analysis oftheproteincompositionandorganization ofthese tendons andconsiderableeffort hasbeendirectedtowards the matrix thatconnectstheintercostalmusclestoribs. the monolayeroftendoncellsthatsurroundribsandprovide the origins. Anextreme example ofanchoringtendons canbefoundin or shankmusclesmainlyserve toanchorthemusclesattheirskeletal Conversely, theorigintendonsatproximalendofsamearm vectorial transmissionofforce(BenjaminandRalphs,2000). in theformoftightlypacked parallelcollagenfibers, enablingthe generate acomplex andremarkablyorganized extracellular structure perform theirbiomechanicalroles,theseforce-transmittingtendons serve totransmitmuscleforcespecific skeletal positions.To tendons arethelongchord-like tendonsofthelimbsandtail,which much attentioninstructuralandanatomicalstudies.Themostvisible tissues demonstrateconsiderablediversity, whichhasnotreceived fulfill theirrolesthroughoutthemusculoskeletal system,tendon architecture andthebiomechanicsofmusculoskeletal system.To muscle contraction.Thetendonsarethuscrucialforboththe tendons, whichtransmittotheskeleton theforcegeneratedduring mechanical units;musculartissue,whichgeneratesforce;and skeletal tissue,whichprovides structuralsupportanddefines the The musculoskeletal systemconsistsofthreemajorcomponents: INTRODUCTION persisted in condensation oftendonprogenitorstogiverisedistincttendonswasfirstdetectedatembryonicday(E)13.5.Inthe Accepted 15May 2007 3 2 1 KEY WORDS:Scleraxis,Tendon, Connectivetissue,Mousemutant to intermixingoftenocytesandendotenoncells.Thephenotype poorly organizedtendons.Tendon progenitorsappearednormalin dramatic failureofprogenitordifferentiation resultinginthelossofsegmentsorcompletetendons,toformationsmall mutants. Theforce-transmittingtendonsofthelimbsandtailvariedinseveritytowhichtheywereaffected, rangingfrom determine Scxfunction,weproducedamutantnullallele. The scleraxis( and RonenSchweitzer Nicholas D.Murchison anchoring tendons distinguishes force-transmittingtendonsfrommuscle- Regulation oftendondifferentiation byscleraxis (2007)doi:10.1242/dev.001933 Development 134,2697-2708 at Dallas,TX75390,USA. *Author forcorrespondence (e-mail:[email protected]) Oregon HealthandScienceUniversity, Portland,OR97239,USA. which ismainlytoanchormusclestheskeleton,werelessaffected andremainedfunctional,enablingtheviabilityof the differentiation ofallforce-transmittingandintermusculartendonswasdisrupted,othercategoriestendons,thefunctio manifested inadrasticallylimiteduseofallpawsandbackmusclescompleteinabilitytomovethetail.Interestingly, represents thefirstmolecularinsightintoimportantprocessoftendondifferentiation. Department ofMolecularBiology, UniversityofTexas MedicalCenter Southwestern Department ofGenetics,Harvard MedicalSchool,Boston,MA02115, USA. Shriners HospitalforChildren, Research Division,Portland,OR97239,USA. Tendon studieshave focusedmostlyonforce-transmitting Scx Scx –/– ) ,encodingabHLHtranscriptionfactor, isexpressedintheprogenitorsandcellsofalltendontissues.To mutants, wefoundareducedandlessorganizedtendonmatrixdisorganizationatthecellularlevelthatled 4 Department ofCellandDevelopmental Biology, 1 1,4, , BrianA.Price * 1 , DavidA.Conner Scx –/– mice wereviable,butshowedseveretendondefects,which 2 Scx Scx ( tendon progenitors, Tabin, 2004;Schweitzeretal.,2001).Asthefirst robust marker for stage ofspecification ofthislineage(Brentetal.,2003;Brentand tendon/ligament lineageandappearstobeinducedattheearliest Schweitzer etal.,2001).Scxisahighlyspecific marker ofthe progenitors, anddifferentiated cells(Cserjesietal.,1995; scleraxis (Scx)isadistinctmarker fortendonand ligament differentiation andpatterning. tendon, todate,thereisnodescriptionofmutationsaffecting tendon obvious importanceofthestructuralelementsthatmake upthe (Mao andBristow, 2001).Althoughthesestudieshighlightthe Ehler-Danlos syndrome,inwhichthecollagenIgene ismutated in patientswithconditionsaffecting matrixmolecules,suchasthe fibers inthetendons.Tendon disruptionshave alsobeendescribed Young, 2002)resultinirregularities inthediameterofcollagen proteoglycan genesdecorin( 2005). For example, mutationsinthesmallleucine-rich result indiscretetendonphenotypes(reviewed inTozer andDuprez, attained viatheanalysisofmiceharboringgeneticmutationsthat deposition. tendon specification, differentiation, patterningormatrix littleiscurrentlyknown aboutthemoleculesregulating and ontogeny ofthetendonshasreceived far lessattention, the (Benjamin andRalphs,2000;Zhangetal.,2005).Bycontrast, provides