gene trap screening as an effective approach for identification of wnt-responsive genes in the mouse...

RESEARCH ARTICLE

Gene Trap Screening as an Effective Approachfor Identification of Wnt-Responsive Genes inthe Mouse EmbryoYoshifumi Yamaguchi,1,2 Satoshi Ogura,1,2 Mio Ishida,1,2 Mika Karasawa,3 and Shinji Takada1,4*

In this study, we examined whether gene trap methodology, which would be available for systematicidentification and functional analysis of genes, is effective for screening of Wnt-responsive genes duringmouse development. We screened out two individual clones among 794 gene-trapped embryonic stem celllines by their in vitro response to WNT-3A proteins. One gene was mainly expressed in the ductal epitheliumof several developing organs, including the kidney and the salivary glands, and the other gene wasexpressed in neural crest cells and the telencephalic flexure. The spatial and temporal expression of thesetwo genes coincided well with that of several Wnt genes. Furthermore, the expression of these two genes wassignificantly decreased in embryos deficient for Wnts or in cultures of embryonic tissues treated with a Wntsignal inhibitor. These results indicate that the gene trap is an effective method for systematicidentification of Wnt-responsive genes during embryogenesis. Developmental Dynamics 233:484–495, 2005.© 2005 Wiley-Liss, Inc.

Key words: gene trap; Wnt, ES cell; organogenesis; tubulogenesis; kidney; salivary gland; neural crest

Received 15 November 2004; Revised 20 December 2004; Accepted 23 December 2004

INTRODUCTION

The development of multicellular or-ganisms requires a concerted and se-quential expression of a variety ofgenes. Secreted signal proteins, in-cluding bone morphogenetic protein(BMP), fibroblast growth factor (FGF),and Wnt, activate various cellular sig-naling pathways, which then triggerintracellular molecular networks ofgene expression during animal devel-opment. To gain more insight into themechanism underlying a variety of de-velopmental events in which thesesignalings are involved, further iden-

tification and functional analysis ofcomponents in the molecular cascadesand networks activated by these sig-nal are required.

Canonical Wnt signaling is involvedin several aspects of development,such as axis determination, cell differ-entiation, and cell proliferation (Cadi-gan and Nusse, 1997; Nelson andNusse, 2004). WNT proteins, whichare cysteine-rich secreted molecules,bind to their transmembrane recep-tors, the Frizzled proteins, and its co-receptor, the LDL receptor-relatedprotein 5 and 6 (LRP5/6; Mao et al.,

2001a,b), and transduce a signal intothe cell, leading to the translocation of�-catenin to the nucleus and to subse-quent activation of target genes bytranscriptional complex of �-cateninand TCF/LEF-1 (Behrens et al., 1996;Huber et al., 1996; Schneider et al.,1996). For an understanding of themolecular mechanism governing eachdevelopmental phenomenon regulatedby the Wnt signaling, identificationand functional analysis of Wnt-re-sponsive genes are indispensable.Thus far, many Wnt-responsive geneshave been identified in vertebrates;

1Okazaki Institute for Integrative Biosciences, National Institutes of Natural Sciences, Myodaiji, Okazaki, Aichi, Japan2Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto, Kyoto, Japan3Department of Cell Biology, The Cancer Institute, Kamiikebukuro, Toshima-ku, Tokyo, Japan4The Graduate University for Advanced Studies (SOKENDAI), Myodaiji, Okazaki, Aichi, JapanGrant sponsor: Ministry of Education, Science, Culture, and Sports of Japan; Grant sponsor: Japan Science and Technology Corporation;Grant sponsor: Mitsubishi Foundation.*Correspondence to: Shinji Takada, Okazaki Institute for Integrative Biosciences, National Institute of Natural Sciences, 5-1Higashiyama, Myodaiji, Okazaki, Aichi, 444-8787, Japan. E-mail: [email protected]

DOI 10.1002/dvdy.20348Published online 18 March 2005 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 233:484–495, 2005

© 2005 Wiley-Liss, Inc.

however, only a few of these geneshave been characterized in the contextof embryogenesis (Danielian and Mc-Mahon, 1996; Yamaguchi et al., 1999;Lickert et al., 2000; Kratochwil et al.,2002; Kioussi et al., 2002; Morkel etal., 2003).

The gene-trap methodology is apowerful strategy for the systematicidentification and functional analysisof genes in the postgenomic era, be-cause this methodology offers theidentification of a novel gene, theanalysis of its expression pattern, andthe generation of its functional muta-tion in a single experimental approachas described below. In this methodol-ogy, random insertion of a gene-trapvector leads to the tagging, and fre-quently to the disruption, of genesacross the genome (Friedrich and So-riano, 1991; Joyner et al., 1992; Skar-nes et al., 1992; Hicks et al., 1997;Stanford et al., 2001). Therefore, ifembryonic stem (ES) cells are used forthe generation of insertion events, em-bryonic and adult whole bodies con-taining tagged and disrupted allelescan be produced. Such an insertionevent affords the following advan-tages for identification and functionalanalysis of a trapped gene: (1) Nucle-otide sequences of a trapped tran-script and an insertion site can be de-termined by 5� rapid amplification ofcDNA ends (RACE) and plasmid res-cue method, respectively. The processof identification of a trapped gene hasbeen substantially eased by the recentcompletion of the genome database.(2) The expression pattern of atrapped gene during development canbe easily monitored by the expressionof protein tag derived from a gene-trap vector in embryos generated fromtrapped ES cells. (3) An insertionevent has the potential to be muta-genic. Because of these strong advan-tages, the gene trap methodology hasbeen applied for several studies, in-cluding large-scale insertional mu-tagenesis programs (Wurst et al.,1995; Stoykova et al., 1998; Stanfordet al., 2001; Hansen et al., 2003;Stryke et al., 2003).

Although the gene-trap methodol-ogy basically involves the random in-tegration of a gene-trap vector, thisstrategy has also been expected to beavailable for identification and char-acterization of genes regulated by par-

ticular signals. In a modified gene-trap strategy, called induction genetrap, genes are selected by their re-sponse to specific secreted signal pro-teins, including BMP2, activin, nodal,and FGF, added to the culture of gene-trapped cells (Forrester et al., 1996;Harrison and Miller, 1996; Stoykovaet al., 1998; Mainguy et al., 2000;Akiyama et al., 2000; Medico et al.,2001; Kluppel et al., 2002; Tateossianet al., 2004). Although the in vivo cor-relation between selected genes andsignals has remained unclear in thesecases, in vitro prescreening of trappedES cell lines with secreted signal pro-teins would be effective for enrich-ment of genes regulated by specificsignals even in normal development.

