the expression, andinvivochromatinimmunoprecipitationdemonstratesadirectinteractionbetweenthe prehypertrophic chondrocytesbyWnt9a,becauseembryosdouble-heterozygousforWnt9aand Ihh-signaling activityatE12.5-E13.5.ThecanonicalWnt/ hedgehog ( suppressing theirchondrogenicpotential.Furthermore,weshowthatWnt9aisatemporalandspatialregulatorofIndian playing acrucialroleindirectingbi-potentialchondro-synovioprogenitorstobecomesynovialconnectivetissue,byactively metaplasia insomejoints.ThisphenotypecanbeenhancedbyremovalofanadditionalWntgene, Informatics), Wnt4,Fgf18,growth differentiation factors (Gdf5, parathyroid-hormone relatedpeptide(Pthrp;Pthlh–MouseGenome 2001). Inaddition,theinterzoneexpresses factors suchas 2005; Gongetal.,1999;Guo2004;HartmannandTabin, chondrogenic natureofthesecells(Brunetetal.,1998;Debeer al., antagonist Noggin,whichareimplicatedinregulating thenon- molecules suchasWnt9a(formerlycalledWnt14)andtheBMP II (RalphsandBenjamin,1994).Theinterzonealsoexpresses I andIII,comparedwithchondrocytes, whichproducecollagentype cells. Jointinterzonecellsproducedifferent typesofcollagens,type so-called interzone,whichisdenselypacked andcontains flattened cartilage elements.Cellswithintheprospective jointregion formthe while neighboringcellscantake onthisfate andcontribute tothe prospective jointregion fromdifferentiating intochondrocytes, the firststepinjointformationistoinhibitcellswithin forming pre-cartilaginouscondensations.Ithasbeenproposedthat a processstartingwiththecondensationofmesenchymalcells The limbskeletal elementsareformedbyendochondralossification, formation anddifferentiation ofskeletal elements aretightlylinked. bodies) jointscanbedistinguished.Inthelimb,processofjoint sutures intheskull)andcartilaginous(e.g.jointsbetweenvertebral Three different types,synovial (e.g.jointsinthelimb),fibrous within theopposingskeletal elements(Francis-West etal.,1999). are importantsignalingcentersthatcontrolchondrocyte maturation Joints, whichseparateadjacentskeletal elementsfromeachother, INTRODUCTION KEY WORDS:Wnt,Synovialjoint,Chondrocyte maturation,Ihh,Mouse experiments inchickenandmouse.We showthatlossof in shortenedelements.Wnt9ahaspreviouslybeenimplicatedasbeingaplayerjointinduction,basedongain-offunction endochondral ossificationandfortheultimatesizeofskeletalelements,asprematureordelayedmaturationresultspredominant elements bycontrollingproliferationandmaturationofchondrocytes.Accuratechondrocyteiscrucialfor Joints, whichseparateskeletonelements,serveasimportantsignalingcentersthatregulatethegrowthofadjacentcartilage Hartmann Christine and Daniela Später of Wnt9a signalingisrequiredforjointintegrityandregulation Development 133,3039-3049(2006)doi:10.1242/dev.02471 DEVELOPMENT ANDDISEASE Accepted 2June2006 † 2 1 Pennsylvania CancerCenter, 421CurieBoulevard, Philadelphia,PA19104,USA *Present address: DepartmentofCellandDevelopmentalBiology, Universityof MA 02115,USA. Author forcorrespondence (e-mail:[email protected]) Department ofGenetics,Harvard MedicalSchool,77Avenue LouisPasteur, Boston, Institute ofMolecularPathology, DrBohr-Gasse Austria. 7,A-1030Vienna, Ihh Ihh promoter. Ihh during chondrogenesis ), acentralplayerofskeletogenesis.LossWnt9aactivityresultsintransientdownregulation 1 , TheoP. Hill 1,† 1 , RoderickJ.O’Sullivan ␤ Wnt9a -catenin pathwayprobablymediatesregulationof 1 , MichaelaGruber does notaffect jointinduction,butresultstosynovialchondroid secreted molecule, Pthrp,thatnegatively regulates chondrocyte osteoblastogenesis: Ihhsignaling controlstheexpression ofanother skeletogenesis coordinating cartilagegrowth and hypertrophic chondrocytes andplaysessentialrolesin molecule skeletal element.Akey regulator fortheseprocessesisthesignaling and maturationiscrucialfor determining thefuturesizeof undergo apoptosis.Accurate controlofchondrocyte proliferation hypertrophic chondrocytes, whichmaturefurtherandeventually chondrocytes, fromproliferative, topostmitoticprehypertrophic, and others,areimportantregulators forthematurationof Wnt9a signalingisinvolved injointinduction. Hartmann andTabin, 2001). Basedonthis,ithasbeenproposed,that regions (Garciadiego-Cazares etal.,2004; Guo etal.,2004; characteristic forthejoint-interzoneandformationofjoint-like of Wnt9asignalingleadstotheinductionmolecularmarkers By contrast,disruptionofintegrin signalingandectopicactivation Niswander, 2000;StormandKingsley, 1999;Tsumakietal.,2002). Johnson, 1998;Merinoetal.,1999;Pathi etal.,1999; Pizetteand of thosefactors issufficient toinduce jointformation(Capdevila and 1998; Settleetal.,2003;StormandKingsley, 1996).However, none and Nogginhave beenimplicatedinjointformation (Brunetetal., well understood.Various signalingmolecules,suchasGdf5,Gdf6 2003; Rountreeetal.,2004). joint capsule,originatefromtheinterzoneregion (Archer etal., synovial cells,articularpermanentchondrocytes andcellsofthe diverse celltypespresentinthematuresynovial joints,suchas al., 1994;StormandKingsley, 1996).Recentdatasuggestthatthe Tabin, 2000;Merinoetal.,1999;Ohbayashi2002;Storm adjacent cartilageelements(Francis-West etal.,1996;Hartmannand proteins (BMPs)thatregulate growth anddifferentiation ofthe Gdf6 andGdf7),various membersofthebonemorphogenetic Factors secretedbycellsadjacenttothejoint,suchasPthrp,Fgf18 The molecularmechanismsunderlyingjointformationarenotyet Ihh , whichisproducedbyprehypertrophic/early 1, *, DavidA.Conner ␤ -catenin showreduced Wnt4 RESEARCH ARTICLE ␤ -catenin/Lef1 complexand , suggestingthatWntsare 2 Ihh expression in Ihh and reduced Ihh 3039 ly