thephysicalpropertiesnecessaryfortendon function tendons andtowards understandinghow thisorganization tendon progenitors, thesyndetome,was lateridentified at the ectoderm (Schweitzeretal.,2001), andasomiticcompartmentof identified atendonprogenitor poolinthelimbsubjacentto to follow theorigin andregulation oftendoncellfate. We , DouglasR.Keene Fmod Several yearsago,wefoundthatthebHLH transcriptionfactor Some insightintotherequirementsfortendonformationhasbeen –/– –/– mutants emphasizesthediversityoftendontissuesand embryos andaphenotyperesultingfromfailureinthe ) (Svensson etal.,1999)andbiglycan( Scx 1 , EricN.Olson expression provided auniqueopportunity Dcn ) (Zhangetal.,2006),fibromodulin RESEARCH ARTICLE 3 , Clifford J.Tabin Bgn ) (Ameye and Scx –/– although and that n of 2697 2

DEVELOPMENT cW Scx5 ScxWT cfo Scx5 Scxflox homozygous foratargeted involved inregulating thetendoncellfate. tobeatranscriptionfactor thatis is none-the-lessalikely candidate in alltendoncellsfromprogenitorstagesthroughdevelopment, Scx (Schweitzer etal.,2001),asabHLHtranscriptionfactor expressed sufficient toalterthenormalprogressionoftendonformation has notbeenelucidated.Although kb genomicclone(Brown et al.,1999).The5 to re-evaluate thephenotypeofasimple . We thereforegeneratedanew loss-of-functionalleleof effect oftheneomycin-resistance(Neo)cassette on neighboring were affected in equally. Whereasallforce-transmittingandintermusculartendons tail. Surprisingly, of thebackmusclesandcompletelossabilitytomove the the forelimbpaw, thelimiteduseofallpaws, reducedfunctionality disruption oftendondifferentiation, manifestingindorsalflexure of generate aproduct thatspansthe5 the wild-typeand The ScxNullPCRextendsacross thesequencesoffirstexon Forwardprimer(5 Scx5 ScxNull PCR Table 1.Genotypingof Hin The vector totarget the Generation of and, intendonsthatpersist morphologies. Scxhowever, hasadditionalrolesintendonformation of tendonprogenitorsthatresultinemergence ofdiscreettendon in alltendons,concurrentwiththecondensationanddifferentiation affected. Thetendondefectswerefirst detectedatorclosetoE13.5 severity), themuscle-anchoringtendonsandligamentswerenot powerful toolintendonstudies,but al., 2005). signaling fromtheadjacentmyotome(Brentetal.,2003;Brent dorsolateral edgeoftheearlysclerotome,arisinginresponsetoFgf 2698 MATERIALS ANDMETHODS the transcriptionalnetwork regulating thetendoncellfate. required fortendonformation,andopensthedoortoanalysisof tendon tissues,itlaysthefoundationforunraveling thegenes differentiation phenotype.Whileemphasizingthediversity of of thetendonswas disrupted. matrix was reducedanddisorganized, andthecellularorganization we foundthatthereported of Scxintendondifferentiation (Brown etal.,1999).Surprisingly, embryogenesis, thusprecludingtheabilitytoaddressapossiblerole embryonic stemcell(ES)clonesassayed,approximately60weretargeted an (FRT)-Neo cassetteappended witha3 and thefirst exon of In aprevious attempttoexplore Scxfunction,embryos Analysis of This isthefirst reportofageneticalterationproducingtendon Homozygous Eco dIII sitewas introducedintoa RI restrictionsiteinthesingleintronof RESEARCH ARTICLE GCGCCCCATCAGCGGTAGTTG add10%DMSO AAGCCCGTGTTCACGCTGTTGG TGGCGGCCCCACTCCAGTCC GCGCCCCATCAGCGGTAGTTG add10%DMSO AAGCCCGTGTTCACGCTGTTGG TGGCGGCCCCACTCCAGTCC AAGCTGAAAGC GAGGGGTAGTGGCACATCAGC CACACGGCCTGGCACAAAAGACC Scx Scx Scx Ј Ј Ј TA ScxExon1rev:40 UTRA: TA ScxExon1rev:40 UTRA: gen: Scxint1rev: 40 flox Scx Scx Scx mutant mice expression hasthusproven tobeanextremely Scx alleles. –/– Scx was flanked byinsertingtheFLPrecombinasetarget –/– mice wereviablebut showed adramatic loss didnotaffect alltendoncategories locus was generatedusingsequencesfrom a12 Scx mutants (althoughtheeffects varied in Scx Scx Ј alleles loxP siteinthe Scx Sac KO KO –/– II restrictionsiteinthe5 phenotype was largely duetoan Ј Ј allele diedintheearlystagesof -3 Scx loxP site(Meyers etal.,1998)into mice, wefoundthatthetendon Ј Reverseprimer(5 ) function intendonformation Scx Ј Scx Scx loxP sequencefollowed bya flox misexpression isnot mutation. allele. Inaheterozygous mouse, Scx gene. Outof180 Scx Ј missing inthe UTR of Scx Scx Scx – allele. TheScxfloxandScxWTPCRsboth makeuseofthesameprimersetand Ј strepavidin-Cy5 fordetection. terminal transferasereactionwithabiotin-16-dUTP, followed by For apoptosis,astandardTUNELprotocolwas followed performingthe alternating sectionsandmerged withtheBrdUsignalinPhotoshop(Adobe). Pharmingen, #550803).TheScxGFPsignalwas