Here, we used the induction gene-trap approach to identify Wnt-respon-sive genes during mouse development.We screened 794 trapped ES lines andrecovered two ES cell lines that con-tained trapped genes responsive toWNT-3A protein. The embryonic ex-pression, as well as the requirement ofWnt genes for their in vivo expression,was precisely examined in these twoclones. These results strongly indicatethat this approach is a powerful strat-egy for identification, and probably forfunctional analysis, of genes respon-sive to Wnt signaling during embryo-genesis. Our results are the first clearindication that induction gene trap-ping can be used for identification ofgenes regulated by secreted signalproteins during normal development.

RESULTS

Isolation of Wnt-ResponsiveGene-Trap Lines

To determine the culture period ad-equate for screening Wnt-responsivegenes in ES cells, we first examinedthe time course of response to Wntsignal after the addition of mediumconditioned by Wnt-3a– expressingcells (Wnt-3a C.M.; Shibamoto et al.,1998). Because Wnt-3a is known tobe a typical signal to induce the ca-nonical Wnt pathway, stimulationby Wnt-3a C.M. would be represen-tative of that by many Wnt mole-cules that can induce this pathway,for instance, Wnt-1, Wnt-3, Wnt-8,Wnt-7b, and some other Wnt familymembers. The response to the Wnt

signal was examined by monitoringEGFP reporter expression, whichwas driven from a promoter contain-ing a tandem repeat of seven TCF-binding sites (Ueda et al., 2002). Inthe absence of Wnt-3a C.M., as wellas in the presence of control C.M.,the enhanced green fluorescent pro-tein (EGFP) expression level wasvery low in almost all cells, indicat-ing that the endogenous activationlevel of the canonical Wnt signalingis very low in ES cells (Fig. 1). Onthe contrary, the number of cellsthat expressed the EGFP signalgradually increased after 6 hr of in-cubation with Wnt-3a C.M. andreached its maximum after 24-hr in-cubation (Fig. 1). In parallel to thisactivation, the expression ofbrachyury, which has been shown tobe activated by Wnt signaling in EScells (Arnold et al., 2000), was alsogradually induced (data not shown).Thus, a 24-hr incubation was consid-ered to be long enough for screeningWnt-responsive genes with maxi-mum sensitivity in ES cells.

For screening Wnt-responsivegenes by the gene-trap approach, weestablished several ES cell lines inwhich gene-trap vectors were ran-domly integrated into the chromo-somal DNA. The vector used in thisstudy, called pLSA�geo, contains anengrailed-2 splicing acceptor se-quence linked to the �geo reportergene (a lacZ-neomycin phospho-transferase fusion gene) with an in-ternal ribosome entry sequence(IRES) at its 5� end. ES cells wereelectroporated with this trap vector,and then G418-resistant ES cell col-onies were picked up and split intotwo 96-well plates. The cells on thefirst plate were frozen for storage,whereas those on the second platewere allowed to grow, after whichthey were further divided into twogelatinized plates. For screeningthese clones in terms of their re-sponse to Wnt proteins, the dividedcells on the second plates were cul-tured for 24 hr with either Wnt-3aC.M. or control C.M. Before the ad-dition of the C.M., the ES cells on thesecond plates were allowed to differ-entiate for 24 hr without the addi-tion of leukemia inhibitory factor(LIF), because we aimed at obtainingWnt-responsive genes that play roles

Wnt-RESPONSIVE GENE SCREENING BY GENE TRAP 485

in early differentiated cells. As a re-sult of screening of 794 individualtransfectants, we obtained two celllines (clone 43 and clone 5) in which�-gal activity was strongly increasedby Wnt-3a C.M. (Fig. 2; Table 1). Onthe other hand, �-gal activity wasdecreased by Wnt-3a C.M in 1 cellline, whereas its level was main-tained without any drastic change in694 other cell lines (Table 1).

Molecular Characterizationof the Trapped Genes

To identify the trapped genes molec-ularly and to confirm that their ex-pression was actually regulated byWnt signal, we cloned the trappedgenes responsible for Wnt-depen-dent expression in clones 43 and 5.Genomic Southern blot analysis us-ing a lacZ probe, which can detectrestriction fragments containingjunctions between the trap vector

and chromosomal DNA, indicatedthat the trap vector was integratedinto a single locus of the genome inboth trapped lines (data not shown).Then, the integration sites were de-termined by the plasmid rescuemethod, and transcripts from thetrapped loci were characterized by5�-RACE on RNA isolated from eachtrapped ES cell line.

In the case of clone 43, the trapvector was inserted in the second in-tron of the CP2-related-transcrip-tion-repressor-1 (CRTR-1) locus (Fig.3A; Rodda et al., 2001). Character-ization of a transcript from the clone43 trapped allele by 5�-RACE dem-onstrated that a fusion transcripthad been generated by a splicing be-tween the splicing donor site at theend of exon 2 of CRTR-1 and theengrailed-2 splicing acceptor in thetrap vector.

In the case of clone 5, the gene-trapvector was inserted in chromosome 13(Fig. 3A,B). Upon this insertion event,the splicing acceptor and a part of the5� region of the IRES sequence hadbeen deleted from the vector, whereasthe lacZ gene remained intact. Thus,the lacZ gene in this trapped allelewas supposedly expressed withoutsplicing. The genomic DNA aroundthe insertion site was also deletedupon this insertion. A modified 5�-RACE method that can capture only5�-capped mRNA revealed that a tran-script in this trapped allele was tran-scribed starting 24-bp upstream fromthe integration site without splicing(Fig. 3B). In addition, reverse tran-scriptase-polymerase chain reaction(RT-PCR) analysis indicated that atranscript containing a sequence fromthe initiation site to 247 bp down-stream to the integration site was gen-erated from this locus in E11.5 em-bryos as well as in the wild-type EScells (Fig. 3B–D). One presumptiveopen reading frame encoding a proteinwith a molecular weight of 5.8 kD wasfound in this transcript, and this pro-tein showed no similarity in aminoacid sequence to known proteins inthe data base.