DEVELOPMENT MATERIALS ANDMETHODS catenin. and chondrocyte maturationbyWnt9aismediatedthrough 2005). in themesenchymeweregeneratedasdescribedbyHilletal.(Hill al., targeting constructwas generatedbyintroducinganSA-IRES- was ligatedintoanApaLIsite67bp downstream ofexon 2.The Spe promoter withan3 follows: aFRT-flanked neomycinresistancegene(neo)driven bythePGK The targeting constructoftheconditionalWnt9aallelewas generatedas Generation ofWnt9amutantalleles transcriptional controlby differentiation (Kronenberg, 2003). roles; itstimulateschondrocyte proliferationandosteoblast maturation. Furthermore,IhhhasadditionalPthrp-independent 3040 For incorporation were performedasdescribedpreviously (McLeod,1980). ethanol. AlizarinRed/AlcianBlueorAlcianstainingoftheskeletons Newborn pups(P0)andembryoswereskinned,eviscerated andfixed in95% Skeletal analysis Huelsken etal.,2001;Stark1994).Limbslacking performed byPCRaspreviously described(Huelsken etal.,2000; Genotyping of Wnt4 Mouse strains buffer containing0.2%glutaraldehyde,2mMMg minutes andskinnednewborns werefixed for1hour in0.1Mphosphate washed threetimes in0.1Mphosphatebuffer containing 0.01% (129/Sv) backgrounds. on mixed, randombred(Swiss-Webster), F1(129/Sv;C57Bl6/J)andinbred SV40pA-FRT- chromatin immunoprecipitationthattheregulation of Furthermore, weshow bygeneticinteractionandinvivo conditional alleleanda we have targeted the primarily inashorteningoftheskeletal elements. affect growth anddifferentiation oftheskeletal elements,resulting expression ofsecretedfactors controllingthecentral regulator Ihh Fgf andBmpsignaling(Mininaetal.,2002).Modulationinthe and itexpression levels areregulated inanantagonisticmannerby homozygous foreitherofthetwo by PCR(primersequencesavailable uponrequest).Phenotypesforembryos deleted inthegermlineusingPrx1Crefemales.Genotypingwas performed the recombinantalleles(onlyoneforconditionalallele).Exon2was used togeneratechimeras(two foreach allele),threeofwhichtransmitted (Hendrickson etal.,1995).Four independentlytargeted ES-cellcloneswere in 25theC1EScellline)andintroducedintomouseblastocysts external 5 Positively targeted EScellswereidentifiedbySouthernblotanalysesusing expression in The shorteningisduetoatemporarydownregulation of found that probably notrequiredforinducingjointformation.Inaddition,we that Wntsareessentialtomaintainjointintegrity, but thatthey are augmented in joints. Thephenotypesassociatedwithsynovial jointsare tarsal elementsandchondroidmetaplasiainsynovial andfibrous mutants dieatbirth.They displaypartialjointfusionsofcarpaland ␤ -Galactosidase staining,histology, insituhybridization andBrdU In ordertoaddresswhether I restrictionsite357bpupstreamofexon 2.Adouble-strandedloxPoligo ␤ -galactosidase staining,embryosE9.5-E13.5 werefixed for15-30 heterozygous micewerepurchasedfromJacksonlaboratory. RESEARCH ARTICLE Ј and 3 Wnt9a PGK Wnt9a;Wnt4 Ј Wnt9a Wnt4 probes on Ј mutants have shortenedappendicularlongbones. - located loxPsite(Sunetal.,2000)was inserted intoan neo- and FRT cassetteintothe Wnt9a lacZ mutants atembryonicdaysE12.5-13.5. Eco Runx2 ␤ double mutants.Ourdatademonstrate -catenin alleles(lacZandfloxed) was knock-in allele. RV-digested genomicDNA (frequency: 1 Wnt9a locus andgeneratedtwo alleles,a Wnt9a and is necessaryforjointinduction, alleles ( Runx3 Ihh Wnt9a ⌬ (Yoshida etal.,2004), 2 expression isunder Cl, 5mMEGTA onice, Sma or lacZ I siteofexon 2. ␤ loss-of-function -catenin activity Ihh ) wereidentical expression lacZ lacZ Ihh ␤ - - anti- immunohistochemical stainingonparaffin sectionswas performedusingthe ABC kit(Vector labs)andDAB (Sigma)asasubstrate. antibody (dilution1in250;Vector Laboratories)incombinationwith the (Iowa). Thesignalwas detectedusing abiotinylated anti-mousesecondary type III(3B2supernatant1:30)fromtheDevelopmental HybridomaBank antibodies againstcollagentypeII(II-II6B3supernatant,1:30)and with PBS,10%FCSand0.1%Triton-X100; andincubatedwiththeprimary were subsequentlywashed threetimeswithPBS;blocked for30minutes above. citrate buffer antigeneretrieval. Signaldetectionwas performedasdescribed developmental stage,atleastthreeindependentsampleswereanalyzed. by immunohistochemistry(ZymedLaboratories).For eachanalysisand mice 2hoursbeforesacrifice.BrdUincorporationwas detectedonsections 50 previously described(Hilletal.,2005).For analysisofBrdUincorporation, in DMEM:F12(Invitrogen). Experimentswerecarriedoutintriplicate. formamide, 5mMK stained overnight at37°Cinstainingsolution[1mg/mlX-gal,4%diethyl sequences areavailable byrequest. were analyzedbygelelectrophoresisandmeltingcurves. Allprimer Hprt1 were calculatedusingthecomparative C(t)methodandnormalizedtomouse green 1nucleicacidgelstain(MolecularProbes)andTAKARA Taq. Values produce first-strandcDNA. Real-timePCRwas performedbyusingSYBR ca The RCAS-AP, RCAS-Wnt5a,RCAS-Wnt9a,RCAS-Wnt3aandRCAS- Retroviral workandcultivationofchondrocytes For RT-PCR andreal-timePCRanalysis,1 RT-PCR analysis deoxycholate. 0.02%NP-40and2mMMg were preparedfor ChIPasdescribed(Martensetal., 2005). Approximately minutes, followed byquenchingwith125mM glycine.Whole-cellextracts After aPBSwash, cellswerecrosslinked with1%formaldehydefor10 FCS/DMEM for15minutesat37°C andbyadditionalusageofabouncer. dissociated in500 (FVBN mice:litterswith10-13 embryos). Humeriofonelitterwere For invivo cartilagelysates,humeriweredissectedfrom13.5 dpclimbs Chromatin immunoprecipitation (ChIP) inactivated byincubatingthecellsfor30minutesin1%H in PBS,washed twicewithPBS.Endogenous peroxidaseactivity was were fixed for15minutesatroomtemperaturewith4%paraformaldehyde For immunohistochemicalstainingofculturedcaudalchondrocytes, cells Immunohistochemical staining culture incubatorat37°Cand5%CO top ofNucleporeTrack-Etch Membranes(Whatman)inahumidifiedtissue Glutamine. Limbswereculturedfor24hoursin24-welldishesfloatingon DMSO inDMEM:F12(Invitrogen) supplementedwith10%FCSandL- (Calbiochem)/0.2% DMS0,whiletheotheronewas culturedin0.2% limb ofaforelimbpairwas culturedin thepresenceof25 Forelimbs wereskinnedandremoved fromE12.5andE13.5embryos.One Limb explantcultures incubation withaHRP-conjugatedsecondaryantibody(1:2500;Promega). antibody againstchicken ␮ sternal chondrocytes infectedwithdifferent RCASviruses.Extractsof50 For Western blotanalysis,proteinwas extracted from culturedchicken Western blotanalysis viral supernatantperwell(titers:6-8 six-well plate.Thefollowing daychondrocytes wereinfectedusing5 cultured for1day, collectedandplatedatadensityof5 from thecaudalpartofday18chicksternae(Koyama etal.,1999)were Hartmann andTabin, 2001;Kengaku etal.,1998).Chondrocytes isolated g perlanewereloaded.Luminaldetectionwas performedusingan ␤ For histologyandsectioninsituhybridization,tissuewas treatedas ␮ -cat viruseshasbeenpreviously described(HartmannandTabin, 2000; ␤ g BrdU/gbodyweightwas injectedintra-peritoneallyintopregnant -catenin (BDTransduction Laboratories,1in250)afterheat-induced expression. Primersetsweretestedbydilutionseriesandproducts 3 (Fe(CN) ␮ l of0.3%collagenaseIV/0.1% Trypsin/2% ␤ -catenin (1:800,SigmaC7027),followed by 6 ), 5mMK ϫ 2 10 . 8 4 pfu/ml) andculturedfor3-4days (Fe(CN) 2 Cl atroomtemperature,and ␮ g totalRNA was usedto Development 133(15) 6 )]. ϫ 10 2 O 5 2 cells/well ina in PBS.Cells ␮ M SU5402 ␤ -Catenin ␮ l

