids—UB27 and UD29, U2OS-derivedcell lines with tetracycline (tet)3-repressible WT1(�KTS) orWT1(�KTS) expression, respectively, were maintained as de-

* This work was supported, in whole or in part, by National Institutes of Healthgrants from the Intramural Research Program, NIDDK (to S. B. L.), NationalHuman Genome Research Institute (to D. W. B.), and NCI (to A. O. P.). Thiswork was also supported by the Deutsche Forschungsgemeinschaft (toC. E.) and Grant FG06-11-11 of the 21st Century Frontier Functional HumanGenome Project in Korea (to S. K. Y.).

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental “Experimental Procedures” and Figs. S1–S5.

1 Present address: Dept. of Biochemistry, College of Medicine, The CatholicUniversity of Korea, Seoul 137-701, Korea.

2 To whom correspondence should be addressed: Genetics of Developmentand Disease Branch, 9000 Rockville Pike, Bldg. 10, 9D11, Bethesda, MD20892. Tel.: 301-496-9739; Fax: 301-480-0638; E-mail: [email protected].

3 The abbreviations used are: tet, tetracycline; CM, conditioned media; ChIP,chromatin immunoprecipitation; siRNA, small interfering RNA; DVL,Dishevelled; MO, morpholino oligonucleotides; PTU, 1-phenyl-2-thio-urea; DBD, DVL binding domain.

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scribed previously (21). HEK293, mouse NIH3T3, andmouse Land L/Wnt3a fibroblast cell lines (ATCC, Manassas, VA)were grown in Dulbecco’s modified Eagle’s medium supple-mented with 10% fetal bovine serum. Wnt3a and controlL CM were prepared according to the protocol providedby ATCC. Zebrafish Wnt8a (pCS2P� wnt8 ORF1) andSuper8XTOPFlash plasmids were kindly provided by R.Moon (University of Washington, Seattle). The cytomegalo-virus-driven full-length human WID (pCMVSport6-WID;GenBankTM accession number BC050046) and the humanAXIN1 cDNA (GenBankTM accession number BC044648)werepurchased from ATCC. AXIN1 was subsequently subclonedinto pcDNA3 (Invitrogen).WID deletion mutants were gener-ated by PCR (see supplemental material for primer sequence).Human IDAX cDNA was PCR-amplified and subcloned intopCMVSport6. All PCR-generated cDNAs were verified bysequencing.HumanDVL3 cDNAwas purchased fromOriGene(Rockville, MD) and subcloned into pCMV-3Tag-2A (Strat-agene, La Jolla, CA). FLAG-taggedmouseDvl2 expression plas-mid was kindly provided by X. He (Harvard Medical School,Boston).Generation of Rabbit Polyclonal Antibody against WID—A

portion of mouseWid cDNA containing the C-terminal region(residues 109–317) was amplified by PCR (5�-CCC GGA TCCTAT TGG CCA ATG GTC ATG ACC-3� and 5�-CCC GAATTC ACT GAA ACC ACC GGA AGG C-3�) and subclonedinto pGEX3X plasmid (Amersham Biosciences) to generate aGST-Wid fusion protein. PurifiedGST-Widwas used to obtainaffinity-purified rabbit polyclonal antibodies (Strategic Bioso-lutions, Newark, DE).Quantitative Reverse Transcription-PCR—Total RNAs iso-

lated from UB27 and UD29 cells (� or � tet) were reverse-transcribed, and the expression levels ofWID andGAPDHwereanalyzed by real time PCR using TaqMan probes (Applied Bio-systems, Foster City, CA). Data were analyzed by comparativeCt method using GAPDH as an endogenous control andexpressed as fold differences to a reference value ofWID in thepresence of tet. For the quantification of the zebrafish �-cate-nin target gene (dkk-1), 1 �l of zebrafish cDNA from MO-in-jected embryos was used employing the QuantiTect SYBRGreen real time PCR kit (Qiagen). All samples were measuredas triplicates and normalized to the corresponding amounts ofDanio rerio elongation factor-1� cDNA measured within thesame plate. Relative expression levels were calculated using theCtmethod.Chromatin Immunoprecipitation Assay—UB27 cells were

cultured without tet for 16 h to induce WT1 expression, andchromatin immunoprecipitation (ChIP) was performed asdescribed previously (21). Chromatin immunoprecipitatedwith either rabbit polyclonal�-WT1 antibody (C19, SantaCruzBiotechnology, Santa Cruz, CA) or normal rabbit IgG wasamplified by PCR using primers corresponding to the indicatedenhancer regions (E1–E3) or the N1–N2 regions (see supple-mental material for primer sequence). For quantitative ChIP,precipitated chromatin was amplified with the same primersand SYBR Green PCR Master Mix (Applied Biosystems). Dataare expressed as fold enrichment to a reference value of IgGcontrol.