To examine whether the expressionof the endogenous genes at thetrapped loci was actually up-regulatedby Wnt signaling, we monitored their

Fig. 1. Induction of a Wnt reporter gene in embryonic stem (ES) cells. Wnt-3a conditioned medium(Wnt) and control conditioned medium (C) were added to ES cells stably containing an enhancedgreen fluorescent protein (EGFP) reporter gene that was driven from a promoter containing atandem repeat of seven TCF-binding sites. The response to Wnt signal was examined by moni-toring EGFP reporter expression under the fluorescence microscope at 1, 3, 6, 12, and 24 hr afterthe addition of conditioned medium. Phase-contrast views are shown above fluorescence views ateach time point. The number of cells that expressed the EGFP signal gradually increased after 6 hrof incubation with Wnt-3a C.M. and reached maximum by 24 hr.

Fig. 2. Wnt-responsive expression of genetrapped lines. A–D: Two trapped embryonicstem (ES) cell lines (clone 5, A,B; and clone 43,C,D) were incubated with medium containingcontrol C.M. (A,C) or Wnt-3a C.M. (B,D) for 24hr and were stained with 5-bromo-4-chloro-3-indolyl �-D-galactopyranoside (X-gal) for mon-itoring �-galactosidase activity.

TABLE 1. Summary of Gene Trap Screening in Embryonic Stem Cells

Number of colonies

Resistant for G418 794Positive for �-gal activity 657

Increased �-gal activity by Wnt-3a 2Decreased �-gal activity by Wnt-3a 1Unchanged 654

Negative for �-gal activity 137

486 YAMAGUCHI ET AL.

expression in wild-type ES cells in theabsence or presence of WNT-3A pro-teins by RT-PCR. The mRNA amountsof CRTR-1 and of the clone 5 genewere up-regulated by the addition ofWnt-3a C.M. by 2.5- and 1.5-fold, re-spectively, compared with theiramount obtained with control C.M.(Fig. 3C). Thus, expression of the en-dogenous genes at the trapped lociwas actually induced by Wnt signal-ing as in the case of the trapped al-leles.

Close Correlation Betweenthe Trapped Genes andWnts in Their ExpressionPatterns DuringEmbryogenesis

To examine the expression of thesetrapped genes during embryogenesisand the correlation between their ex-pression and that of Wnt genes, wegenerated mice carrying thesetrapped alleles. Heterozygotes forthese trapped alleles were producedby intercrossing of wild-type femaleswith chimeric male mice that hadbeen generated by injection of thetrapped ES cells into C57BL/6 blas-tocysts. Both lines exhibited partic-ular spatiotemporal lacZ expressionpatterns at mid-gestation stages inheterozygous embryos. Further-more, in the case of clone 43, in situhybridization analysis also indicatedthat the expression pattern of thelacZ reporter was identical to that ofthe endogenous gene as far as wasexamined. On the other hand, in thecase of clone 5, no endogenous geneexpression was detectable by in situhybridization, probably because theexpression level was too low and thelength of this mRNA was too short.Therefore, we further observed theembryonic expression of clone 43 andclone 5 trapped genes precisely bymonitoring LacZ-positive cells in ad-dition to in situ hybridization analy-sis of clone 43.

Although the expression of CRTR-1,the trapped gene in clone 43, in thekidney at E16.5 and in adulthood wasalready described (Rodda et al., 2001),its precise expression pattern has re-mained unclear. Therefore, we exam-ined its expression pattern at several

Fig. 3. Characterization of the trapped alleles and expression of the trapped genes. A:Schematic representation of the two trapped alleles. B: Genomic sequences around theinsertion site of the gene-trap vector in the clone 5 trapped line (cl.5). An underline indicates thesequences obtained by 5�-cap trapper rapid amplification of cDNA ends. An arrowhead pointsat the 5�-cap site. Sequences of the endogenous transcript confirmed by a reverse tran-scriptase-polymerase chain reaction (RT-PCR) experiment (GenBank accession no. AB193402)are enclosed by a box. The presumptive amino acid sequence is indicated below the box.Arrows show sequences used for the RT-PCR in C,D. Bases denoted in gray were deleted uponinsertion of the trap vector into the genome. C: Increase in mRNA of CRTR-1 and cl.5 inembryonic stem cells cultured with Wnt-3a C.M.(WNT) for 24 hr compared with those withcontrol C.M (con) or those before incubation (0h). Because the RT-PCR product of cl.5 isindistinguishable in size from that amplified from its genomic DNA, we examined it by com-parison of with (�RT) and without (�RT) reverse transcriptase in the reaction. Hypoxanthinephosphoribosyltransferase (HPRT) as an internal control. Right panel: Quantitative representa-tion of mRNA of CRTR-1 and cl.5 normalized to HPRT mRNA. The asterisk indicates P vs. 0hr � 0.005, the dagger P vs. control (L.C.M.) � 0.005. D: The endogenous transcript of cl.5 wasdetected with RT-PCR on total RNA from day 11.5 embryonic heads by using primers shownin B.


embryonic stages. The expression ofCRTR-1 was temporarily observed inthe inner cell mass of blastocysts (datanot shown). Later, CRTR-1 was spe-cifically expressed in ductal structuresin the mid-gestation stages. This gene

was expressed during several aspectsof the ductal morphogenesis in kidneydevelopment. The urogenital expres-sion of CRTR-1 was observed first inthe nephric duct and mesonephric tu-

bules at E10.5 (Fig. 4A) and later inthe Wolffian duct, the ureter, the col-lecting duct, and the distal portion oftubules connected to the collectingduct in the metanephros (Fig.4B,C,E). The expression was re-stricted to the epithelium, i.e., was notfound in the mesenchyme, in these or-gans (Fig. 4E,F). CRTR-1 was also ex-pressed in the ductal epithelium of thesalivary glands (submandibular, sub-lingual, and parotid glands) at E 15.5(Fig. 4F,G and data not shown).