DEVELOPMENT reverse, 5 alleles wererecovered atweaningfromheterozygousintercrosses otherwise noted.Nopupshomozygousforeitherofthemutant alleles andthereforewewillrefertothemas–allele,unless translated fromthisallele.We considerbothallelesasbeingnull of acids oftheWnt9aprotein.In therefore consistof65aminoacids,containingthefirst32 premature Stop.Any proteinmadefromthistranscript would mutant embryos(Fig.1C).Sequencingrevealed aframe-shiftand exon 3,couldbeamplifiedbyRT-PCR fromRNA of A truncatedtranscript,resultingfromaberrantsplicingofexon 1to were obtainedbygermlinedeletionofthefloxed exon 2(Fig.1B). Southern blot(Fig.1B).Miceheterozygousforthedeleted( transmission andpropertargeting ofmutantalleleswas verified by lacZ-FRT-neo cassetteinsertedintoexon 2(Fig.1A).Germline flanking exon 2(Fig.1A)andalacZ-knock-inallelewithanIRES- locus inembryonicstemcells:aconditionalallelewithloxPsites Two Generation ofWnt9amutantalleles RESULTS CACT 3 CCAGCACTCC 3 200 Wnt9a GACTGAAGG 3 TCCGGCTGCGACGTGGGTTGC 3 TCF/LEF1 sites,thefollowing primerpairswereused:site1forward, 5 percentage ofinputDNA. For amplificationofthe threepotential research Lightcycler ( real-time PCRusingtheRocheSybrgreenquantitationmethodonaMJ from immunoprecipitates,aswelloftheinputmaterialwas analyzedby H3-K4 methylation(UpstateBiotechnology)antibodies.PurifiedDNA ␮ g Wnt9a ␤ ␮ -catenin (St.Cruz,sc-1496),4 Wnt9a g offragmentedchromatinwas usedinimmunoprecipitationwith4 signaling injointsandchondrocytes Ј ; site3forward, 5 Ј TGGGATGGCAGGGGAGTAGTA 3 was disruptedandthusnofunctionalproteincanbe alleles weregeneratedbytargeting thegenomic Ј Ј ; site2reverse, 5 ; site2forward, 5 n =3). Resultswerenormalizedandpresentedas Ј TGAATCCCGAGCAAGGCGTAG 3 ␮ lacZ Ј Ј g Lef1(St.Cruz,sc-8592)and4 ; site1reverse, 5 GACGGGCACTGCCTGGGAAT- allele, theopenreadingframe Ј Ј . ACTCCCCTGCCATC- Ј CGGCCGGCG- Wnt9a ⌬ Ј ) allele Wnt9a ; site3 ⌬ ␮ / ⌬ g Ј Comparative skeletal analysesof abnormalities Homozygous Wnt9amutantsdisplayskeletal by theabsenceofmilkintheirstomach(Fig.1D). slightly smallerthantheirlittermatesandcanbereadilyidentified kidney andbrain,didnotreveal any obvious abnormalities.They are analyses oftheirmajororgans, heart,lung,liver, intestinaltract, within 12hoursofbirth,forsofar unknown reasons.Histological of Wnt9a joint cellsinWnt9amutants Chondroid metaplasiaoffibrous andsynovial (Fig. 2C,partd-f ectopic cartilagenoduleswerepresentwithinthemidlinesutures the baseofparietalbones(Fig.2C,partc,e-f cartilaginous basewas extended dorsally, particularlynoticeableat shaped andreducedinsize(Fig.2C,partb).Furthermore,the apart (Fig.2C,parta).Thebasioccipitalbonewas abnormally showed reducedmineralizationandthefrontalboneswerefurther were hypoplastic(Fig.2A).Intheskull,supraoccipitalbone ileum andfemur(Fig.2B;datanotshown). Thehyoid boneandatlas prominent intheproximalbones,suchasscapulaandhumerus, of themineralizedregions (Fig.2A).Thesereductionsweremore reduced inlengthandshowed aneven greaterreductioninthesize part a Blue-positive nodulepresentintheelbow region (Fig.2A,Fig.3A, shown). Inallofthe defects but anumberofskeletal abnormalities(Fig.2Aanddatanot heterozygous andwild-typelittermatesrevealed nogrossjoint detected byAlcianBluestainingintheinterfrontalandsagittal fusions ofmajorjoints.However, ectopiccartilaginousmaterialwas Wnt9a Ј –/– ). Inaddition,theappendicularlongboneswereslightly +/ newborns displaynoobvious defectswithrespectto ⌬ or Wnt9a Ј ). Wnt9a obtained from E10.5wild-typeand genomic external probe.genomic external ( mice, respectively, hybridizedwiththe3 intercrosses of+/ wild-type (+/+)E10.5littermatesfrom lacZ/lacZ), heterozygous (+/ digested genomicDNAfrom mutant( stomach (arrow). side) andthattheydonothavemilk inthe (indicated bytheshorterblackline ontheright littermates. Mutantsare slightly smaller (WT) and cassette intoexon2.The5 transcript in transcript inwildtypeandashorter900bp embryos, showingthepresence ofa1.1kb Eco loxP sites. 11. Floxedallele(fl):exon2(Ex2)flankedby into theWnt9alocusonmousechromosome targeting constructs,whichwere introduced analysis ofmutants. Fig. 1.ConstructionofWnt9aallelesand map. Restrictionenzymes:B, probes are indicatedbelowthegenomiclocus +/lacZ RI; RV, mice. Homozygousmutantpupsdied mutants, therewas anectopicAlcian Wnt9a lacZ Eco Wnt9a RV. ( allele: insertionofanIRES-lacZ RESEARCH ARTICLE Wnt9a ⌬ ⌬ / ⌬ B and +/lacZheterozygous ⌬ ) Southern-blot on ) Southern-blot mutant newborn / ⌬ –/– mutants. ( ( A ) Schematicofthetwo newborns withtheir C Ј ) RT-PCR usingRNA ⌬ and 3 Bam or +/lacZ)and Ј D ). Inaddition HI; N, Ј ) Wild-type external Wnt9a ⌬ Eco / Ј Nhe ⌬ or RV- ⌬ 3041 / ⌬ I; RI,

DEVELOPMENT detectable asearly asE15.5byinsituhybridization [showing metaplasia inthehumeral-radial joint(HRJ)of or synovial chondromatosis. Theonsetofsynovial chondroid cartilaginous nodulesisreferred toassynovial chondroidmetaplasia cells differentiating intochondrocytes forminglooseectopic 3A, partc part a revealed thatthechondrocyte markers skulls (Fig.2C,parte).Insituhybridizationofcoronalskullsections mineralized regions werefurtherapartinmutantthanwild-type skull growth (Opperman,2000).Van Kossa stainingshowed thatthe vault, whichserve asmajorsitesforboneexpansion duringpostnatal 3). Suturesarefibrousjointsbetweentheflatbonesofcranial appearance (Fig.3A,partb revealed thatcellswithinthesynovial foldhadachondrocyte-like 3042 humeral-radial spaceofall parts f,f expression domainsatthebasewereexpanded dorsally(Fig.2C, (Fig. 2C,partse ectopically expressed incellswithinthefrontalandsagittalsutures (Fig. 2C,partsd,e suture regions, separatingfrontalandparietalbones,respectively An ectopicAlcianBlue-stainednodulewas observed inthe Ј ). HistologicalanalysesonsectionsofP0 RESEARCH ARTICLE Ј ). Ј ; datanotshown). In humans,thisphenotypeofsynovial Ј Ј ,f ,f Ј Ј ; datanotshown). Inaddition,theirnormal ; datanotshown) andintheelbow joint(Fig. Wnt9a Ј ) andexpressed mutant newborns ( Col2a1 Sox9 Wnt9a Wnt9a and and n =24; Fig.3A, Col2a1 –/– mutants was Sox9 forelimbs were (Fig. expression ofthechondrogenicmarker within theHRJregion (Fig.3A,partd Additional jointabnormalitieswereobserved