Luciferase Reporter Assays for Enhancer Regions of WID—The enhancer regions (E1–E3) were subcloned into a pGL3plasmid containing a minimal promoter (21) in both sense andantisense orientations. NIH3T3 cells were transfected with 0.4�g of pcDNA3-WT1(�KTS) or the empty vector alongwith 0.1�g of the enhancer-reporter and 0.01 �g of Renilla luciferase(Promega, Madison, WI) plasmids using FuGENE 6 (RocheApplied Science). After 48 h, firefly and Renilla luciferase activ-ities were measured using Dual-Luciferase kit (Promega).Renilla luciferase activity was used to normalize transfectionefficiency.Luciferase Reporter Assays for WNT/�-Catenin Signaling—

To measure WNT-dependent T cell factor/�-catenin activ-ity, 0.4 �g of pCMVSport6-WID, IDAX, or the empty vectorwas transfected into HEK293 cells together with 0.1 �g ofSuper8XTOPFlash and 0.01 �g of Renilla luciferase plas-mids. After 46 h, cells were serum-starved for 6 h, stimulatedwith either control L or Wnt-3a CM for 6 h, and luciferaseactivities weremeasured. To examine the effects ofWT1 on theWNT/�-catenin pathway, UB27 cells were transfected with 0.1�g of Super8XTOPFlash and 0.01 �g of Renilla luciferase plas-mids, and the following day, tet was removed to induce WT1expression for 24 h. Cells were serum-starved for 6 h and stim-ulated with either control or Wnt3a CM for 6 h, and luciferaseactivity was measured as described. For the siRNA knockdownofWID in UB27 cells, cells were transfected twice with humanWID siRNAs (see supplemental material) or control scrambledsiRNA using Lipofectamine 2000 (Invitrogen) along withSuper8XTOPFlash and Renilla luciferase plasmids. After 24 hpost-transfection, tet was removed to induce WT1 expressionfor 24 h. Cells were stimulated with either control or Wnt3aCM for 6 h, and luciferase activity was measured.siRNA Knockdown of Wid—Mouse L fibroblasts were trans-

fected with two different Wid siRNAs (see supplementalmaterial) or control scrambled siRNA using Lipofectamine2000 (Invitrogen) along with Super8XTOPFlash and Renillaluciferase plasmids. After 46 h, cells were serum-starved for 6 hand stimulated with either control or Wnt-3a CM for 6 h, andluciferase activity was measured. For the analysis of �-catenin,L cells were transfected with siRNA against Wid or controlscrambled siRNA using Lipofectamine 2000 (Invitrogen). At48 h, cellswere serum-starved for 6 h and treatedwithWnt3a orcontrol CM for 1 h, and total cell lysates were analyzed byimmunoblotting with antibodies against �-catenin (Santa CruzBiotechnology), WID, and actin (Sigma). To visualize nuclearlocalization of �-catenin, L cells cultured on chambered cover-glass (Nunc, Rochester, NY) were transfected with siRNAagainst Wid or control siRNA and treated with Wnt3a CM asdescribed. Cells were fixed, immunostained with antibodies toWID and �-catenin (BD Biosciences), and visualized withAlexa 594 anti-rabbit IgG and Alexa 488 anti-mouse IgG(Invitrogen). Confocal images were captured with the AxioObserver Z1 inverted laser-scanning microscope (Zeiss,Thornwood, NY) equipped with LSM 5 Live confocal scanner(Zeiss).Immunoprecipitation and Immunoblot Analyses—HEK293

cells transfected with pcDNA3-AXIN1, FLAG-Dvl2, andpCMVSport6-WID were lysed in buffer (20 mM Tris-HCl (pH

WT1 Target Gene Inhibits WNT/�-Catenin Signaling Pathway

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7.5), 150 mM NaCl, 1% Nonidet P-40, 10% glycerol) containingprotease inhibitors (Roche Applied Science). The supernatantwas immunoprecipitated with either �-WID or �-FLAG anti-bodies (M2, Sigma) and immunoblotted with �-AXIN (SantaCruz Biotechnology), �-FLAG, or �-WID antibodies. For de-tecting the endogenous WID-DVL2 interaction, HEK293 celllysates were immunoprecipitated with �-WID antibody followedby immunoblotting with �-Dvl2 antibody (kindly provided byMikhail V. Semenov, Children’s Hospital Boston, Boston).Identification of the DVL-binding Domain of WID—Myc-

DVL3, wild type WID, and the WID deletion mutants (WID-�DBD, WID-�CXXC, and WID-�CXXC-DBD) were syn-thesized in vitro using [35S]methionine (PerkinElmer LifeSciences) and TNT-coupled reticulocyte lysate systems (Pro-mega). Similar amounts of 35S-labeled wild type WID or thedeletion mutants andMyc-DVL3 were mixed in binding buffer(20 mM Tris (pH 7.5), 75 mM NaCl, 0.1% Nonidet P-40, 10%glycerol) containing a mixture of protease inhibitors (RocheApplied Science), and the complex was immunoprecipitatedwith �-Myc antibody (Sigma) and analyzed on 12% SDS-poly-acrylamide gel followed by autoradiography.Immunohistochemistry of Mouse Embryonic Kidney—Mouse

embryonic kidneys at 14.5 days post-coitum were isolated,fixed, and embedded in paraffin. After tissue sectioning, slideswere heated in 10 mM citrate buffer, pH 6.0, using a microwaveoven after deparaffinization and rehydration. Serial sectionswere incubated anti-WT1 (BD Biosciences) or anti-WIDantibodies and developed with Vectastain ABC kit (VectorLaboratories, Burlingame, CA) according to themanufactur-er’s recommendations. Tissue sections were counterstainedwith hematoxylin.Zebrafish—For the analysis of headless phenotypes, �50–