Of interest, the expression of sev-eral Wnt genes was also observed inthe ductal structures in whichCRTR-1 was expressed. In urogenitaldevelopment, Wnt-7b, which has beenreported to be expressed in the Wolf-fian duct, the ureter, and the collect-ing duct at E13.5 (Kispert et al., 1996;Patterson et al., 2001), was coex-pressed with this trapped gene (Fig.4C,D). On the other hand, in the sub-mandibular gland (SMG), the expres-sion of several Wnt genes, includingWnt-2, 2b, 3, 4, 5b, 6, 10b, 14, 16, wasdetected by RT-PCR analysis (datanot shown). Among these Wnt genes,Wnt-5b exhibited an expression pat-tern spatially and temporally corre-lated with that of CRTR-1. Wnt-5bwas expressed at mid-gestationstages, for instance E13, in the stalksof the SMG and the sublingual gland(SLG), where CRTR-1 was expressed(Fig. 4G,H). Thus, the ductal expres-sion of CRTR-1 was well correlatedwith the expression of Wnt-7b andWnt-5b in the kidney and the salivarygland, respectively.

The expression pattern of the clone5 trapped gene, also suggested itsclose correlation with that of Wntgenes. The expression of the clone 5trapped gene was first observed atE8.5 in rhombomere 5 (Fig. 5A). AtE9.5, the expression of this gene wasstill detected in rhombomere 5, al-though it was relatively dispersed(Fig. 5B). Rostral to rhombomere 5,the lacZ-positive cells straggled in thesuperior membrane of the rhomben-cephalon. At E10.5–11.5, the expres-sion was observed with strong inten-sity in scattered neural crest cells overthe dorsal diencephalon and mesen-cephalon (Fig. 5C,D), as well as in rel-atively weak intensity over the dorsalspinal cord. Of interest, these scat-tered signals were strong along the

Fig. 4. Embryonic expression of the clone 43trapped gene. A,B: During urogenital develop-ment, the lacZ reporter of clone 43 was ex-pressed first in the nephric duct and meso-nephric tubules at embryonic day (E) 10.5 (A); atE13, it was also expressed in the Wolffian duct,mesonephric tubules, the ureter, and the col-lecting duct (B). C,D: CRTR-1 mRNA (C) wascoexpressed with Wnt-7b in the Wolffian duct(D, arrows). E: In situ hybridization with CRTR-1antisense riboprobe on E14.5 kidney sections.F: A section of 5-bromo-4-chloro-3-indolyl �-D-galactopyranoside (X-gal) -stained submandib-ular gland (SMG) at E15.5. The LacZ reporter ofclone 43 was expressed in the duct of the de-veloping kidney and SMG. G,H: Expression ofCRTR-1 mRNA (G) and Wnt-5b (H) in the SMGand sublingual gland at E13.5.

Fig. 5. Embryonic expression of the clone 5trapped gene. A–E,G: LacZ reporter expressionof the clone 5 trapped gene was examined by5-bromo-4-chloro-3-indolyl �-D-galactopyr-anoside (X-gal) staining of heterozygous em-bryos for the clone 5 trapped allele. A–D: Adorsal view at embryonic day(E) 8.5 (A), a dorsalview at E9.5 (B), a sagittal view at E10.5 (C), anda transverse section around hindbrain at E11(D). E: A sagittal section around the telence-phalic flexure at E11. In the telencephalic flex-ure, the clone 5 trapped gene was expressed inmesenchyme but not in neuroepithelium or sur-face ectoderm. F: For comparison of the ex-pression of the clone 5 trapped gene with thatof Wnt-3a, in situ hybridization with the Wnt-3aprobe was performed. A frontal view of Wnt-3aexpression at E10.5 is shown. Note that thetelencephalic mesenchyme was surrounded bythe cortical hem where Wnt-3a was expressed.G: A transverse section of the hindbrain atE13.5. The clone 5 trapped gene was ex-pressed in the meninx. ov, otic vesicle; di, di-encephalon; te, telencephalon.


dorsal midline adjacent to the roofplate where Wnt-1 and Wnt-3a wereexpressed (Roelink and Nusse, 1991;Parr et al., 1993). The clone 5 trappedgene was also expressed in mesen-chyme in the telencephalic flexure(Figs. 5E, 6A). This mesenchymal ex-pression represents another exampleof correlation between this gene andWnt, because Wnt-3a is expressed inthe neuroepithelium, adjacent to themesenchymal cells, in the telence-phalic flexure (Fig. 5F; Roelink andNusse, 1991; Lee et al., 2000). Theexpression of the clone 5 trapped genewas also observed in the meninx atE13.5 (Fig. 5G). Taken together, theclose correlation between gene expres-sion of the two trapped genes and thatof several Wnt genes strongly suggeststhat the trapped genes screened bytheir in vitro response to WNT pro-teins were also responsive to Wnt sig-nals in vivo.

Requirement of WntSignaling for the Expressionof the Trapped Genes

To examine whether Wnt genes areactually required for the in vivo ex-pression of the trapped genes, a ge-netic analysis would be powerful. Be-cause the expression of the clone 5trapped gene correlated well with thatof Wnt-1 and Wnt-3a, we next exam-ined the expression of this trappedgene in mouse embryos deficient forWnt-1, Wnt-3a, or both of these Wnts.The positive LacZ signal of the clone 5trapped gene was absent in neuralcrest cells migrating from the hind-brain and the spinal cord in the Wnt-1/Wnt-3a double mutant (Fig. 6H). Thisabsence of LacZ-positive cells appearsto have been caused by decreased ex-pression of the clone 5 trapped gene,not by lack of cells that normally ex-press this gene, because the neuralcrest cells were definitely present, al-though the number of migrating neu-ral crest cells was decreased in thismutant (Ikeya et al., 1997). Alterna-tively, it is also possible to speculatethat Wnt-1 and Wnt-3a might act on acertain population of neural crest cellsthrough the activation of the clone 5trapped gene, in which case the ab-sence of these Wnts might result in aselective decrease in this population.In contrast, the lacZ signal was

present in the wild-type and in Wnt-1and Wnt-3a single mutant embryos(Fig. 6E–G). These results stronglysuggest that Wnt-1 and Wnt-3a wereredundantly required for the expres-sion of the clone 5 trapped gene inthis region. In addition, the 5-bromo-4-chloro-3-indolyl �-D-galactopyr-anoside (X-gal) staining in the mes-enchymal cells in the telencephalicflexure was significantly reduced inthe Wnt-3a null mutant, as well as inWnt-1/Wnt-3a double mutant, com-pared with that in the wild-type andWnt-1 single mutant embryos (Fig.6A–D). Thus, the expression of theclone 5 trapped gene in the mesen-chyme was dependent on Wnt-3a.Because Wnt-3a is expressed in theneuroepithelium adjacent to themesenchyme (Fig. 5F), but not in themesenchyme, in the telencephalicflexure, this result suggests an in-ductive interaction between the neu-roepithelium and the mesenchyme.Together, these results indicate thatexpression of the clone 5 trappedgene required the activity of Wnteven in the in vivo situation.