in was restrictedtoafew cellswithinthisregion (Fig.3A,parte Loss of dedifferentiation associatedwithstabilizationof Wnt9a misexpression in chondrocytes leadsto the identityofjointcells. suggest thatWnt9asignalingisrequiredinsomejointstomaintain elements cand3inthewrist(Fig.3A,partsf cuneiform tarsalelementsinthefootandbetweencarpal such aspartialjointfusionsbetweenthenavicular and intermediate constitutively active formof canonicalligandWnt3a,thenon-canonical ligandWnt5a,a the competent avian retroviruses (RCAS)expressing eitherWnt9a, chicken sternalchondrocytes andinfectedthemwithreplication chondrocytes would leadtoalterationsofthecells.We usedprimary hypothesis further, weasked whetherectopic is requiredtosuppresstheirchondrogenic potential.To testthis hypothesized thatWnt9asignalinginsynovial andfibrousjointcells ectopic differentiation intochondrocytes. Basedonthis,we ␤ -catenin Wnt9a which are absentinthewild-typelittermates. thesagittalsuture (asterisks), expressing cellswithin adjacent tothoseshownine,f,showing type and skull. (e cells inthesagittalsuture region ofthe base (arrow) andthepresence ofAlcianBlue-positive (arrowheads ine),dorsalexpansionofthecartilaginous further apartfrom eachotherin (e,f), showingthatthetwofrontal boneplatesare indicated ind.Van Kossa/AlcianBlue-stainedsections type (wildtype= from showingthatin newborns, ( (Mut= regions ofmutantskeletalforelimb elements with regard tothetotallengthandmineralized atlas. ( compared withwildtype.Hypoplastichyoidboneand extend, inulnaandradiusthemutantforelimbs are foundinthescapulaandhumerus,and,toalesser littermates.Smallermineralizedzones (below) newborn Fig. 2.Skeletalabnormalitiesin hyoid bones,atlasfrom WT(ontop)and wild-type and (c) Lateraland(d)dorsalviewofAlcianBlue-stained abnormally shapedbasioccipitalbone(bo)(arrow inb). ossification centerinthesupraoccipital(so)boneandan bones are furtherapart(arrow ina),asmaller skulls ofwild-typeand skull (arrowhead ind).(e,f)Coronal sectionsthrough Alcian Blue-positiveareas inthesagittalsuture ofthe parietal bone(arrowhead inc)andpresence ofectopic expansion ofthecartilaginousbaseinregion ofthe (Het= Red/Alcian Blue-stainedwild-typeand litters. ( and littermatecontrol limbswere collectedfrom seven type/heterozygous elementswassetto100%.Mutant A signaling inHRJandmidlinesuturecellsledtotheir AlizarinRed/AlcianBluestainingofskeletonswild- ) Wnt9a Wnt9a B C Ј ) Table showingquantificationofsizereduction ,f ) Dorsal(a)andventral(b)viewofAlizarin Wnt9a Ј ) Col2a1 +/lacZ lacZ/lacZ Wnt9a Wnt9a –/– ). Average lengthofwild- E17.5 littermates,andofforelimbs, ) incomparisonwiththosefrom wild in situhybridizationonsections ␤ –/– -catenin (ca +/+ Wnt9a newborn heads,showing newborn ) andheterozygous Ј )], while –/– Sox9 Development 133(15) newborns, atthelevels newborns, Wnt9a Wnt9a Wnt9a Wnt9a ␤ in abroaddomain Col2a1 -cat) oralkaline Ј Wnt9a ,g Wnt9a Wnt9a –/– mutants Col2a1 Wnt9a Ј ). Thesedata the frontal mutants. signaling in –/– expression mutant mutants, - –/– heads Ј ).

DEVELOPMENT Wnt9a ( However, micedoublemutantfor mutants donothave any jointabnormalities(Starketal.,1994). Hartmann andTabin, 2000;HartmannandTabin, 2001). genes, Fig. S1inthesupplementary material). Atleasttwo otherWnt mutants, despitethefact that Surprisingly, onlyafew synovial jointswereaffected in joint integrity Wnt4 andWnt9aactcooperativelyinmaintaining into fibroblast-like cellsproducingadifferent typeofcollagen. ␤ or Wnt5a-infectedcellextracts (Fig.3C). chondrocytes infectedwithWnt9aorWnt3aviruscomparedAP extracts revealed thatthe produced bythecontrolcells(Fig.3B).Western blotsfromwhole-cell (Col3)instead,whichwas not stained positively forcollagentypeIII and ceasedtoproduceCol2(Fig.3B,datanotshown). Thesecells either Wnt9a,Wnt3aorca for collagentypeII(Col2;Fig.3B).Bycontrast,cellsinfectedwith chondrocytes retainedtheirtypicalcuboidalshapeand stained positive phosphatase (AP)ascontrol.InAPandWnt5a-infectedcultures,the Wnt9a -catenin levels, andthatthisincreasecan transform chondrocytes These datashow thatgainofWnt9asignalingleadstoincreased Wnt4 –/– signaling injointsandchondrocytes ; Wnt4 and –/– Wnt16 ) developed synovial chondroidmetaplasiaintwo , areexpressed injoints (Guoetal.,2004; ␤ -cat viruseshadafibroblasticappearance ␤ Wnt9a -catenin levels wereincreasedin is expressed inalljoints (see Wnt9a and Wnt9a Wnt4 Wnt4 suggested apossible delayofchondrocyte maturationin elements formedbyendochondral ossification(Fig.2A,B) The reductionofthemineralization/ossification centersinskeletal mutants Altered chondrocyte maturationinWnt9a owing totheabsence ofWnt9aandWnt4activity. formed andthatthefusionofskeletal elementsoccurssecondarily, These observations stronglysuggestthatthejointsareoriginally fused andjointmarker expression was lost(Fig.4B,partsg,h). 4B, partsb,f;datanotshown). However, atE15.5theelementswere mutant wristsatE13.5,showing identicalmarker expression (Fig. Those canbedistinguishedasseparateelementsinwild-type and E15.5 wrists,focusingprimarilyatthecarpalelements2,cand 3. affected bythelossof part g). of anectopiccartilagepieceinaligament(seeasteriskFig. 4A, ( cuneiform) (Fig.4A,partsf,g)andwrist(carpalelements2,c3) in thefoot(calcaneusandcuboid,navicular andintermediate d,e). Inaddition,fusionsoftarsalandcarpalelementswereobserved additional majorjoints,theankleandkneejoint( of various markers n =4/4; Fig.4B,parte).Inonespecimen,weobserved the presence In ordertoaddresswhetherjointformationormaintenancewere Col2a1 catenin levels. expressing Wnt9aorWnt3ahaveincreased showing thatcellsinfectedwithaRCASvirus using protein extractsfrom infectedchondrocytes, Wnt5a, RCAS-Wnt9aandRCAS-ca chondrocytes infectedwithRCAS-AP, RCAS- type IIandcollagenIIIonchickensternal ( elements (arrow) inthemutant. intermediate cuneiformandnaviculartarsal II-V. (g navicular; t,tarsal;metatarsalelementsofdigits cuneiform; i.c.,intermediatenc, wild type;Cal,calcaneus;cub,cuboid;l.c.,lateral Schematicdiagramofthetarsalelementsin (g) b sections ofwild-type(d,e)and instead ofsynovialcellswithintheHRJfold.Serial (b elbowregion; (a Wild-type (a) forelimbs stainedwithAlcianBlue/AlizarinRed. chondrocytes infibroblast-like cells. joints, andectopicWnt9acantransform Fig. 3.LossofWnt9aleadstodefectsin U, radiusandulna.