capturedfromdirectly null allele(seeFig.1A).For genotypingconditions,seeTable 1. Cre activity infemalesfromthisline(Loganetal.,2002)togeneratethe mated with effects ontendonformation(Brown etal.,1999).We therefore GGCTTGCCT-3 GCCTGAGTTCACACCG-3 the geneusingfollowing primers:Col14for, 5 2001). TheprobeforcollagenXIVwas amplified fromthefourthexon of ScxGFP transcript(Pryceetal.,2007)andtenomodulin(Brandau http://genepath.med.harvard.edu/~cepko/protocol/insituprotocol.pdf. http://genepath.med.harvard.edu/~cepko/protocol/ctlab/ish.ct.htm and protocolsfoundontheTabin laboratorywebpages: using and aMicroPublishercooledCCDcamerafromQImaging. Images werecapturedusingaNikon EclipseE800compoundmicroscope from HaroldErickson,Duke University MedicalCenter, Durham,NC). antibody (1:50;R&DSystems)andtheanti-tenascinC(1:100;gift chain, anti-collagenXIIantibody(Gregory etal.,2001),anti-EphA4 monoclonal antibody(mAb;1:400,Sigma)todetectthemyosinheavy camera (Nikon). For sections,12 captured onanMZFLIIIdissectingmicroscope(Leica)withaDXM1200 and observed directlyorafter2-6hoursfixation in4%PFA. Pictureswere For whole-mountvisualizationofthe Immunohistochemistry andinsituhybridization blot usingthe to the but but might beimportantfortendonformation(Schweitzeretal.,2001), Scx Generation ofan RESULTS tenocytes/section inmutantembryos. of 97FDPtenocytes/section inwild-typeembryosand56FDP were countedintensectionsfromfive different embryosandgave anaverage with DAPI tohighlight thenucleiandhelpidentifyindividual cells.Cells stained withanantibodytobeta-cateninhighlightthecellmembranesand mutant embryosatE18.5(Fig.4B,E,whitearrowhead). Thesectionswere tendons insectionsatthelevel ofthedigitvinculumbothwild-typeand ScxGFP Counting tenocytesinacross sectionoftheFDPtendon al., 2000)toproducetheconditional generated byblastocyst injectionandmatedwithFLPeRfemales(Farley et -3 Scx Ј proliferationwas detectedusingthe BrdUIn-SituDetectionKit(BD Probes includedScx(Brown etal.,1999)thatwas alsousedtodetectthe Whole-mount andsectioninsituhybridizationswereperformed C odtosProduct PCRconditions ) Scx flox/+ expression inearlytendonprogenitorcellssuggestedthatit Scx KO , thisPCRreaction wouldtherefore result intwobandsrepresenting both -positive cellswere countedinflexor digitorumprofoundum(FDP) locus and27retainedbothloxPsites,asdeterminedbySouthern embryos diedbeforeE8.5,precludingtheevaluation ofthe prx1Cre Hin Ј ϫ ϫ ϫ . dIII siteinthe5 9°,1mn 3C i;7°,1mn 451bp (94°C, 1min;63°C,72°C, 1 min) 9°,1mn 3C i;7°,1mn 485bp (94°C, 1min;63°C,72°C, 1 min) 9°,1mn 2C i;7°,1mn 578bp (94°C, 1min;62°C,72°C,min) females, takingadvantage ofthestochasticubiquitous Scx Ј ; Col14rev, 5 -null allele ␮ m cryosectionswerestainedwithMy32 Ј Scx loxP site.Asingle ScxGFP flox allele. Ј -CAGGAGTGTGTTTCAG - signal, tissueswereskinned Scx Development 134(14) flox males, inturn,were Ј Scx -TCCTGATCA - fneo male was Scx -

DEVELOPMENT It isthereforelikely thatthelethalityobserved in biogenesis(Strezoskaetal.,2000;Strezoska2002). of proliferation1( instance. Theentire explanation forthedramaticeffect ofthe Neocassetteinthis organization ofthegenomic locusof (Muller, 1999;Olsonetal.,1996;SartorelliandCaretti,2005).The neighboring geneshave beendemonstrated innumerouscases embryonic lethality. suggesting thattheNeocassetteinthislocusissufficient tocause of the organization ofthe for negativeselectionduringtargetinginembryonicstemcells(seeMaterialsandmethodsdetails).( the ( methods). Homozygous is theNeocassetteembeddedinsingleintronof E10.5 (datanotshown). Theonlydifference betweenthetwo alleles the phenotypeofasimpledeletion the presenceofNeocassette.We thereforedecidedtore-evaluate likely thatthephenotypeobserved inthatstudywas alsoaffected by also introducedaNeocassetteinsimilargenomicposition, itis surprisingly, homozygous Scx Cre recombinaseinthegermlinetogenerateaconditionalallele, Scx Scx allele thatwehave generated. Scx enable selective removal oftheNeocassette(Fig.1A).Founder loxP sites)andusedthe the first exon, whichincludesmostoftheopen readingframe,with decided togenerateaconditionalalleleinthe Scleraxis isessentialfortendondifferentiation Contrary totheearlylethality of disruption offorce-transmitting tendons embryos atE15.5. Fig. 1.Generationofan could notbedistinguished fromtheirlittermates (datanotshown), the is causedbyaneffect onthetranscriptionlevels and/orsplicingof Bop1 Effects oftheNeoresistancecassetteonexpression of fneo Bop1 –/– flox loxP –/– ), whichistranscribedintheoppositeorientation.