To investigate whether Wnt sig-naling is also required for the ex-pression of CRTR-1 during organo-genesis, we cultured submandibularand sublingual glands isolated fromE13 embryos with or without CKI7,a chemical inhibitor of canonicalWnt signaling (Peters et al., 1999).In this culture, the expression ofCRTR-1 was markedly diminishedin comparison with the expression ofkeratin18, which is also expressed inthe ductal epithelium, in the duct ofthe SMG and the SLG treated withCKI7 (Fig. 7A–D). Quantitative PCRexperiments showed that the expres-sion of CRTR-1 mRNA in cultureswith CKI7 was decreased to 40% ofthat of control cultures, whereasthat of keratin18 was not signifi-cantly changed in this condition(data not shown). Thus, Wnt signal-ing was required for the normal ex-pression of CRTR-1 in the SMG andthe SLG. Taken together, our resultsindicated that the in vivo expressionof the two trapped genes, which werescreened by their in vitro response toWnt, was also dependent on Wnt ac-tivity.

Evidence That a Gene TrapEvent Is Mutagenic

To examine whether the trapped genewas required for proper developmentof cells or organs where it was ex-pressed, we generated homozygousmutants for the CRTR-1 trapped al-lele by crossing the heterozygousmales and females. In homozygotesfor this allele, the expression of thisgene was almost completely dimin-ished, whereas the expression of atruncated transcript fused to the�-geo gene in the trap vector wasstrongly detected (Fig. 8A). Of inter-est, 70% of the mice homozygous forthe CRTR-1 trapped allele died before5 weeks after birth. Most of these miceexhibited hypoplasia of the kidney atpostnatal day 30, whereas the het-erozygous and wild-type mice in thesame litters showed no obvious defectin their kidney (Fig. 8B,C). Further-more, in homozygous mice, tubules inthe cortex were occasionally dilatedand the papilla appeared apart fromthe ureter. Thus, CRTR-1 is likely tobe required for proper development ofthe kidney, in which this gene is ex-pressed. More extensive analysis ofthis mutant phenotype at molecularand physiological levels will be de-scribed elsewhere.

Thus, the in vivo expression of thetwo trapped genes, which werescreened by their in vitro response toWnt, was also dependent on the Wntactivity. Furthermore, homozygotesfor a trapped allele showed a morpho-logical phenotype in the kidney, wherethe trapped gene was expressed over-lappingly with Wnt-7b. These resultsindicate that an inductive gene trap inES cells is likely to be effective forscreening and functional analysis ofgenes induced by Wnt signaling dur-ing embryogenesis.

DISCUSSION

Gene Trap Screening as anEfficient Approach forIdentification and FunctionalAnalysis of Wnt-ResponsiveGenes

To identify genes regulated by Wntsignaling during embryogenesis, weestablished a gene-trap screening sys-tem, in which gene-trapped ES cell


lines were selected in terms of theirresponse to Wnt proteins. Among 794clones screened, two clones exhibited

a strong increase in in vitro reportergene expression in response to Wnt-3aC.M. (Fig. 2). We examined temporal

and spatial expression of these genesin embryos and found that the expres-sion patterns of these trapped genescorrelated well with those of severalWnt genes (Figs. 4, 5). Furthermore,the expression of the trapped geneswas regulated by Wnt signaling notonly in vitro but also in vivo (Figs. 6,7). Thus, the gene-trap approach cou-pled with a primary screening withWNT proteins appears to be effectivefor identification of genes actually reg-ulated by Wnt signaling during em-bryogenesis.

Furthermore, the gene-trap ap-proach has the strong advantage ofnot only being a screening method,but also being a tool for mutagenesis.Mutant mice can be generated fromtrapped ES cells if integration of thetrap vector blocks the production of agene product with normal function.Actually, homozygotes for the clone 43trapped allele showed abnormal de-velopment of the kidney (Fig. 8). Thus,the gene-trap approach is a powerfuland systematic strategy not only foridentification but also for functionalanalysis of genes regulated by Wntsignals during mouse development.Further extensive studies should re-veal the precise function of this genein kidney development and its rela-tion to Wnt -7b, which is overlappinglyexpressed with CRTR-1 in the devel-oping kidney.

Variety of Genes That CanBe Obtained by Gene-TrapScreening With Wnt-TreatedES Cells

Wnt signaling plays roles in severaldifferent aspects of embryogenesisand is operative even after birth. Dif-ferent sets of genes should be inducedor repressed by this signal, dependingon the cellular context. Thus, the va-riety of Wnt-responsive genes that wecan obtain by gene-trap screeningwould reflect the characteristics ofcells used for the screening. ES cells,which we used for screening of Wnt-responsive genes in this study, pos-sess self-renewing activity as stemcells, as well as pluripotency to differ-entiate into all cell types. Under the invitro culture conditions used in thepresent study, brachyury and cdx-1,both of which are known to be inducedby Wnt signals in mesodermal cells in

Fig. 6. Down-regulation of the expression of the clone 5 trapped gene in Wnt-3a and Wnt-1/Wnt-3a mutants. A–D: Frontal views of the expression of the lacZ reporter for the clone 5 trappedgene in the telencephalic flexure (arrow) at embryonic day (E) 11.5. E–H: Dorsal views of theexpression of the lacZ reporter for the clone 5 trapped gene in the hindbrain at E11.5. A–H:Wild-type (A,E), Wnt-3a null mutant (B,F), Wnt-1 null mutant (C,G), and Wnt-1 and Wnt-3a double-null mutant (D,H).