(f element; randu,radialulnarR carpal elements1-5inwildtype.c,central metacarpal elementsofdigitsI-Vanddistalrow of Schematicdiagramofthecarpalelements, (f) of cellsexpresses region express E15.5, showingthatinthemutantcellsHRJ Serial sectionsofwild-type(b,c)and present inthehumeral-radialjoint(HRJ)(arrow). which anectopicAlcianBlue-stainednoduleis collagen typeIII.( producing collagentypeIItheysynthesize change theirmorphology, andthatinsteadof that Wnt9aandca carpal elementscand3(arrow) in B Wnt9a Ј Ј Immunohistochemicalstainingforcollagen ) ) and ,c Ј ) P0forelimbs, showingAlcianBlue(arrow in , Ј Col3 ) Partialjointfusionbetweenthe Col2a1 and , Wnt4 Gdf5 Sox9 -positive (arrow inc RESEARCH ARTICLE C Col2a1 ) Westernblotfor ␤ , , weanalyzedtheexpression Gli3 -cat-infected chondrocytes (d Ј ) Partialfusionbetween Ј ), andthatasmallcluster (arrow ine and Ј n Wnt4 ) Wnt9a =4; Fig.4A,parts Wnt9a Ј Wnt9a ␤ ) chondrocytes Ј Wnt9a in E13.5and -cat, showing ). ␤ –/– -catenin –/– ( (d A elbow, in –/– Ј ) P0 Wnt9a ,e –/– . ␤ 3043 Ј - ) at

DEVELOPMENT in proximallong bones. suggests thatWnt9asignalingtemporally andspatiallyregulates but notindigits(seeFig. S2B,Cinthesupplementarymaterial).This was alsoobserved in (compare Fig.5Dwith6C). Temporary downregulation of chondrocytes (Fig.5D) and resembledwild-typeexpression atE13.5 separated byapopulation of moremature, in wild-typehumeri.Nevertheless, the Fig. S2Ainthesupplementarymaterial). generated fromE12.5andE13.5wild-typemutanthumeri (see independently confirmedbyreal-timePCRanalysisoncDNA pronounced atE13.5(Fig.5C).Downregulation of in mutants(Fig.5B)andthisdownregulation was even more differentiation marker also notaltered(datashown). Furthermore,thechondrocyte not shown). Proliferationrate(BrdU)andapoptosis(TUNEL)were and fusionofthethree carpalelements2,cand3in E13.5andE15.5embryos.(a,e)Vannewborns, Kossa/AlcianBlue/Eosinstaining,showingnormalarrangementofcarpalelementsin elements (arrow) andsynovialchondroid metaplasiainthejointcapsuleligamentofdigitI(asterisk).( mutants. (f)Fusionbetweentheintermediatecuneiformandnaviculartarsalelements(arrow). (g)Fusionbetweenthecalcaneus (a,d), knee(b,e)andfootregion (c,f,g)ofwild-type(= Fig. 4. 3044 E12.5, however, wild-type orheterozygouslittermates(Fig.5A;datanotshown). At in adomainofsimilarsizeandatlevels equal tothosedetectedin bone anlagenbetween and situ analysesatE11.5usingtheearlychondrogenicmarkers bones atdifferent embryonicstages.Whole-mountandsectionin histological andmolecularmarker analysesonthedeveloping long mutants. Inordertodeterminethenatureandonset,weperformed fused in (c) Footregion. (d,e)Synovialchondroid metaplasiaintheanklejoint(d,arrow) andinthejointcapsuleofknee(e,arr 2, cand3are separatedinwildtype(b)and are separatedinwildtype(d)butare fusedinthe Surprisingly, atE14.5 Col2a1 Wnt9a RESEARCH ARTICLE Wnt9a revealed nodifference intheoverall sizeofthelong and –/– ;Wnt4 Ihh Wnt4 Wnt9a expression levels wereslightlydownregulated –/– Ihh Wnt9a mutants (h). Ihh act redundantly inmaintainingjointintegrity. was expressed in mutant radiiandulnae(datanot shown), expression levels weresimilartothose mutants andlittermatecontrols(data Ihh Wnt9a Wnt9a domain was notyet Wnt9a –/– Wnt9a Wnt9a ;Wnt4 mutant humeri –/– Ihh ;Wnt4 –/– –/– +/– -negative ;Wnt4 Ihh ; Wnt4 mutants atE13.5(f).(c,g) –/– Sox9 was mutants (g).(d,h) Ihh Ihh –/– +/– mutants (e).(b,f) ) and proliferation rate withintheflattenedzoneatE13.5 ( incorporation revealed a7%reductionofthechondrocyte al., 2000).Consistentwiththe reductionin chondrocyte proliferation inaPthrp-independentfashion (Karpet parathyroid hormonereceptor1( Wnt9a al., 1999;Vortkamp etal.,1996),showed areducedexpression in which functionsalsoasareceptor forIhh,and also affected. AnalysisoftheIhhtarget genespatched1( analyzed whetherIhhsignalingandregulated processeswere Because Wnt9a mutantlongbones Temporal downregulation ofIhhsignalingin E13.5. signaling iscrucialforchondrocyte maturationaroundE12.5- humeri (Fig.5H).Thismarker analysisshowed thatWnt9a Col10a1 not detectableatE13.5(Fig.5G;datashown). AtE14.5the mutant humeriatE12.5(Fig.5F),whileitwas eitherreduced or hypertrophic chondrocytes, was slightlyexpanded in collagen 10a1(Col10a1) chondrocyte maturation(Fig.5E;datanotshown). The in notinmagnitudeatE13.5andE14.5,reflectingadelay extent Informatics), whichoverlaps with Wnt9a ( A By contrast,theexpression domainofthegeneencoding ) Van Kossa/AlcianBlue/Eosin-stainedsectionsthrough theankle –/– –/– Col2a1 humeri atE13.5(Fig.5I,J).Ihh signalingalsoregulates ;Wnt4 domain inmutantsresembledthatofE13.5wild-type Ihh Gdf5 Gdf5 expression was temporarilydownregulated, we –/– staining. Thecarpalelementsinwildtype(d)butare staining onwristsections.Thethree carpalelements staining onwristsections.Thethree carpalelements newborn littermates.(a)Anklejoint.(b)Knee newborn B ) Sectionsthrough thewristregions of expression domain,whichmarks Ppr Ihh ; Pthr1 ow) of , was reducedonlyin Development 133(15) Pthrp – MouseGenome Wnt9a and cuboidtarsal Ihh wildtype(a) (St-Jacques et P levels BrdU –/– <0.05; data ;Wnt4 Ptch1 Wnt9a –/– ),

DEVELOPMENT domains atE14.5(D)in E12.5 (B)andE13.5(C),delayedseparationofthe perichondrium/periosteum. E13.5. ( significant difference atE11.5(A),downregulation of Wnt9a periosteal markers, suchas (Long etal.,2004;St-Jacques1999).Analysesofvarious required forosteoblastogenesisintheperichondrium/periosteum not shown). detected inmutantcomparedwithwild-typehumeriatE14.5(data not shown). Nosignificantdifference inproliferationratecouldbe wild-type and In situhybridizationonserialsectionsshowingthehumerusregion of Fig. 5.Temporal regulation of E14.5 ( humeri. ( E12.5, E13.5andE14.5inwild-typeWnt9a expression domainin (shoulder joint)( chondrocytes andperichondrium( the temporarydownregulation of 1998). Therefore,weaddressed whetherFgfsignalingisinvolved in (Chen etal.,2001;Li1999; Mininaetal.,2002;Naski of proximalskeletal elements. thereby remainsnoticeableeven atbirth,reflectedintheshortening around E14.5,thematuration delay cannotbecompensatedand osteoblast maturation.Although This ultimatelyleadstoadelayby~1dayinchondrocyte and Wnt9a activity resultsinatemporaryreductionofIhhsignaling. at E13.5(datanotshown). Thesedatademonstratethatlossof E14.5, expression ofthesemarkers was comparablewithwildtype