( Scx , andanullallele mice survived toterm.Indeed,homozygous mice werematedwithexpressing FLPrecombinase and mice are viableanddisplaysevere (green boxes)and gene. Becausetheoriginal gene, andopenboxesrepresent the5 Bop1 Scx Scx locus. Thetwoexonsofthe ; Fig.1B),ahousekeeping generequiredfor FRT gene islocatedinthethirdintronofblock Scx FRT ,Scx Scx Scx null allele. -Neo cassette(Meyers etal.,1998)to (orange boxes)sites,respectively, are shown.Thebrown boxrepresents theneomycin-resistance (Neo)cassetteused fneo/fneo – flox/flox (Fig. 1AandseeMaterials Scx Scx embryos diedatapproximately KO/KO KO mice wereviable,but, ( A allele (Brown etal.,1999) ) Schematicrepresentation ofallelesgeneratedinthe Scx Scx embryos, homozygous Scx Ј and 3 gene usingthe Scx provides alikely Scx locus (byflanking fneo/fneo Scx Ј gene (A)are locatedwithinthethird intron of a secondgenecalledblockofproliferation 1 C untranslated regions (UTRs).Therecognition sequencesfortheCre andFLPrecombinases, ) –/– Scx Scx embryos embryos in situhybridizationoncross sectionsthrough thedigitsofwild-type(WT)and gene, Scx – of tendonformationin al., 2007).Crossingthistransgeneintothe in strongGFPexpression inalltendonandligamentcells(Pryceet which theexpression ofGFPisregulated by advantage of suggesting thatthedeltoidtuberositymayalsobemissingin formation isdependentontheactivity oftheattachedmuscles– the deltoidtuberosity–alateraloutgrowth ofthehumeruswhose found nosignificant skeletal defects,withtheexception ofaloss impairments couldbecausedbyskeletal malformations,but we Movie 1inthesupplementarymaterial).Inprinciple,some ofthese very difficult toturnover whenplacedlyingontheirback(see a severe impairmentintheuseoftheirbackmusclesandfoundit expression todetectthetendonsin morphology inthemutants.Becausewecouldnotuse that causethesephenotypes,wewanted toexamine tendon unpublished results).To characterizepossibletendondisruptions mice becauseofatendonphenotype(Fig.2B)(E.ZelzerandR.S., on theirwrists(Fig.2A).Furthermore, of theforelimbwas locked inadorsalflexure, andthemicewalked material). Inmutantsexamined immediatelyafterbirth, theautopod severe tendondisruption(seeMovie 1inthesupplementary mutant micewereviable,they displayedphenotypesthat suggesta by insituhybridizationwithan and hencetheiridentitywas verified byPCR(datanotshown) and were completelyimmobile.Inaddition, the cagerailingwithpaws oftheforeorhindlimbsand theirtails not shown). Fambrough, 1984)and by insituhybridizationforcollagenI(data staining forthetendonstructural proteintenascinC(Chiquetand mutants. Thetendonphenotypewas additionallycorroborated by enabled visualizationoftendonsandtendonprogenitorsin the missing in significant lossoflimb tendons;sometendonswerecompletely Using the Scx ScxGFP ScxGFP, –/– mutants, whereas othersappearedsmalland Scx reporter, we indeedfoundasevere disruption a transgenictendonreportermouseline,in Scx locus. Blackboxesrepresent codingregions B –/– ) Representation oftheunique mutants atE18.5.We first observed a Scx probe (Fig.1C).Although Scx RESEARCH ARTICLE Scx –/– Scx mutant embryos,wetook –/– –/– Scx mutants couldnotgrip mutants demonstrated enhancers, resulting Scx –/– background Scx Scx Scx 2699 Scx –/– –/– –/–

DEVELOPMENT the subscapularismuscleatE18.5seeninaventralviewofscapula.( myosin heavychain(MHC).Pinkarrowheads showshort-rangetendons.Yellow arrowheads showlongtendons.( fibrosus. ( direct illumination(G)orbyfluorescence ofthe carpi ulnaris,respectively. ( Yellow arrowheads showtheextensordigitoriumcommunis(EDC).Longandshortpurplearrowheads showtheextensordigitiquinti mouse atpostnatalday(P)3are lockedinadorsalflexure. ( alizarin red. Blackarrowheads pointtothedeltoidtuberosity, whichismissinginthemutant.( detected inthe digits(Fig.2E,F).Even moredramatically, the tendons wereapparentinthezeugopod andonlysomeflexors were more severely thantheextensors; noteven rudimentaryflexor yellow arrowheads). In along eachdigit,finally insertingatthetipofdigit (Fig.2C, close proximityuptothewrist,wherethey separatedandcontinued originated intheEDCmusclesclosetoelbow andextended in of theforelimb,extensor digitoriumcommunis(EDC), F). For example, inwild-typeembryos,themajor extensor tendons rudimentary comparedwiththoseofwild-typelittermates(Fig. 2C- embryo. diaphragm tendonfrom awild-typeandmutantembryo.(Q,R)Expression oftenascinCandcollagenXIIinthediaphragmtendon middle tendonofthediaphragm.