Fig. 7. Down-regulation of CRTR-1 expression in cultured salivary gland by a Wnt inhibitor. Aninhibitor for the Wnt signaling, CKI7, caused down-regulated CRTR-1 expression. A,B: In situhybridization with the CRTR-1 probe of embryonic day 13 submandibular gland and sublingualgland cultured for 12 hr with dimethyl sulfoxide (DMSO, A) or CKI7 (B). C,D: The expression ofkeratin18, which was detected in the ductal epithelium, was not altered between cultures treatedwith DMSO (C) and CKI7 (D).

Fig. 8. Defect in the kidney of homozygotes for the clone 43 trapped allele. A: Northern blotanalysis to indicate the expression of CRTR-1 mRNA in the kidney of wild-type (�/�), heterozygote(�/tra), and homozygote (tra/tra) for the clone 43 trapped allele at postnatal day (P) 0. In homozy-gotes, CRTR-1 mRNA was almost diminished, whereas the expression of truncated transcript, inwhich the first two exons were fused to �-geo gene, was strongly detected. B,C: Histologicalsections of kidneys of a heterozygote (B) and a homozygote (C) for the clone 43 trapped allele atP0. Most of in the CRTR-1 homozygous mice exhibited hypoplasia.


early embryonic stages (Yamaguchi etal., 1999; Arnold et al., 2000; Lickertet al., 2000; Ikeya and Takada, 2001;Prinos et al., 2001), were induced byWNT proteins (data not shown). Inaddition to these mesodermal genes,we also detected Wnt-induced expres-sion of the light polypeptide of neuro-filaments, which is expressed in neu-ral cells from an early differentiatedstage (data not shown). Thus, the cul-ture conditions used for ES cells inthis study appear to mimic someevents at early embryonic stages andto be effective for obtaining genes re-sponsive to Wnt signaling during earlyembryogenesis. Actually, CRTR-1 wasexpressed in the inner cell mass of blas-tocysts, and the clone 5 trapped genewas first expressed in rhombomere 5 atE8.5, suggesting that biological eventsin blastocysts and in early neural devel-opment seem to be reproduced at leastto some extent under the in vitro cul-ture conditions used for this screening.However, it also seems true that thesein vivo events are not exactly repro-duced under this in vitro condition, es-pecially in terms of the temporal regu-lation of gene expression. For instance,expression of the clone 5 trapped genewas increased in ES cells cultured for24 hr with Wnt-3a C.M.; although itsearliest in vivo expression was not de-tected at the appropriate time expectedby the in vitro expression, but later, atE8.5. Thus, the in vitro culture condi-tions for this screening might accelerateand/or bypass some events in early de-velopment; thus, the screening strategyin this study may have a bias for a va-riety of certain genes. In addition to thecharacteristics and culture conditions ofthe cells, the characteristics of the gene-trap vector affects the variety of genesthat can be obtained. The gene-trap vec-tor used in the present study can cap-ture genes expressed in undifferenti-ated ES cells, whereas it cannot thoseexhibiting no expression in these cells.Further improvement of cell cultureconditions and design of the gene-trapvector should be effective for obtaininggenes with different characteristics, forinstance, those expressed in late embry-onic stages.

One of the important applications ofthe present gene-trap approach is aselective screening of genes involvedin particular biological phenomenaregulated by Wnt signal. As men-

tioned above, ES cells possess pluripo-tency to differentiate into all celltypes. Recently, several lines of evi-dence have suggested that activationof Wnt signaling in human and mouseES cells leads to inhibition of differen-tiation and maintenance of pluripo-tency (Aubert et al., 2002; Kielman etal., 2002). Thus, by modifying the cul-turing conditions of gene-trapped ESclones, some Wnt target genes in-volved in the machinery for maintain-ing pluripotency might be identified.On the other hand, several cultureconditions in which ES cells efficientlydifferentiate into particular cell types,including neuronal, mesodermal, andendodermal cells, have been estab-lished (Nishikawa et al., 1998; Ka-wasaki et al., 2000; Mizuseki et al.,2003; Kubo et al., 2004). Thus, a gene-trap approach with more refined andlineage-restricted in vitro screeningcould be available for identifying Wnttarget genes in some particular celllineages.

Roles of the Trapped Genesin Clone 43 and Clone 5During MouseEmbryogenesis

The trapped gene of clone 43 was iden-tified as CRTR-1, a member of the CP2/LSF/Grh transcription factor family(Rodda et al., 2001; Venkatesan et al.,2003). In vertebrates, this family con-sists of several members, includingCRTR-1 (known as LBP-9 in human;Huang and Miller, 2000), CP2 (alsoknown as LBP-1c and LSF; Lim et al.,1992; Shirra et al., 1994), NF2d9(known as LBP-1a in human; Sueyoshiet al., 1995; Yoon et al., 1994), mamma-lian Grainyhead (MGR), brother ofMGR (BOM; Wilanowski et al., 2002),sister of MGR (SOM)/GET1 (Kudryavt-seva et al., 2003; Ting et al., 2003b), buttheir developmental roles have beenlargely unknown (Ramamurthy, 2001;Ting et al., 2003a). We found thatCRTR-1 was specifically expressed inthe ductal epithelium of the kidney andthe salivary glands (Fig. 4). The devel-opmental processes of these organsshare many similarities; for instance,epithelial–mesenchymal interaction,branching morphogenesis, and lumenformation. Thus, CRTR-1 may be com-monly involved in the development of

different organs by playing a role insome of these phenomena. Of interest, aDrosophila member of the CP2/LSF/Grh family, Grainy head (Uv et al.,1994), controls luminal elongation ofthe airways. Overgrowth of the apicalmembrane in grainy head mutantsleads to lumen elongation without af-fecting epithelial integrity, whereasoverexpression of the gene limits lumi-nal growth (Hemphala et al., 2003).Thus, CRTR-1 may have a role in lumi-nal development during vertebrate or-ganogenesis, as in the case of its ho-molog in Drosophila.