four markers was reduced(Fig.5K,L;datanotshown). However, at Informatics) and Genome Informatics),osteopontin( Ihh playsathirdroleduringendochondralboneformation;it is Fgf signalinghasalsobeenshown tonegatively regulate H F signaling injointsandchondrocytes ) in ) Increased G ) Strongly reduced Wnt9a Wnt9a J ), andof Bmp3 –/– Col10a1 –/– Wnt9a humeri. Reducedexpression levelsof littermates. ( Wnt9a , atE13.5revealed thattheexpression ofall Runx2 –/– expression domainatE12.5in Col10a1 –/– humeri compared withwildtypeat Runx2 Ihh ( humeri. ( K I ), of ) and Ihh A-D Ihh expression byWnt9asignaling. expression domainatE13.5and , osterix( Pthrp in Wnt9amutants byassaying Op ) Ihhexpression atE11.5, levels arebacktonormalat Osx E ; ) Sizereduction ofthe Spp1 ( in thearticularregion L ) inthe –/– ; showingno Osx – MouseGenome Ihh ; Ihh Sp7 expression at expression Ptch1 Wnt9a – Mouse in Ppr –/– Ihh in Wnt9a signalsthrough contribute totheobserved downregulation of maturation. Mostimportantly, ourdatasuggestthatFgfsdonot developmental stagesisapositive regulator ofchondrocyte Based onouranalysis,wewould concludethatFgfsignalingatearly slightly decreased(seeFig.S3Ginthesupplementarymaterial). wild-type andheterozygoushumeri, humeri (Fig.6A).In cells atE13.5was onlyslightly reducedinthedoubleheterozygous E12.5 (Fig.6A). expression was observed inhumeriof G380R) (Naskietal.,1998).However, nodownregulation of constitutive active Fgfr3signalinginchondrocytes ( exception of and Fgf20-22)usingreal-timePCRatE12.5E13.5.With the for altered while the at E12.5incontrolandinhibitor-treated limbs(datanotshown), in thesupplementarymaterial).No Ihh Expression levels anddomainsizeof the canonical Gain-of-function experiments suggestedthatWnt9asignalsthrough chondrocyte maturation expression of at E13.5.To furtherinvestigate thispossibility, weanalyzedthe This increasecouldpotentiallycontribute todownregulation of Fgf21 or E13.5( expression levels offive Fgfgenes( material; datanotshown). InterestinglyatE13.5 therelative expression levels atE12.5(seeFig.S3Ainthesupplementary analyzed showed any significantincreaseordecreaseinrelative Col10a1 with SU5402didnotnotablyincreasethesizereductionof at E13.5(seeFig.S3Ginthesupplementarymaterial).Treatment Wnt9a Wnt9a maturation atE12.5-E15.5inembryosdoubleheterozygous for between (this work). To assess thisfurther, welooked atgeneticinteraction in thesupplementarymaterial).In while inE13.5limbsonlyasizereductionwas visible(seeFig.S4F cultured humeri( expression levels of SU5402 resultedinastrongreductionthedomainsizeand inhibitor specifictoFgfrs(Mohammadietal.,1997).Treatment with pairs wereculturedinthepresenceofDMSOorSU5402,akinase would rescue littermates totestwhetherinhibitingFgfsignalingin forelimbs from Fgfr3, weperformedlimbexplant culturesusingE12.5 andE13.5 signaling mightpromotechondrocyte maturationaroundE13.5. supplementary material).Thissuggeststhatactivation ofFgfr3 E13.5 comparedwithwild-typelittermates(seeFig.S3Dinthe expression domainsof contrast, weobserved onaverage aslightexpansion ofthe wild-type or levels werereducedindouble-heterozygous embryoscomparedwith As theupregulated Fgfsmightnotnecessarilysignal through Ptch1 expression was even furtherdownregulated ( ) wereincreased(seeFig.S3Binthesupplementarymaterial). –/– and ; expression incomparisonwith domain alreadypresentin Col10a1 ␤ n Wnt9a Fgf =10) (seeFig.S3C,Dinthesupplementarymaterial).By -cat ␤ Fgf4 -catenin. AtE12.5,but notatE13.5, Wnt9a ␤ Ihh expression of19Fgfgenes(Fgf1-13,Fgf15,17,18 -catenin pathway (Dayetal.,2005;Guo2004) +/– Ihh n Ptch1 , whichwas downregulated, noneoftheFgfgenes expression levels. Limbsofcorresponding limb Wnt9a in comparisonwith =5; seeFig.S3Einthesupplementarymaterial), expression domainwas notsignificantlyaffected and +/– and Wnt9a Ihh Ihh littermates (Fig.6A,anddata not shown). expression inchondrocytes andperiosteal ␤ -catenin withregard tochondrocyte Col10a1 mutant, heterozygousandwild-type in wild-typeandheterozygousE12.5 (by ~8%)and –/– ; ␤ ␤ -catenin regulating -cat Wnt9a Col10a1 RESEARCH ARTICLE in humeriofembryoswith +/– Wnt9a Fgf1 Fgfr3ach Ihh Wnt9a humeri, afurtherreduction –/– Col10a1 Wnt9a Col10 , E12.5 andE13.5humeri, were furtherreducedin Fgf6 –/– expression was detected Ihh humeri, but asinthe –/– mice atE12.5( n single mutantswas , (by approx.6%)at in =3; seeFig.S3E,F Fgf15 single mutantsat expression was Wnt9a Ihh Wnt9a , expression Fgf20 Fgfr3ach mutants. mutants 3045 n and =5) Ihh Ihh

DEVELOPMENT wild type, (arrowheads). ( and even furtherreducedin domain was alsoreducedinsizethedoubleheterozygous at E13.5 above. Similar chondrocytes (bracket)in closer togetherin stabilization of Consistent withtheobservation thatWnt9asignalingledto wild-type and indicate reduced expression ofPtch1.( cat particularly scapulaandhumerus, was reducedinsize embryos, wherethemineralized region inthelongbones, mineralization was alsonoticeable inskeletal preparationsofE15.5 maturation. Thisdelayin chondrocyte maturationand wild type(Fig.6B),demonstratingadelayinchondrocyte Col10a1 double heterozygous mutantscompared with size reduction ofthemineralizedregions ofscapulaand humerusin 3046 strongly expressed (arrowheads inFig.6A). The primarily noticeableintheperiosteum,where showing reduced humeri compared withWT. ( Col10a1 Fig. 6.Geneticinteractionof 6A). AtE15.5,theexpression domainsof cat chondrocyte maturation. +/– +/– , Wnt9a to asimilarextent as in RESEARCH ARTICLE expression domainsare closertogetherin on alternating sectionsofhumerifromon alternating wildtype, Wnt9a –/– Wnt9a ␤ D -catenin levelsare foundintheperiosteum and ) AlcianBlue/AlizarinRedstainedforelimbs ofE15.5 ␤ +/– ␤ -catenin, nuclear -catenin levelsinflattenedandprehypertrophic ; Wnt9a ␤ Wnt9a +/– -cat ; ␤ +/– -cat Wnt9a +/– –/– ( and C A ; +/– ; ␤ ) ImmunohistochemicalstainingatE13.5, ␤ ) Insituhybridizationfor Wnt9a -cat -cat Wnt9a humeri, showingthatthe Wnt9a mutants incomparisonwithwildtype +/– +/– ␤ –/– mutant littermates.Arrowheads Wnt9a B -catenin levels werereduced in mutant humericomparedwith –/– and ) InsituhybridizationonE15.5 ; ␤ embryos, showingasimilar -cat ␤ –/– -catenin in Wnt9a +/– Ihh embryos (Fig.6D). mutant humeri(Fig. Col10a1 Wnt9a and single mutants. Ptch1 Ihh Col10a1 +/– Ihh , Wnt9a Wnt9a Ptch1 ; expression ␤ and -cat was less +/– +/– and +/– were ; ␤ ; ␤ - - This couldbeduetothepresenceofanotherWntgene, in different typesofjoints.Nevertheless, notalljointsareaffected. suppress thechondrogenicpotentialofsynovial andother jointcells showed thatWnt9aandWnt4signalingiscooperatively requiredto synovial chondroidmetaplasiaintheHRJ.Doublemutant analyses suture, topartialjointfusionsofcarpalandtarsalelements, Wnt9a leadstoectopicdifferentiation ofcartilageinthesagittal All jointsareinitiallyformedin chondrogenic potentialofjointcells Wnt signalingisrequired tosuppress the DISCUSSION transcription andthatWnt9aistherelevant ligand. propose thatthecanonicalWntpathway directlyregulates interaction with to theputative transcriptionalstartsiteshowed thestrongest state. Real-timePCRanalysisrevealed thattheTCF/Lefsiteclosest H3K4 confirmedthatthechromatinwas inatranscriptionallyactive sites (Fig.7B).PerformingChIPassayswithanantibodyagainst of the ␤ lysates fromE13.5wild-typeskeletal elements.Antibodiesagainst shown byinvivo chromatinimmunopreciptation(ChIP)usingcell potential TCF-bindingsiteswithinthe catenin/TCF-complex. Thisissupportedbythepresenceofthree Our datasuggestedthat Regulation ofIhhexpression by canonical Wnt/ regulating chondrocyte maturationand Wnt9a prehypertrophic chondrocytes andinarticularchondrocytes in maintaining joint integrity. This strong pro-synovial anti- essential tosuppresstheir chondrogenic potentialthereby development, andthatcontinuouscanonicalWntsignalingis potenial synovioprogenitors presentatlaterstagesofjoint postulate thatinthesynovium andjointcapsuletherearestillbi- integrity ofthejointatlater stages.Basedonouranalyseswewould region, therebyenabling jointformationandmaintainingthe suppress thechondrogenicpotentialofcellsinpresumptive joint Wnt/ chondroprogenitors (Dayetal.,2005;Hill2005),canonical Thus, itislikely that,analogoustoitsfunctionin osteo- chondroprogenitors (Nalinetal.,1995;Nishimura1999). Previous work hasshown that synovial tissuecontains in partialfusionsatlaterstages(Guoetal.,2004)(datanotshown). expression was lostandthejointsnever properlyformed,resulting of thosejointmarkers. However, aspreviously reported,their suggesting thatcanonicalWnt-signalingisnotrequiredforinduction joint regions atE11.5(seeFig.S4inthesupplementarymaterial), such as (Hill etal.,2005).Thisrevealed thatearlyjointinterzonemarkers development inmicelacking examined theexpression ofjointmarkers duringearlystagesoflimb (Guo etal.,2004;HartmannandTabin, 2000),thereforewe initial jointformation.BothWntsmightusethe Wnt9a;Wnt4 fusion ofthethreecarpalelements(2,cand3)presentin 2001). Thisisfurthersupportedbytheobservation thateven the as previously proposed(Guoetal.,2004;HartmannandTabin, analysis suggeststhatWntsmightnotberequiredforjointinduction Binding ofa -catenin andLef1couldimmunoprecipitatethreedifferent regions ␤ Ihh -catenin signalingisrequiredinanautocrinefashion to –/– Gdf5 skeletal elements(Fig.6C).Thus,Wnt9aisprobably promoter, eachcontainingoneofthepotentialbinding , double mutantnewborns was notduetoadefect inthe Wnt4 ␤ ␤ -catenin/TCF-complex tothe -catenin pathway. ␤ -catenin andLef1(Fig.7B).Therefore,we and Gli1 Ihh were stillexpressed inthepresumptive could beadirecttarget forthe ␤ -catenin inthelimbmesenchyme Wnt9a Ihh mutants; however, lossof Ihh ␤ Development 133(15) expression throughthe -catenin/Lef-1 promoter (Fig.7A). Ihh ␤ -catenin pathway promoter was Wnt16 . Our Ihh ␤ -

DEVELOPMENT and site3withthelowest( numbers below)revealed thatsite1isboundwiththehighestaffinity Real-time PCRquantification(normalizedtoinputandindicatedbythe of allthree sitesinthe from E13.5dissociatedwild-typehumeri,showingimmunoprecipitation osteoblastogenesis byapproximately1day andultimatelyin proliferation, andadelay of chondrocyte maturationand immunoprecipitation for relative tothetranslationalstartsite(+1).( showing thepositionofthree potentialTCF/Lef1-bindingsites Wnt9a Temporary downregulation of of skeleton. Itisrequiredaround E12.5-E13.5tomaintainhighlevels temporally andspatiallyregulates individual skeletal elements.Here,weshow thatWnt9asignaling predominantly leadtosizereductionofallorsometimes only 2003). Mutationsaffecting chondrocyte maturation orproliferation central regulator ofendochondralboneformation(Kronenberg, way tocontrolsizewould bethroughmodulationof Ihh signaling,a signals fromthejointmightbeinvolved inthesizeregulation. One how thisisregulated islargely unknown, but there isevidence that chondrogenesis. Skeletal elementsdiffer insizeandshape;however, In addition,wediscovered aroleforWnt9a signalingin size ofskeletalelements Wnt9a atemporalregulator ofIhhcontrolling the seem tobeinvolved innormaljointinduction. Hartmann andTabin, 2000).However, thisWntactivity doesnot redirect earlyskeletal condensationstoajointfate (Guoetal.,2004; to transformchondrocytes intosynoviofibroblast-like cellsandto progenitors isalsoreflectedintheabilityofectopicWnt9aactivity chondrogenic activity ofcanonicalWntsignalingonchondro- promoter. Fig. 7. Ihh expression, but seemstobedispensablefromE14.5onwards. ␤ signaling injointsandchondrocytes -Catenin andLef1physicallyinteractwiththe ( A ) Schematicviewofthe2.0kb Ihh ␤ -catenin, H3-K4tri-methylationandLef1 promoter, butnoneofthe n =3). Ihh Ihh results inreducedchondrocyte expression intheappendicular B ) Chromatin Ihh promoter region, tubulin Ihh promoter. as co-repressors(Brantjesetal.,2001).Intriguingly, repressor in display apostnatal,temporaryreductionin potentially compensate atlaterstagesfortheloss of explained bythepresence ofotherWntgenes,whichcould The fact thattheeffect of A Wntcanoncontrolling skeletogenesis growth ofskeletal elements. development, therebycontrollingchondrocyte maturationandthe level of published observations, suggestthatWntsignalingregulates the Runx2 couldcooperatively regulate further dependenton in actually contribute torestoringnormalchondrocyte maturation E12.5. OurFgfsignalinganalyses,instead,suggestthatFgfsmight in theexpression levels of E12.5 andE13.5limbsof Marie, 2002).However, potentially mediatetheeffect seenin shortening oftheproximallongbones. Groucho 5( activators suchasgroucho5(Vega etal.,2004;Wang etal.,2004). modulated byinteractionwithco-repressorssuchasHDAC4 andco- Hu etal.,2005).Interestingly, Runx2activity