(O,P,S,T) ComparisonofthesignalScxGFP(O,P)andinsituhybridization withacollagenI embryos atthelevelofdiaphragm,carrying Fig. 2.Thelongforce-transmitting tendonsandintermuscularare severely disruptedin 2700 mutant littermateswere visualizeddirectly inskinnedtissuesusingthe respectively). In counterparts (Fig.2C,D,long andshortpinkarrowheads, but werestillsignificantly reducedcomparedwiththeirwild-type and theextensor carpiulnaris tendonswerecompleteinthemutant, (Fig. 2D,yellow arrowheads). Bycontrast, theextensor digitiquinti detected inthedigits,but thetendonsdisappearedat thewristlevel RESEARCH ARTICLE I , J , M , N ) Tendons ofthetrunkatE18.5.(I,J)Dorsalviewskinnedtrunks.(M,N)Sagittalsectionsstainedwithanantibodyto Scx –/– Scx mutants, theflexor tendons wereaffected E , F –/– ) Theventralflexortendonsoftheforelimb inwildtype(E)andmutants(F).( embryos, however, theEDCcouldbe ScxGFP ScxGFP reporter (H).Yellow arrowheads showthetailtendons. Pinkarrowheads showtheannulus B reporter andcounterstainedwithanMHCantibody. Yellow arrowheads showthe ) Forelimbs ofP14wild-type(WT)and ScxGFP O-T affected in 2M,N). myosin heavy chain(MHC) tovisualizetheaxialmusculature(Fig. 2I,J) andinsectionscounterstained withanantibodydirectedagainst arrowheads), aswas seenbothinwhole-mountGFPimages(Fig. the longtendonswerecompletelyabsent(Fig.2,yellow possibly somewhat disruptedorreduced(Fig.2,pinkarrowhead), in thetrunk(Fig.2I,J,M,N):whereasshortaxialtendonswere (Fig. 2G,H,yellow arrowheads). Finally, wefoundasimilarscenario and thecellsinthesetendonscouldbeseenby collagen fibers ofthetendonscanbedetectedbydirect illumination loss ofalltailtendons,asvisualizedin2-week-oldmice,when inability ofthemutantstousetheirtailswas theresultofa complete Intermuscular tendons thatwereaffected includedthelateral are tendinoustissuesinterconnecting two musclesegments. ) Frontal sectionsthrough thetrunkofE18.5wild-typeand rngncrpre CL.( transgenic reporter (C-L). Interestingly, anothercategory oftendonsthatwas severely Scx –/– C mutants was the‘intermuscular tendons’,which , D ) Thedorsalextensortendonsoftheforelimb. Scx –/– A mutant micestainedwithAlcianblueand Scx G ) Theforelimbs ofan , H –/– ) Tail tendonsatP14visualizedby mice. K , L ) Theintermusculartendonsof Tendons inwild-typeand Development 134(14) ScxGFP probe (S,T)inthe Scx –/– of amutant andextensor newborn fluorescence Scx –/–

DEVELOPMENT of wild-typeand detected inany ofthesetendonsin DIC opticsandoverlaidwithScxGFPsignalintheannulusfibrosis. (H,I)ScxGFPsignalintheannulusfibrosis athighmagnifi osteoblasts andinthetendinousattachmentlayer(yellowarrowheads) ofwild-typeandmutantembryos.( (A,B) ScxGFPsignal(green) attheintercostal muscleattachments.(C,D)Sectioninsituhybridization(ISH)detectingcollagen electron microscope imagesofamedialsagittalsectionthrough thetailofanE18.5 muscle; Vm,vastusmedialismuscle. along thesectionplane(K)andperpendicularto(L).AF, annulusfibrosus; NP, nucleuspulposus;P, patella; the annulusfibrosus (AF, red arrowheads). (K,L)Theannulusfibrosus atahighermagnification.Thecollagenfibers(purplear whichisflankedlaterallybycartilagecellsanddorsalventraltightlystacked pulposus (NP;theintervertebraldisc), 2O,P, yellow arrowheads). scapula (Fig.2K,L),andthemiddletendonofdiaphragm (data notshown), thesubscapularismusclesatventral sideofthe tendinous stripesintherectusabdominismuscleofabdomen Scleraxis isessentialfortendondifferentiation intermuscular tendons, levels (Fig. 2S,T). expression was also maintained,althoughatdrasticallyreduced diaphragm tendonofmutantembryos(Fig.2Q,R),andcollagen I such astenascinCandcollagenXII,was notaffected inthe the mutants.Indeed,wefoundthatexpression ofmatrixproteins, therefore indicatesthatafunctionalconnective tissuewas formedin which isessentialfornormalbreathing.Theviabilityofmutantmice diaphragm isaflattenedmusclebisectedbybroadmiddletendon, Fig. 3.Short-rangeanchoringtendonsandligamentsare notaffected in type (WT)and significant changeinthedistribution of to bonesisnotdisruptedinthe mutants. Indeedwedidnotdetecta testament tothefact thatthe capacityoftendonstoattachmuscles arrowheads showthepatellarligament;purplearrowheads showtherectus femoristendonandvastusmedialistendon.