It has been reported that severalWnt genes are expressed in the duc-tal epithelium where CRTR-1 wasexpressed. In the SMG and the SLG,Wnt-5b was expressed in the stalk(Fig. 4H), where CRTR-1 was ex-pressed, whereas Wnt-2b were ex-pressed in the mesenchyme (Lin etal., 2001). On the other hand, in thedeveloping kidney, Wnt-6 andWnt-7b (Kispert et al., 1996; Patter-son et al., 2001; Itaranta et al., 2002)are expressed in the collecting duct,whereas Wnt-2b is expressed in thesurrounding mesenchyme aroundureteric buds. Also, Wnt-4, which isrequired for tubulogenesis, is ex-pressed in the condensed mesen-chyme and in the newly formed dis-tal tubule (Stark et al., 1994).Among these Wnt genes, Wnt-7bshowed expression tightly coincidentwith that of CRTR-1 in the Wolffianduct, the ureter, and the collectingduct (Patterson et al., 2001). SinceWnt-7b has been reported to inducecanonical Wnt signaling in mamma-lian cells in culture, the overlappedexpression of CRTR-1 with Wnt-7bsuggests that the CRTR-1 expres-sion induced by Wnt-3a, a typical in-ducer for the canonical Wnt signal-ing, was also induced throughcanonical Wnt signaling by Wnt-7b(Zhang et al., 2004). Thus, Wnt-7bseems to be the best candidate signalto regulate CRTR-1 expression inthe developing kidney. However, be-cause the role of Wnt-5b and Wnt-7bin salivary gland and kidney devel-opment remains unclear, we cannotnow speculate on the role of CRTR-1based on studies of Wnt genes.

The clone 5 trapped gene was ex-pressed in a part of the migratingneural crest cells caudal to the dien-


cephalon and in the mesenchyme inthe telencephalic flexure. In neuralcrest development, Wnt signalingplays several important roles. Wnt-1and Wnt-3a, which are expressed inthe roof plate of the neural tube, re-dundantly regulate proper expan-sion of neural crest precursor cells,and �-catenin–mediated Wnt signal-ing also regulates the specification ofsubtypes of neural crest cells (Ikeyaet al., 1997; Dorsky et al., 1998; Leeet al., 2004; Lewis et al., 2004). Be-cause the expression of the clone 5trapped gene in the hindbrain andaround the dorsal neural tube in thetrunk was significantly reduced inthe Wnt-1/Wnt-3a double mutant(Fig. 6H), this gene may be involvedin some aspect of neural crest devel-opment activated by both Wnt-1 andWnt-3a. In addition, the expressionat a later stage was found in themeninx, which may originate fromthe neural crest (Fig. 5G; Couly etal., 1993), suggesting that the clone5 trapped gene might be involved inthe development of the meninx. Onthe other hand, clone 5 expression inthe mesenchyme in the telencephalicflexure of the Wnt-3a null mutantand in the Wnt-1/Wnt-3a double mu-tant was significantly reduced com-pared with that in wild-type embryoand Wnt-1 null mutant, respectively(Fig. 6A–D). This finding clearlyshowed that the expression of theclone 5 trapped gene in the mesen-chyme required Wnt-3a, which is ex-pressed in the neuroepithelium ofthe telencephalic medial wall (Ro-elink and Nusse, 1991). It has al-ready been described that Wnt-3aacts locally in the telencephalic flex-ure as an autocrine signal to regu-late the expansion of the neuroepi-thelial cells from which thehippocampus develops (Lee et al.,2000), but our results have revealeda new role of Wnt-3a, as a paracrinesignal in this region.

The results of this study stronglysuggests that the induction genetrap approach in ES cells is an effec-tive one for screening the down-stream target genes of Wnt signalingduring embryogenesis. We have beengenerating and analyzing mice ho-mozygous for each trapped gene toinvestigate their function in vivo.Extensive studies with these mu-

tants should reveal the roles of theseWnt-responsive genes and molecularmachinery activated by Wnt signalin several aspects of vertebrate de-velopment.

EXPERIMENTALPROCEDURES

Gene Trap Vector, Selectionof Wnt-Responsive CellLines, and Generation ofMice

pLSA�geo was modified from SA-IRES�geo (Mountford et al., 1994).Briefly, lox71 sequences (Araki et al.,2002) were inserted into a BamHI siteimmediately upstream of the en-2splicing acceptor.

CJ7 ES cells were maintained onprimary embryonic fibroblasts inDMEM containing 15% fetal calf se-rum (FCS) and LIF (1,000 U/ml). EScells (1 � 107) were electroporatedwith 25 �g of linearized gene-trap vec-tor DNA in 1 ml of phosphate bufferedsaline (PBS), by applying two pulsesof 0.23 V, 500 �F. After 5-min incuba-tion on ice, the cells were plated in10-cm dishes and allowed to recoverfor 24 hr before adding 200 �g/mlG418 (Invitrogen) for selection of neo-mycin-resistant colonies. After 7–10days, single neomycin-resistant colo-nies were picked and grown in dupli-cate 96-well dishes: one for freezingand the other for screening.

For screening of Wnt-responsiveclones, the cells from the screeningplate were split into two gelatin-coated dishes and allowed to grow for24 hr without LIF. The medium con-ditioned by either Wnt-3a–expressingL cells or parental L cells (Shibamotoet al., 1998; Muroyama et al., 2004)containing 5% FCS was then added toindividual dishes. After an additional24 hr, lacZ expression was detected bystaining the cells for �-galactosidaseactivity and examining them micro-scopically. Clones in which the expres-sion of the lacZ reporter was activatedby treatment with Wnt-3a C.M. werechosen, recovered from frozen stocks,and re-tested repeatedly for their re-sponsiveness to Wnt-3a C.M.. Theconcentration of Wnt-3a protein inWnt-3a C.M. was 400 �g/ml (Shi-bamoto et al., 1998), and 100 �l ofC.M. was added to each well.

The selected ES cell lines were usedfor generating mouse chimeras byblastocyst injection. Chimeric micewere checked for germ-line transmis-sion and used for generation of trans-genic mice heterozygous for thetrapped gene. For examining the timecourse of response to Wnt signal, anES cell line stably containing anEGFP reporter gene whose expressionwas driven from a promoter contain-ing a tandem repeat of seven TCF-binding sites (Ueda et al., 2002) wasestablished.