inchondrocytes is 2002). Thus,itisconceivable thatthe unpublished) (Akiyamaetal.,2004;Hu2005). activity dependingontheCre-deleterline(T.P.H. andC.H., delayed expression inskeletal elementsofmicelacking Ihh catenin doubleheterozygousanimalswereslightlyreducedandthat observations that canonical/ in The levels of canonical Wnt9a regulates Ihhexpression through the size ofindividual appendicularskeletal elements. bone formationprocess,couldbeacrucialmechanismtocontrolthe on theregulation of sequential signalinginputthroughdifferent pathways, converging developmental timepoints.Therefore,itistemptingtospeculatethat negatively regulated byWntsignalingandFgfatdifferent upregulation ofsomeFgfs.Thus, implicated innegatively regulating changed attheonsetof chondrogenic Wnt/ Runx2 Runx3 Runx2 in thehumerusandfemurisalsotranscriptionallydependenton altered in al., 2004).Furthermore, signaling(Huetal.,2005;Long expression isdependentonIhh stages isprobablyduetothedownregulation of mutants (datanotshown). Theslightdecreasein dependent regulation of Lef1 associatewiththe of Wnt9a ␤ Wnt9a -catenin invitro.This,togetherwiththefact that expression varies fromdownregulation totemporarylossor ␤ , andinmoredistalbonesitrequirestheactivity of -catenin signalinginosteoblasts(Gauretal.,2005);however, has beenrecentlysuggestedtobedirecttarget forcanonical (Inada etal.,1999;KimYoshida etal.,2004). Ihh mutants, while ␤ ␤ mutants fromE14.5onwards, concomitantwithan -catenin mutantlimbs(Dayetal.,2005;Hill ␤ expression temporallyduringembryonicandpostnatal -catenin pathway. This is furthersupportedbythe Grg5 ␤ ␤ ctnnTFsgaig niegoco1-4,whichact -catenin/TCF signaling,unlike groucho -catenin arereducedinprehypertrophicchondrocytes -catenin pathway Runx2 ; Aes Ihh Ihh Runx2 – MouseGenomeInformatics)actsasade- expression levels werenotsignificantly expression levels inhumeriof Ihh Runx2 , whichisattheheartofendochondral Wnt9a Ihh Wnt9a Fgfr3ach Ihh Fgfs promoter invivo, suggeststhatWnt9a- Ihh levels (Wang etal.,2004;Wang etal., expression was notdownregulated in downregulation atE12.5in expression was alsonotsignificantly overexpression leadstoanincrease regulation isonlytemporary canbe was observed in is probablymediatedviathe mice andnosignificantincrease RESEARCH ARTICLE Ihh Ihh ␤ Wnt9a Ihh Fgf -catenin/Lef1 complex and . Ourdata,togetherwith expression isprobably Ihh signaling hasalsobeen expression andcould mutants (Ornitzand expression, whichis Runx2 Wnt9a Wnt9a Ihh Ihh ␤ Grg5 levels atlater -catenin and , as expression Runx2 mutants at Wnt9a; ␤ -catenin activity. –/– Wnt9a Runx2 3047 mice and ␤ -

DEVELOPMENT genes ( Interestingly, whenweanalyzed theexpression levels ofvarious Wnt 3048 Akiyama, H.,Lyons, J.P., Mori-Akiyama,Y., Yang, X.,Zhang,R.,Z., References CT-2003-504468). was supportedbyBoehringerIngelheim andtheNoECellsintoOrgans(LSHM- Wagner andLatifaBakiriforcriticalcommentsonthe manuscript. Thiswork Christian TheusslforblastocystinjectionsofWnt9aLacZEScells,andErwin embryos, andGailMartinNaomiFukaiforreagents. We alsothank mouse strains,Andrea Vortkamp andManuelaWülling fortheFgfr3ach reagents andfortheC1ES cells,Walter Birchmeier andMalcomLoganfor support andgenerosity. Furthermore, wethankJohnSeidmanforBAC We thankCliff Tabin, inwhoselaboratory thisworkwasstarted,forhis 2000). Although positive regulator ofchondrocyte maturation(HartmannandTabin, reduction of supplementary material);however, thisdidnotresultinfurther Wnt9a/Wnt4 Concomitantly, chondrocyte maturationwas furtherdelayedin (see Fig.S5Cinthesupplementarymaterial;datanotshown). E12.5 andE13.5in real-time RT-PCR, wefoundthat and et al.,2004;HartmannandTabin, 2000),suggeststhatboth function analysis,togetherwithprevious gain-of-function data(Guo mutants (seeFig.S5Binthesupplementarymaterial).Ourloss-of- chondrocyte maturationintheappendicularskeleton ofsingle phenotype (Starketal.,1994),wedidobserve aslightdelayin regulates chondrogenesis: canonical Wnt/ joint destruction.Thus,itwillbeimportanttoexamine whetherthe synovial membrane,causingpain,jointdysfunctionandultimately originates fromchondroidmetaplasiaofconnective tissueofthe implicated indiseasepredisposition(Hopyan etal., 2005). It However, deregulation ofhedgehogsignalinghasrecentlybeen chondromatosis. Theetiologyofthisdiseaseislargely unknown. cartilaginous nodulesareknown inhumansassynovial Pathological alterationsofthejointwithspontaneous formation of canonical Wntpathway isaffected in defects (St-Jacquesetal.,1999)andrecentwork hasshown thatthe differentiation. Interestingly, maintain jointintegrity byactively suppressingchondrocyte catenin. interesting totestwhetherthosephenotypesdependinparton mice arevery similar(Yang etal.,2003).Therefore,itwillbe Surprisingly, thephenotypesof to attenuationof 2003). Part ofthe proliferation andmaturationindependentlyof and prehypertrophicchondrocytes, controlschondrocyte levels. Similarly, Wnt5a,whichisexpressed intheperichondrium prehypertrophic tohypertrophicchondrocytes, but doesnotalter regulates chondrocyte maturationatthetransitionfrom (see Fig.S5Dinthesupplementarymaterial).Wnt4signaling is expressed inprehypertrophicchondrocytes fromE12.5onwards addition, Wnt9asignalingseemstonegatively control regulating thepaceofchondrocyte proliferationandmaturation.In Genes Dev. Interactions between Sox9andbeta-catenincontrol chondrocyte differentiation. Deng, J.M.,Taketo, M.M.,Nakamura,T., Behringer, R. R.etal. Therefore, weproposethefollowing modelfor Wntsand In addition,ourwork shows thatWntsignaling isrequiredto Wnt4 RESEARCH ARTICLE Wnt4 Ihh are positive regulators ofchondrocyte maturation. 18 Ihh , , 1072-1087. levels, probablyviathecanonicalpathway, thereby Wnt5a double mutanthumeri(seeFig.S5Ainthe ␤ Wnt9a expression levels. -catenin pathway isalteredinaffected individuals. ␤ Wnt5a -catenin-mediated activities (Topol etal.,2003). Wnt4 Wnt9a , Wnt5b signals fromtheperichondriumorjointsand mutant micehave noreportedskeletal loss-of-function phenotypemightbedue mutant comparedwithwild-typehumeri and Ihh Wnt4 Wnt6 Wnt5a Wnt4 mutant micealsodisplayjoint Ihh ) bysemi-quantitative and expression was elevated at has beensuggestedtobea mutants (Huetal.,2005). loss- andgain-of-function Ihh (Yang etal., Wnt4, (2004). 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