100 pg of each DNA, zebrafish Wnt8a expression plasmid(pCS2P� wnt8 ORF1) and pCMVSport6-WID or pCMVSport6-IDAX, was injected through the chorion into the blastomereof one-cell wild type zebrafish embryos. For coinjectionof two DNA, plasmids were mixed (1:1) prior to injection andinjected into the 1-cell stage at 50 ng/�l (50–100 pg/embryo).After 5 days postfertilization, injected embryos were observedand scored for the headless, small eye, or rescued (wild type)phenotypes. The experiment was repeated three to four times.For the analysis of zebrafish kidney, we used the wt1b::GFPtransgenic zebrafish line with pronephros-specific green fluo-rescent protein expression (22). Embryos were raised at 28.5 °Cand staged according to Kimmel et al. (23). Morpholino anti-sense oligonucleotides (see supplemental material for se-quence) against either the first splice acceptor site (intron 1 toexon 2) of zebrafish wid pre-mRNA (called splice MO) or thewid start codon (called ATG MO) were dissolved in water (1mM) and injected into the yolk of embryos in the 1–4-cell stage.A gradient of different injection volumes was tested for eachmorpholino to find the lowestmorpholino amount, which leadsto a consistent and reproducible phenotype (�0.6 nl (0.6 pmol)for the wid splice and 0.3 nl (0.3 pmol) for the wid ATG mor-pholino, respectively). Embryos treated with 1-phenyl-2-thio-urea and anesthetized with Tricaine (0.016%) were embeddedin 1% low melting point agarose (Biozym, Hessisch Oldendorf,Germany) with their back facing the bottom of a �-dish (ibidi

GmbH, Munchen, Germany) and photographed using aninverted fluorescence microscope (Axiovert 200, Zeiss).

RESULTS

Identification of a Novel WT1 Target Gene—We previouslyperformed kinetic expression profiling analysis to identifyWT1-regulated genes thatmight play important roles in kidneydevelopment (21). This analysis led us to identify a numberof genes and novel ESTs induced by WT1(�KTS). Amongthem, three ESTs (GenBankTM accession numbers AK001782,BC006428, and BC002490) were derived from a single geneencoding a novel transcript termed CXXC5 in the data base(herein designatedWID, see below for detail), which belongs toa recently identified gene family characterized by aCXXCmotif(X is any amino acid) (24). We first confirmed that WID is adirect transcriptional target of WT1 by examining the expres-sion of endogenous WID upon induction of WT1(�KTS) orWT1(�KTS) using tet-repressible WT1-expressing cell lines(21). Following WT1(�KTS) expression, levels of the endoge-nousWID transcript increased rapidly and gradually decreasedover time (Fig. 1A). Upon WT1(�KTS) expression, however,only a marginal increase inWID transcript levels was observedat 4 h. We also observed a concomitant increase in the levels ofthe endogenous WID protein shortly after the induction ofWT1(�KTS) using a rabbit polyclonal antibody raised againstWID (Fig. 1B).Next, we examined theWID proximal promoter region (�5

kb upstream from exon 1) to determine whether WT1 mightdirectly activate its transcription, but we found no evidence forWT1-mediated transactivation of the proximalWID promoterin a reporter assay (data not shown). Thus, we searched forconserved regulatory regions by comparing the genomic se-quences of WID from human, rat, and mouse and identifiedthree highly conserved regions, designated E1–E3, each con-taining multiple potential GC-rich WT1-binding sites (25)located �10 kb upstream of the transcriptional start site (Fig.1C and supplemental Fig. S1). To determine whetherWT1 wasrecruited to these sites, ChIP analysis was performed. Theresults demonstrated that WT1(�KTS) was present in theputative enhancer regions E1 and E3 but not at E2 nor inthe adjacent regions designated N1 and N2 (Fig. 1C). Anindependent quantitative ChIP analysis confirmed this find-ing (Fig. 1C, lower panel). Each putative enhancer region wasthen inserted upstream of a minimal promoter-luciferasereporter plasmid and tested in the reporter assay. The E1region, positioned in either direction,was activated 5–6-fold byWT1(�KTS), whereas the E2 and E3 regions were only mod-estly activated (Fig. 1E). To further examine WT1-mediatedtransactivation of the enhancers, we deleted at least three tofour putative WT1-binding sites from each of the enhancerregions and tested in the reporter assay. Deletion of multiplepotential WT1 sites in the E1 had no effect on the ability ofWT1 to activate the reporter, suggesting the existence of otherWT1-binding sites (supplemental Fig. S2). This is not surpris-ing given the presence of high GC-rich sequences throughoutthe enhancer (supplemental Fig. S1). Interestingly, deletion offour putative binding sites in the E3 region resulted in 40%reduction in theWT1-mediated activation, demonstrating that