( Fluorescent antibodysignalswere mergedwiththeISHimageinPhotoshop.Yellow arrowheads showthecruciate ligamentsofthe sections through thetailofwild-typeand Despite thesevere disruptionintheirforce-transmittingand Scx Scx –/– –/– mro tE85sandwt ysnhaycan(MHC;red) antibodytodetectintercostal muscleattachmentstothe ribs. embryos atE18.5stainedwithamyosinheavychain embryos atE18.5.CollagenIwasdetectedbysectionISHfollowedantibodiestoMHC(red) andcollagenII(green). Scx –/– ScxGFP mice wereviableandmobile, a -positive cellscouldnotbe Scx Scx ScxGFP –/– –/– mutant embryos.The embryos atE18.5thatare carryingthe -positive cellsin Scx collagen fibers. We thereforeexamined theintegrity ofligamentsin ligaments inthepatterning,shape,compositionandstructureof their 2001). Thereare,however, distinctdifferences betweentendonsand tendons andalsoexpress patella totherectus femorismuscle,was almost entirelymissingin respectively). Conversely, therectusfemoristendon,connecting shown) andofcollagen I(Fig.3E,F, yellow andblackarrowheads, normal inbothmorphology, andinexpression of patellar ligamentthatconnects thepatellatotibiaappeared embryos, wefoundthatthecruciate ligamentsofthekneeand Scx expression ofcollagenI(Fig.3C,D)intheintercostaltendons a normaldistribution oftendoncells(Fig.3A,B) andnormal muscles totheribs(Fig.3A-D).Asectionthroughshowed not shown) orthetendinouslayerthatanchorsintercostal anchoring tendons,suchastheorigintendonsoflimbmuscles(data Ligaments, whichconnectbonetobone,arecloselyrelated to –/– Scx –/– embryos. –/– mutants. Insagittalsections through thekneeof Scx mice. ScxGFP –/– embryo. (J)Lowmagnificationreveals thenucleus ( A-D reporter. (G)Thecartilageoftailbonesvisualizedby ) Frontal sectionsthrough theribcageofwild- Scx E , from earlystages(Schweitzeretal., F ) Sagittalsectionsthrough theknees RESEARCH ARTICLE layers ofligamentcells G-I cation. ( Rf,rectus femoris Iexpression inrib rowheads) are aligned ) Superficialsagittal ScxGFP J-L knee; black ) Transmission (data not Scx 2701 –/–

DEVELOPMENT of wild-type(WT)and (EDC);white,flexorvinculum(B,E)orthepalmarmetacarpalligament(J).(B-G)Sectionsthr pink, extensordigitoriumcommunis ( tendons (blue).Sectionlevelsare markedbyblacklines:(1)digitlevel,(2)metacarpal-phalangealjoint,(3)midmetacarpal, onset oftheFDPphenotypeoccursatE13.5.( at E13.5inmutantmice.(J,M)Sectionsthrough wild-type(J)and Scx Fig. 4.Theearliesttendonphenotypein 2702 the boxedareas inRandT, respectively. E12.5 andE14.0.(I,L) (D,G). (H,K) magnification inbothwild-typeand mutantembryos(Fig.3H,I). directionality ofcellsinthe AFbecamedistinctatahigher longitudinal sectionthroughthe tail(Fig.3G),andthealternating pattern. TheAFhasadistinct morphologyinasuperficial alternating perpendiculardirectionality togenerateacriss-cross organized inlayerswhichthecellsandcollagenmatrixhave connects adjacentvertebra. TheAFismadeupofelongatedcells organized ligament,theannulus fibrosus (AF)–theligamentthat examined ingreaterdetailthestructuralfeaturesofanotherhighly arrowheads). phenotypic effects ontendonsandligaments(Fig.3E,F, purple positive cells.( Enlargementsoftheboxedareas inNandP, respectively. sectiontohighlightthetendons.(O,Q) Arrowheadsalternate indicate directly proximal tothewrist.BrdU wasdetectedusingaDABstainandtheimageswere overlaidwiththeScxGFPsignalfrom an ( B-M A Schematic representation ofthemajorflexortendonsforelimb: FDPtendons(green), theFDS(red), andthelumbricalmu ) Surprised tofind nophenotypeintheligamentsof knee,we –/– ) Arrowhead assignmentsare: yellow, flexordigitorum profoundus (FDP);red, flexordigitoriumsuperficialis(FDS);blue,lumbr RESEARCH ARTICLE mutants, emphasizingthesharpcontrastbetween Scx R-U in situhybridizationlimbsatE12.5(H)andE14.0(K),showingthat,wild-typemice,overttendonformationoccursbetwe ) TUNELstaininginsectionsatdigitlevel(level1A)ofwild-type(R,S) and ScxGFP Scx –/– embryos atE18.5highlightingtheflexorandextensortendons.Thesectionswere takenatlevels1(B,E),2(C,F)and3 detected byinsituhybridizationwild-type(I)and Scx N-Q –/– ) Cellproliferation intendoncellsofwild-type(N,O)and embryos isdetectedatE13.5,concurrently withtheovertformationoftendons. Scx –/– (M) forelimbs atE13.5aproximal metacarpallevel(level4inA).The used medialsagittalsectionsthroughthetailofanSc To examine theorganization ofthecollagen matrixintheAF, we the arrowhead). along orperpendiculartothe sectionplane(Fig.3K,L,purple matrix, withlayersinwhich the collagenfibers wereoriented magnification revealed theintact organization ofthecollagen inter-vertebral disc(Fig.3J,redarrowheads). Higher tightly packed layersofligamentcellsintheAF, surroundingthe Low-magnification transmissionelectronmicroscopy revealed the ug n nthekidneys (Pryceetal.,2007). Majorstructuraldefects lungs andin the sertolicellsoftestes(Muir etal.,2005),inthebronchiof In additiontoitsexpression inthetendonandligamentlineages, Scx Scx gene isexpressed inanumber ofothertissues,includingin –/– (L) forelimbs atE13.5.TheEDCphenotypeisfirstdetected Scx –/– (T,U) embryosatE14.5.(S,U)Enlargementsof Scx –/– (P,Q) embryosatE14.5insections (4)proximal metacarpal. Development 134(14) tendons withBrdU- ough theautopod immediate ical tendons; scles and x –/– embryo. en