�-Galactosidase Staining andIn Situ Hybridization

Cells were washed once in PBS, fixedfor 10 min in 0.2% glutaraldehyde inPBS, washed twice in PBS, andstained in staining solution [PBS con-taining 1 mg/mmol X-gal, 5 mMK3Fe(CN)6, 5 mM K4Fe(CN)6, and 2mM MgCl2] at 30°C overnight. Em-bryos were dissected and fixed in asolution consisting of 1% formalde-hyde, 0.2% glutaraldehyde, 0.02%NP40, 2 mM MgCl2, 5 mM ethyl-enediaminetetraacetic acid (EDTA) inPBS (for embryos older than E12.5,0.4% NP40 was used and 0.1% sodiumdeoxycholate was added) for 10 min(�E11.5) to 60 min (E16.5); andwashed twice in PBS containing 2 mMMgCl2 for 10–60 min. They were thenstained in a solution containing 20mM Tris pH 6.8 (for embryos olderthan E12.5, 0.4% NP40 and 0.1% so-dium deoxycholate were added) at30°C until sufficient color had devel-oped. Whole-mount in situ hybridiza-tion was performed as described(Wilkinson, 1992). Full-length cDNAsfor Wnt-7b (Kispert et al., 1996) andWnt-5b (Gavin et al., 1990) were la-beled with digoxigenin to prepare ri-boprobes.

Identification of TrappedLocus

RNA preparation was performed withan RNeasy kit (QIAGEN). Cloning offusion transcripts by 5�-RACE wasperformed with a Smart RACE kit(BD Clonetech) or GeneRacer kit (In-vitrogen) according to the manufac-turer’s instructions. The gene-specificprimer sequences were as follow:GSP1, 5�-TGGCGAAAGGGGGATGT-


GCTG-3�; nested GSP1, 5�-GATGT-GCTGCAAGGCGATTAAG-3�; nestedGSP2, 5�-CTCAGCCTTGAGCCTCT-GGAGCTGCTC-3�. RACE–PCR prod-ucts were cloned and sequenced.

Plasmid rescue was done as follows:Briefly, genomic DNA was extractedfrom ES cell clones with lysis buffer(0.1 M Tris-HCl pH 8.0, 5 mM EDTA,0.2% sodium dodecyl sulfate, 0.2 MNaCl with 0.2 mg/ml of proteinase K).Ten micrograms of DNA was digestedwith NcoI overnight, cleaned, and li-gated at a concentration of 5 �g/ml.The reactions were incubated over-night at 16°C. Transformation ofDH10B (GIBCO) was done by electro-poration with 1 �g of ligated DNA.Colonies were selected for ampicillinresistance, and the rescued plasmidswere sequenced. In addition to theplasmid rescue method, DNA WalkingSpeedUp Kit (Seegene) was used forcloning of the transition site betweenthe gene-trap vector and genomicDNA according to the manufacturer’sinstructions.

Organ Culture

The SMG rudiments at E13 were dis-sected in Hanks’ balanced salt solu-tion (Umeda et al., 2001). The rudi-ments were placed on Millipore filtersand cultured for 12 hr with DMEM/10% FCS containing 0.3mM of CKI7(Seikagaku Kogyo, Japan) dissolvedin dimethyl sulfoxide. Total RNA usedfor cDNA synthesis was extractedfrom two rudiments with Trizol (In-vitrogen), treated with DNase, andcleaned up by use of an RNeasy minikit (QIAGEN).

Quantitative RT-PCR

Quantitative RT-PCR was done withLightCycler (Roche). ComplementaryDNA was created from DNase-treatedtotal RNA by using SuperScriptIII(Invitrogen) with random oligo (6mer)primer. Minus RT controls were alsoprepared similarly. One microgram ofRNA was included in each reaction ina total volume of 20 �l. The amplifica-tion reaction was performed using thefollowing thermocycler conditions:95°C for 10 min followed by 40 cyclesof 95°C for 15 sec, 55 °C for 5 sec, and72°C for 15 sec. Plus RT, minus RT,and no template controls were tested

for primer and MgCl2 concentrationoptimization. The combination of for-ward and reverse primer concentra-tions was selected based on the pres-ence of a single band only in the plusRT sample. Expression levels of allsamples were normalized to the levelof mouse hypoxanthine phosphoribo-syltransferase (HPRT) in each sample.The primer sequences used were asfollows: CRTR-1 (forward), 5�-ATCT-TCCTGGAAGAGCTGAC-3�; CRTR-1(reverse), 5�-TCAGGATGATGTGGTA-GCCATC-3�; clone 5U(forward) 5�-TTCCCTAAAGACCAATCAGT-3�;clone 5L(reverse), 5�-CCACATG-GAGCCAGATCAATG-3�; HPRT (for-ward), 5�-GCTGGTGAAAAGGAC-CTCT-3�; and HPRT (reverse), 5�-CACAGGACTAGAACACCTGC-3�.Each experiment was performed intriplicate.

Northern Blot Analysis

Ten micrograms of total RNA pre-pared from P0 kidney of the wild-type,heterozygous, and homozygous micefor the clone 43 trapped allele wereseparated by agarose gel electrophore-sis according to standard procedures.Blotting and hybridization were per-formed according to the manufactur-er’s protocol (Roche). The riboprobe forCRTR-1 was generated from an EcoRIfragment of CRTR-1 cDNA containingexons 1 to 15.

Histology

For histological examination, kidneysfrom 30-day-old mice were fixed inBouin’s fixative, dehydrated, embed-ded in paraffin, and sectioned at 6 �m.The sections were dewaxed, rehy-drated, and stained by the periodic ac-id/Schiff reaction according to stan-dard procedures.

ACKNOWLEDGMENTSWe thank W. Wurst for the gift ofpGT1 vector, A.P. McMahon forWnt-7b and Wnt-5b riboprobes, M. Hi-jikata for the reporter plasmids, andH. Niwa for the EB5 cell. We alsothank Y. Hieda for expert help in ex-plant cultures, H. Hijikata and R.Takada for preparing Wnt-3a–ex-pressing L cells, N. Takeda and H.Watanabe for blastocyst injection, M.Ogawa for technical advice, and M.

Futamata and M. Sawada for techni-cal support. We thank all the mem-bers of the S.T. lab and the Takeichilab for helpful discussions. This workwas supported by a grant-in-aid forscientific research from the Ministryof Education, Science, Culture, andSports of Japan and grants from theJapan Science and Technology Corpo-ration, and Mitsubishi Foundation toS.T.

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gene trap screening as an effective approach for identification of wnt-responsive genes in the mouse...

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