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WT1, at least partially, activates E3 enhancer through one ormore of the deleted WT1 sites (supplemental Fig. S2). BecauseWT1 was not recruited to the E2 region by ChIP (Fig. 1, C andD), a modest activation from the E2 region by WT1(�KTS)might be due to an indirect mechanism in a manner similar tothe WT1-SF1-mediated activation of the Mullerian-inhibitingsubstance promoter, which is independent of WT1 bindingto the DNA (26). Thus, deletion of three putativeWT1-bind-ing sites in E2 had no effect on the reporter activity

(supplemental Fig. S2). Collectively, these results suggest thatWT1(�KTS) activates the transcription of WID through theupstream enhancer region.WID Encodes a Negative Regulator of the WNT/�-Catenin

Signaling Pathway—Sequence analysis of WID by a BLASTPsearch identified another CXXC family protein, CXXC4, alsoknown as IDAX (Inhibitor of Dishevelled and Axin) (27), with ahigh degree of homology to the C terminus of WID (sup-plemental Fig. S3). The C-terminal regions of WID and IDAX

FIGURE 1. WID is a WT1(�KTS) target gene. A, quantitative reverse transcription-PCR analysis of WID. WID transcript level was measured by quantitativereverse transcription-PCR at indicated times after removal of tet in UB27 and UD29 cells. Data are the means � S.D. of three independent experiments.B, Western blot analysis. Total cell lysates were prepared from UB27 cells (� or � tet) and immunoblotted with �-WT1, �-WID, and �-actin antibodies. C, ChIPanalysis. UB27 cells were induced to express WT1(�KTS), and the cross-linked chromatin was immunoprecipitated with �-WT1 or rabbit IgG antibodies,followed by PCR amplification with primers corresponding to the WID enhancer regions E1–E3 (black boxes) or to the adjacent regions N1 and N2 (gray boxes)located �10 kb upstream of the transcriptional start (�1). Distance between regions is as follows: E1–E2 (420 bp), E2–N1 (1540 bp), N1–E3 (2170 bp), and E3–N2(1610 bp). Quantitative PCR analysis of ChIP was performed independently using SYBR Green, and the result is presented as the fold increase over IgG (lowerpanel). D, luciferase (LUC) reporter assay for WID enhancer regions. NIH3T3 cells were cotransfected with plasmids containing the E1, E2, or E3 regions in eithersense (SE) or antisense (AS) orientations and with either pcDNA3-WT1(�KTS) or empty vector and Renilla luciferase. Data represent the mean � S.D. from threeindependent experiments.




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contain the CXXC domain as well as highly conserved se-quences flanking the CXXCmotif. The N-terminal half ofWIDdoes not share significant homology with any other protein andcontains an unusually high number of serine, alanine, and gly-cine residues (comprising 51.6% of the first 167 residues).Because IDAX has been shown to inhibit WNT/�-cateninsignaling (27), we tested whetherWIDmight also function asa negative regulator of the WNT pathway by using aSuper8XTOPFlash luciferase reporter assay to measure theactivation of theWNT/�-catenin pathway (28). Upon transfec-tion of the reporter plasmid into HEK293 cells and treatmentwith Wnt3a CM, a 10-fold induction of the luciferase activitywas observed as compared with the control CM (Fig. 2A). Incontrast, cotransfection ofWIDwith the luciferase reporter led toa severe repression (�5-fold reduction) of Wnt3a-mediatedreporter activity. As expected, coexpression of IDAX also led tothe inhibition ofWNT/�-catenin signaling.We observed a sim-ilar inhibition of Wnt3a signaling by WID in NIH3T3 mousefibroblast, mouse L-fibroblast, andU2OS human osteosarcomacell lines (data not shown).We next examined the effects of Wid depletion on the

activation of the WNT/�-catenin pathway using theSuper8XTOPFlash reporter assay. Suppression of endoge-nousWid inmouse L fibroblasts using twodifferent small inter-fering RNAs (siRNA) resulted in a significantly higher level ofthe Wnt3a-mediated luciferase activity than the controlsiRNA-transfected cells (Fig. 2B). To further study the inhibi-tory effect of WID on the WNT/�-catenin signaling pathway,we examined the level of �-catenin after reducing endogenousWid levels by siRNAs. In the absence of Wnt3a, suppression ofWid led to a slight increase in �-catenin levels compared withthe control siRNA-transfected cells (Fig. 2C, compare lane 1with lanes 3 and 5). In the presence ofWnt3a, depletion ofWidresulted in a greater accumulation of �-catenin compared withthe control (Fig. 2C, compare lanes 2, 4, and 6). This was furtherconfirmed by confocal microscopy, which showed about 2-foldincrease in the nuclear localization of �-catenin in Wnt3a-treatedWid depleted cells compared with the control (Fig. 2D).Together, these observations clearly demonstrate the role ofWID as the negative regulator of theWNT/�-catenin signalingpathway.WID Is a Negative Regulator of WNT Signaling in Zebrafish—