DEVELOPMENT (J,K) and detected byScxGFPfluorescence inawholetailatE18.5(E)andcross sectionatE15.5(I).( tendon phenotypein detection ofexpression ofthe extensor tendonsin tendon activity. We thereforedecidedtoanalyzetheflexor and activity, suchthatdorsalextension isnotcounteractedbyflexor forelimb paw, canbeexplained byaspecific lossofflexor tendon ScxGFP flexors werecompletely absent,replacedbycloudsofunorganized phalangeal joint(Fig.4C,F).However, at metacarpallevels, both sections throughthedigits(Fig. 4B,E)andatthemetacarpal- rudimentary FDStendonswhen comparedwithwild-typeincross autopod. In excellent indicatorsfor theproximal-distalpositionwithin organization ofthesetendons incrosssectionsaretherefore metacarpal level (Fig.4A,red).Themorphology andrelative subsequently fusingtoasingletendonventral totheFDPat second digitaljoint,andbifurcatetowraparoundtheFDPtendons, digitorium superficialis (FDS)tendonsinsertmoreproximally, atthe proximal endofthemetacarpals(Fig.4A,green).Theflexor metacarpals, fusingtoaseeminglycontinuoustendonsheathat the the distaljointofeachdigitandextend alongthedigitsand in Fig.4A.Theflexor digitoriumprofundus(FDP)tendonsinsertat are two majorflexor tendonsintheautopod,shown schematically A majorfeatureofthe embryos were notobserved inthesetissues Scleraxis isessentialfortendondifferentiation progenitors isdisruptedinthelimbofScx The differentiation andcondensationoftendon upon closerexamination infuturestudies. shown), but potentialrolesforScxinthesetissuesmightberevealed Fig. 5.Theonsetofthetailtendonphenotypein with BrdU-positive cells. sectiontohighlightthetendons.(K,M)Enlargements oftheboxedareasimmediate alternate inJandL,respectively. Arrowhea idtp W)ebys (A,B) wild-type (WT)embryos: fibrosus, pinkarrowheads showtailtendonsandwhitearrows showtheinsertionofindividualtendons.(A-D,H)Thedevelopment later stages(C-E,H,I).Therostral endofthetailispointingrightinA,B,F,G andpointingupinC,D,E.(C,E,H,I)Yellow arr at E13.5. -positive cells(Fig.4D,G,yellow arrowheads). Conversely, Scx ( A-I –/– Scx nst yrdzto odtc h c rncit(A,B)andScxGFPtranscript(F,G) intailsatE12.5(A,F)andE13.5(B,G)a ) InsituhybridizationtodetecttheScxtranscript (L,M) embryosatE13.5.BrdU wasdetectedusingaDABstainandtheimageswere overlaidwiththeScxGFPsignalfrom an –/– mutants, wefoundsmallerFDP tendonsandonly Scx Scx –/– –/– Scx embryos mutants indetail.Inwildtypemice,there –/– ScxGFP Scx phenotype, thedorsalflexure ofthe nst yrdzto fwoetis lc rohasso h pitn ftesneoea 1.;(C,D) in situhybridizationofwholetails.Blackarrowheads showthesplittingofsyndetomeatE12.5; . (F,G) Insituhybridizationoftailstodetectthetranscript reporter inskinnedtailsatE18.5(C)andP14(D);(H)cross sectionofatailatE15.5.(E,F,G,I) Thetail Scx –/– mutants (datanot Scx –/– –/– embryos occursduringthetransitionfrom progenitors toorganizedtendons be detected. by E13.5(Fig.4I)and,earlyE14.04K),distincttendonscan sharp transitioninvolving thecondensationofprogenitorsoccurs highly dynamicuptoE12.5(Fig.4H)(Schweitzeretal.,2001).A type mice,thedistribution oftendonprogenitorsinlimbbuds is hra h xesr eedaaial smallin dramatically whereas theextensors were progenitors toform thelongtendonsoflimb. requirement forScxactivity duringthecondensationoftendon mesenchyme (Fig.4M).Both phenotypesindicateaspecific to form,andthetendonprogenitors remainedaslooselyorganized (Fig. 4J,whitearrowhead). Inthemutant,thesecondensationsfailed (Fig. 4J,yellow arrowhead) andthepalmarmetacarpalligament two distinctcondensedlayers (Fig.4J)thatwillgive risetotheFDP in wild-typemice(Fig.4A,level 4),theventral progenitorssplitinto cross sectionsofE13.5forelimbsattheproximalmetacarpallevel phenotype atthemetacarpallevel was alsoinitiatedatthisstage.In EDC phenotypedescribedabove (Fig.2C,D).The dramaticflexor (Fig. 4L,pinkarrowhead), representingthefirst manifestationofthe triangular EDCtendonblastemadidnotforminthemutantatE13.5 distinct EDCtendonsbyE14.0(Fig.4H,I,K,pinkarrowheads). The intoatriangulartendonblastemaatE13.5andthe transformed the wristlevel originatedfromapatch ofprogenitorsthat observed atE13.5.For example, inwildtype,theEDCtendonsat from thatofwildtypeupuntilE12.5,anddistinctdefectswere first arrowheads). Theabsenceofflexor activity in they persistedthroughouttheautopod(Fig.4C,D,F,G, pink in be anunderlyingcauseforthedorsalflexure oftheforelimb paw. To determinewhichstepsoftendonmorphogenesisareaffected Scx –/– mutants, weexamined thelimbsatearlierstages.Inwild- ScxGFP J-M expression in ) Cellproliferation intailtendonsofwild-type ScxGFP reporter. (E,I)Thelatertailphenotype, Scx RESEARCH ARTICLE –/– owheads showtheannulus embryos was notdifferent Scx ds showtendons –/– of tailtendonsin Scx mice maythus nd ScxGFPin –/– embryos, 2703