Next, we examined whether WID inhibits WNT signaling invivo. To this end, we used a previously described zebrafishmodel in which ectopic expression of zebrafish Wnt8 cDNAdriven from a constitutive promoter causes either a headless or asmall eye phenotype (29). When zebrafish Wnt8 cDNA wasinjected into fish embryos, approximately two-thirds of theinjected embryos displayed either the headless (�60%) or thesmall eye (�17%) phenotype (Fig. 2E). Coinjection of Wnt8 andWID cDNAs, however, resulted in less than 5% of the embryoswith the headless phenotype, and�70% of the embryos displayednormalheaddevelopment,demonstrating thatWIDfunctionsasanegative regulator of theWNTpathway in zebrafish. As expected,coinjection of IDAX also reversed the Wnt8-induced headlessphenotype but less efficiently thanWID (Fig. 2E).WT1 Inhibits WNT/�-Catenin Signaling Pathway via WID—

Because WT1(�KTS) activates endogenous WID expression

(Fig. 1), a potent inhibitor of WNT/�-catenin signaling, wetested whether WT1(�KTS) expression alone can inhibitWNT/�-catenin signaling. To this end, we transfected theSuper8XTOPFlash plasmid into the WT1(�KTS)-induciblecell line and examined the reporter activity with or withoutWT1 expression. In the absence of WT1(�KTS) expression(�tet), Wnt3a activated the luciferase reporter to �5-fold (Fig.3A). In contrast, expression ofWT1(�KTS) (�tet) resulted in amarked decrease (�2-fold) in the Wnt3a-mediated reporteractivity, demonstrating that WT1(�KTS) can negatively regu-

FIGURE 2. WID inhibits the WNT/�-catenin signaling. A, TOPFlash luciferasereporter assay. Super8XTOPFlash plasmid was transfected into HEK293 cellseither with pCMV-WID, IDAX, or empty vector. Luciferase activity wasmeasured following treatment with control L (white bar) or Wnt3a CM(black bar) for 6 h. B, luciferase reporter assay after siRNA knockdown ofWid. Super8XTOPFlash plasmid was transfected into mouse L cells eitherwith scrambled (control) or Wid siRNAs, and luciferase activities were mea-sured. Data represent the mean � S.D. from three independent experiments.Student’s t test. *, p value � 0.017; **, p value � 0.019. C, stabilization of�-catenin after Wid depletion. L cells were transfected with either scrambled(control) or Wid siRNAs, treated with L (�) or Wnt3a CM for 1 h, and total celllysates were immunoblotted with antibodies against �-catenin, WID, andactin. D, nuclear localization of �-catenin after Wid depletion. L cells grown onchambered coverglass were transfected with control or Wid siRNAs andtreated with Wnt3a CM as in C. Fixed cells were double-immunostained withantibodies against WID and �-catenin. Images were captured under an oilimmersion objective. Scale bar, 20 �m. Lower panel, percentage of nuclear�-catenin from 3 to 4 randomly chosen fields was calculated from three inde-pendent experiments. Data represent the mean � S.D. Student’s t test. *, pvalue � 0.008; **, p value � 0.016. E, inhibition of WNT signaling by WID inzebrafish. Plasmids containing zebrafish wnt8a alone or together with eitherWID or IDAX were injected into 1-cell zebrafish embryos and scored for theheadless, small eye, or wild type phenotypes. Representative phenotypes ofzebrafish embryos are shown.




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late theWNT/�-catenin signaling. Reduction ofWID by siRNAfollowing WT1(�KTS) expression resulted in a partial loss ofthe WT1-mediated inhibition of WNT/�-catenin signaling ascompared with the control siRNA-transfected cells (Fig. 3B),suggesting that WT1-induced repression of WNT/�-cateninsignaling is at least partially mediated through WID. Westernblot analysis confirmed the induction ofWT1 and reduction ofWID expression (Fig. 3B, bottom panel).Physical Interaction of WID and DVL—Activation of WNT/

�-catenin signaling ismediated by the direct binding of Dishev-elled (DVL) to a complex containing AXIN-APC-GSK3� (30).A previous study showed that IDAX might inhibit WNT/�-catenin signaling by directly interacting with DVL (27). Todetermine whether WID can physically interact with DVL, weintroduced FLAG-tagged DVL2, and WID into HEK293 cells

followed by immunoprecipitation with an anti-WID antibody.As shown in Fig. 4A,WID readily formed a complex withDVL2but not with AXIN. A reciprocal immunoprecipitation with ananti-FLAG antibody also demonstrated the interaction ofDVL2 andWID.We further confirmed the interaction betweenendogenous WID and DVL2 in HEK293 cells by immunopre-cipitating the endogenous WID, followed by immunoblottingwith an anti-DVL2 antibody (Fig. 4B).Identification of the DVL-binding Domain of WID—A

“KTXXXI” motif (X is any amino acid) within IDAX has beenidentified as the minimal DVL binding domain (DBD) (31). TheKTXXXI motif, as well as the immediate surrounding residues(herein designated as the DBD), is perfectly conserved in WID(supplemental Fig. S3). To examine whether the KTXXXI motif isrequired for binding toDVL,wedeleted theputativeDBDdomainofWID (WID-�DBD, residues 284–296) and tested the ability oftheWID-�DBDmutant to bindDVL in vitro. As controls, we alsogenerated two additional WID deletion mutants, one lacking theCXXC motif (WID-�CXXC, residues 263–281) and the otherlacking both theCXXCand theDBDmotifs (WID-�CXXC-DBD,residues 263–296). Similar amounts of in vitro 35S-labeled wildtype WID or the deletion mutants were mixed with 35S-labeledMyc-DVL3 followed by immunoprecipitation with �-Myc anti-body.Comparedwith thewild typeWID, theWID-DVL3 interac-tion was markedly reduced with the deletion of DBD, but the�DBDmutant still retained some binding to DVL (Fig. 4C, com-pare lanes 6 and7). In contrast, deletionof theCXXCmotif hadnoeffect on the WID-DVL3 interaction. Interestingly, deletion ofbothDBDandCXXCdomains (�CXXC-DBD)resulted inanearlycomplete lossof theWID-DVL3 interaction (Fig. 4C, lane9), dem-onstrating that both domains are required for the full interactionwith DVL.DBD and the CXXC Domains Are Essential for WID Func-

tion—We next examined the ability of the WID deletionmutants to inhibit WNT/�-catenin signaling using theTOPFlash reporter assay. As expected, expression of wildtypeWID led to a significant inhibition ofWnt3a-stimulatedTOPFlash luciferase reporter (Fig. 4D). However, expression ofWID-�DBD mutant failed to inhibit the Wnt3a-stimulatedTOPFlash luciferase activity, indicating that the WID-DVLinteraction is critical for the inhibition of the WNT signalingpathway. Surprisingly, the WID-�CXXC mutant completelylost the ability to inhibit WNT signaling (Fig. 4D), despiteretaining the ability to bind to DVL. In fact, bothWID-�CXXCandWID-�DBD-CXXC mutants resulted in a stronger WNT-reporter response than either the control (empty vector) or theWID-�DBD mutant, suggesting a possible dominant-negativeeffect on the endogenousWID. These results demonstrate thatthe WID-DVL interaction is necessary but not sufficient toinhibit WNT signaling and further highlight the importance ofthe CXXC domain in addition to the DBD. Western blottingdemonstrated that theWIDdeletionmutantswere expressed ata higher level than wild type WID (Fig. 4E), indicating that theinability of theWIDmutants to inhibitWNT signaling was notdue to a difference in expression levels.Wt1 and Wid Are Coexpressed in Podocytes of Developing

Kidney—Expression of WT1 in kidney is restricted to meta-nephric mesenchyme, renal vesicles, the proximal portion of

FIGURE 3. WT1 inhibits the WNT/�-catenin signaling. A, luciferase reporterassay. Super8XTOPFlash and Renilla luciferase plasmids were cotransfectedinto UB27 cells, and luciferase activity was measured with (�tet) or without(�tet) WT1 expression. B, siRNA knockdown of WID. Super8XTOPFlash andRenilla luciferase plasmids were transfected into UB27 cells with either scram-bled (control) or WID siRNA, and WT1 expression was induced (�tet) or unin-duced (�tet), and luciferase activity was measured. Data represent themean � S.D. from three independent experiments. Cell lysates were immu-noblotted with antibodies against WT1, WID, and actin.




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S-shaped bodies, and finally podocytes, which are specializedvisceral epithelial cells that form the glomerular filtration bar-rier in nephrons (1). To determine whether Wt1 and Wid arecoexpressed in developing kidney, we examined the expressionof Wt1 and Wid in the developing mouse kidney. In the devel-oping kidney, Wt1 expression can be seen in the condensedmesenchyme, proximal region of S-shaped bodies and podo-cytes of maturing glomeruli (Fig. 5A, arrows, left panel). Immu-nostaining of the serial section of the kidney revealed that Widis coexpressed with Wt1 in the proximal region of S-shapedbodies and the developing podocytes (Fig. 5A, arrows, rightpanel), but Wid expression was also detected in non-Wt1-ex-pressing cells of the tubules (Fig. 5A, white arrowheads).Inactivation ofWid in Zebrafish Leads to Kidney Defect—We

searched for WID orthologs in the NCBI data base and identi-fied a hypothetical protein in zebrafish (GenBankTM accessionnumber XP_686158) that shares 55% identity and 61% similar-ity with human WID (supplemental Fig. S4). Given such highevolutionary conservation, we examined whether zebrafishwidmight function in kidney development. To this end, we utilized

a transgenic zebrafish expressing green fluorescent proteinunder the zebrafish wt1b promoter, wt1b::GFP (22), whichexpresses green fluorescent protein in the glomerulus andthe pronephric tubule of developing pronephros. A spliceantisenseMO directed against the first splice acceptor site ofwid (supplemental Fig. S5A) or an antisense MO directedagainst the start codon of wid (ATG MO) was used to deter-mine the effects of ablating wid expression in wt1b::GFPembryos. At 48 h post-fertilization, control MO-injectedembryos displayed normal pronephros development, form-ing two glomeruli (arrowheads) and the tubules that extendlaterally (arrows) (Fig. 5B, control panel). Remarkably, injec-tion of either the splice MO or the ATG MO against widresulted in malformed pronephros with large cysts in theglomerular-tubular region in �80% of the injected embryosat 48 h post-fertilization (Fig. 5B, see also Table 1). Theseresults demonstrate that normal pronephric development inzebrafish requires wid expression. The specificity of thesplice MO was demonstrated by the dose-dependent de-crease in the spliced widmRNA by reverse transcription-PCR

FIGURE 4. WID interacts with DVL. A, interaction of WID and Dvl2. HEK293 cell lysate transfected with WID, FLAG-Dvl2, and AXIN1 was immunoprecipitated (IP)with �-WID or �-FLAG antibody and immunoblotted (IB) with �-FLAG, �-AXIN, or �-WID antibodies. B, interaction of endogenous WID and DVL2. HEK293 celllysate was immunoprecipitated with �-WID antibody and immunoblotted with �-Dvl2 antibody. C, in vitro binding assay. In vitro synthesized 35S-labeled WID(wild type or deletion mutants) was incubated with 35S-labeled Myc-DVL3, immunoprecipitated with �-Myc antibody, and analyzed by SDS-PAGE andautoradiography. D, DBD and the CXXC are essential domains. HEK293 cells were cotransfected with Super8XTOPFlash plasmid and either with wild type WIDor WID deletion mutants, and luciferase activity was measured after control or Wnt3a CM treatment. Data represent the mean � S.D. from three independentexperiments. E, Western blot analysis. HEK293 cells were transfected with wild type FLAG-WID or WID deletion mutants, and cell lysates were analyzed byimmunoblotting with �-FLAG and �-actin (loading control) antibody. Because of the lower expression of wild type WID, more cell lysates from the empty vectoror wild type WID-transfected cells were loaded.




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analysis (supplemental Fig. S5B), and the use of two morpho-lino oligonucleotides with unrelated sequences further demon-strates the specificity of the observed phenotypes.

We analyzed the expression levels of several known zebrafish�-catenin target genes after the suppression ofwid and comparedit with the control zebrafish embryos. Among the target genesexamined, we found consistent up-regulation (2–3-fold) of dkk-1mRNAafterwid depletion (Fig. 5C). TreatmentwithwidMOwasvery effective becausewid transcripts were hardly detectable.

DISCUSSION

In Wilms tumors, loss of WT1 often occurs concomitantlywith nuclear localization of �-catenin resulting from gain-of-function (activating) mutations within CTNNB1 itself (7) or byother as yet unknown mechanisms (32). In this study, we dem-onstrate that a novel target of WT1, WID, functions to nega-tively regulate WNT/�-catenin signaling. Recently, a newlydescribed tumor suppressor gene,WTX, was found to be inac-tivated in a subset of Wilms tumors (8–10). TheWTX proteinforms a complex with AXIN-APC-GSK3� and actively pro-motes the degradation of �-catenin (11). Collectively, theseobservations suggest that activation of the WNT/�-cateninpathway may be a common mechanism underlying the forma-tion of Wilms tumors (33). However, preliminary examinationof expression profiling data base for any alteration of WID inWilms tumor and colorectal cancer did not reveal any signifi-cant changes inWID transcript levels (data not shown).Consistent with our findings reported in this study, exoge-

nous expression of WT1 in breast cancer cell line resulted inreduced �-catenin levels (14), and deletion of Wt1 in Sertolicells of the developing testis resulted in increased �-cateninlevels as a result of �-catenin stabilization (12). Another recentstudy also demonstrated that WT1 inhibits WNT/�-cateninsignaling by competing with limited amount of coactivatorCREB-binding protein, which is also required by the T cell fac-tor-�-catenin transcription complex (13). However, a directmechanismbywhichWT1 regulates�-catenin levels or activityhas not been demonstrated. In this study, our results demon-strate a direct role for WT1 in the negative regulation of theWNT/�-catenin signaling pathway by activating the transcrip-tion ofWID, which directly binds to DVL and prevents WNT-mediated stabilization of �-catenin.

During the course of our study, a recent report identifiedWID/CXXC5 as the BMP4-induced inhibitor of WNT/�-cate-nin signaling in neural stem cells (34), but how WID/CXXC5inhibits WNT signaling was not demonstrated. In the samestudy, it was also reported that exogenously expressed WID/CXXC5 is predominantly nuclear. Our results are consistentwith the role of WID in the inhibition of WNT/�-catenin sig-naling; however, our data clearly demonstrate that endogenousWID is predominantly cytoplasmic (Fig. 2D). Curiously, weobserved a small amount of WID translocated into the nucleusupon Wnt3a stimulation (Fig. 2D and data not shown). Ourfindings further demonstrate that WID-DVL interaction re-quires the CXXC and DBD domains of WID and that bothdomains are essential for disabling DVL, which is responsiblefor the stabilization of �-catenin (30). Interestingly, theseresults further imply that the inhibition ofWNT/�-catenin sig-naling byWID requiresmore than just binding to DVL becausethe WID-�CXXC mutant, which retained the ability to bindDVL, was unable to inhibit the WNT/�-catenin signaling (Fig.

FIGURE 5. Depletion of zebrafish wid results in defective kidney develop-ment. A, colocalization of Wt1 and Wid in podocytes of developing kidney. Serialsections of mouse embryonic kidney at 14.5 days post-coitum were immuno-stained with �-WT1 or �-WID antibodies. Arrows indicate pre-podocytes ofS-shaped bodies and podocytes of maturing glomeruli, which are positive forboth Wt1 and Wid. White arrowheads indicate Wt1-negative tubules that expressWid. Scale bars, 100 �m. B, representative images of the pronephros of wt1b::GFPtransgenic zebrafish embryos injected with either the splice (Splice MO) or thestart codon (ATG MO) wid antisense morpholino oligonucleotides are shown.Cystic glomeruli are clearly visible in the wid morpholino-injected animals but notin the control. Arrowheads indicate glomeruli, and arrows indicate pronephrictubules, and asterisks indicate exocrine pancreas. C, quantitative reverse tran-scription-PCR analysis of wid and dkk-1. Total RNAs were collected from 10pooled control morpholino or wid morpholino-injected embryos at the indicatedstage. For comparison, expression in 30-h-old control embryos was set to 1. Dataare the mean � S.D. of three independent experiments.

TABLE 1Phenotypic alterations in wid morpholino-injected embryos

Morpholino na Pronephric cystsb Curved body

Uninjectedc 40 0 0wid splice 37 32 (86%) 34 (92%)wid ATG 32 23 (72%) 30 (94%)

an indicates number of embryos.b Evaluation was 2 days after injection using a fluorescence stereomicroscope.c Nophenotypic differences could be observed between uninjected and controlmor-pholino-injected embryos.




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4, C and D). The precise mechanism by which the CXXCdomain functions to negatively regulate theWNT pathway willrequire further study.We also note that, by virtue of its ability tointeract with DVL, WID might also regulate �-catenin-inde-pendent WNT pathways, such as the planar cell polarity path-way that regulates cell polarity and movement or the Ca2�-related signaling pathway that involves cell adhesion (35).WID orthologs are present in all mammals and in lower verte-

brates such as zebrafish (supplemental Fig. S4). The conservationis especially high in the last 110 residues (�90% identity in allspecies). This region spans the CXXC and DBDmotifs, which areessential for theWID-DVL interaction and for theWID-mediatedinhibition of the WNT/�-catenin signaling pathway. The resultsfrom this study further demonstrated a potential role for WID inkidney development as ablation of zebrafishwiddisruptednormalembryonic kidney development and resulted in the formation oflarge cysts in the glomerular-tubular regions. In this regard, it isinteresting tonote that activatedWNTsignaling also causes cystickidney formation in the zebrafish (36) and the mouse (37). Theseresults suggest that the physiological function ofWID in nephro-genesis might be conserved inmammals.

Acknowledgments—We thank Randall Moon for kindly providingzebrafish Wnt8a and Super8XTOPFlash plasmids, Mikhail V. Sem-enov for the Dvl antibody and Xi He for the FLAG-Dvl2 plasmid. Wealso thankDanielHaber, Rick Proia, andAlanKimmel for advice andhelpful discussion and Kang-Yell Choi for sharing unpublished work.

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LeeKarim Malik, Daphne W. Bell, Christoph Englert, Alan O. Perantoni and Sean BongNathan J. Whye, Yun-Ping Wu, Miguel N. Rivera, Jik Young Park, Ho-Shik Kim,

Myoung Shin Kim, Seung Kew Yoon, Frank Bollig, Jirouta Kitagaki, Wonhee Hur,Signaling Pathway

A Novel Wilms Tumor 1 (WT1) Target Gene Negatively Regulates the WNT

doi: 10.1074/jbc.M109.094334 originally published online March 10, 20102010, 285:14585-14593.J. Biol. Chem.

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A Novel Wilms Tumor 1 (WT1) Target Gene Negatively Regulates