DEVELOPMENT tendons are alsodisruptedin Overt differentiation andelongationoftail for changesincellproliferationlimbtendonsof composed ofindividual tendonsthat originatedatthesacralmuscles tendon bundles were found toextend throughthetail,andeachwas morphology anddevelopment. Inwild-typemice,fourmassive previously described, wefirst characterizedtheirnormal tail tendons.Becausethedevelopment oftailtendonshasnotbeen A strikingfeatureofthe nature oftendonsin possibility thatcelldeathisresponsibleforthegeneralrudimentary death werenotidentified inotherlimbtendons,rulingoutthe in theFDPofmutantembryos(Fig.4T,U). Interestingly, events ofcell in wild-typetendons(Fig.4R,S),but distinctcelldeathwas observed mesenchyme inmutantmice(Fig.4R,T).Celldeathwas notdetected of E14.5embryosidentified theexpected cell deathininterdigital these sizedifferences. TUNELstainingoflimbsectionsatdigitlevels looked forchangesincellproliferationordeaththatmaycause detected assoonthesetendonswereformedatE14.5.We therefore type counterparts(Fig.2D),andthismutantphenotypecouldbe tendons thatformedinthemutantappearedsmallerthantheirwild- 2704 are incorporatedintothetendonsin tendon condensationdefectinwhichasmallernumberofprogenitors reduced tendonsizeinmutantembryosmightalsobeattributable toa similar tothedramatictendonphenotypesdescribedabove, the concomitantly withovert tendonformation,we hypothesizethat, in mutantembryos.Becausethetendonphenotypesemerged cell deathdonotplayamajorroleindeterminingthesizeoftendons in mutants.Inconclusion,theseresultssuggestthatproliferation and would notbesufficient toaccountfortheobserved tendonphenotype tendon phenotypesuggeststhatamerereductioninproliferation rates ofproliferationinmutanttendons,but theearlydetectionof proliferation andthereforecannotexclude thepossibility ofslower of mutanttendons,wecouldnotfaithfully compareratesofcell proliferation intendons.However, becauseoftherudimentarynature arrowheads), demonstratingthatScxisnotessentialforcell limbs frombothwild-typeandmutantembryos(Fig.4N-Q,red signals intendons,wefoundproliferatingcellsthesametendons Interestingly, inallproximal-distallevels thatshowed positive BrdU In additiontothedramatictendonlossdiscussedabove, otherlimb RESEARCH ARTICLE Scx –/– Scx embryos (datanotshown). Next, welooked –/– phenotype was thecomplete lossof Scx –/– Scx embryos. –/– mutants Scx –/– embryos. affected mostintendons of number oftendonrudiments(Fig.5E). leading tothecomplete lossoftailtendons(Fig. 2H). However, thesetenocytes subsequently undergo celldeath(Fig. 6H), limb, thisessentialfeatureof tenocytes isnotregulated by capacity toorganize atendonstructure,suggestingthat,as inthe The smallnumberoftenocytes thatpersistinthetailretaina condensation andincorporation ofprogenitorsintomaturetendons. to themassive tendonsofthetail(Fig.5D). four radialgroups(Fig.5C,H)thatcoalescedafterbirthtogive rise generated completetendonsandthesewereorganized in arrowhead). Bytheendofembryogenesis,atE18.5,eachvertebrae individual tendoncondensationscouldbeclearlyseen(Fig.5H,pink the tailmuscles(Fig.5B).IncrosssectionatE15.5,tight establishing theindividual tendonsthatconnecteachtailbonewith The dorsalandventral tendonselongatedtothebaseof thetail,thus progenitor populationscondensedandextended rostrally (Fig.5B). (Fig. 5A,blackarrowheads). Subsequently, thesyndetomal progenitor populationsofthetwo dorsalandtwo ventral tendons separated intodorsalandventral domainsgiving risetothe the wild-typetail,weobserved thatthesyndetomeissubsequently intersection betweentwo somites(Brentetal.,2003).Inof 2G,H andFig.5D,whitearrows). each tailbone,providing exquisite controloftailmovement (Fig. at thebaseoftailandsequentiallyinsertedrostralend embryogenesis, thetailsof tendons ofmutantembryos(Fig.5J-M).Attheend of proliferation couldeasilybedetectedinalltherudimentary tail from mutantembryosatthesestages(datanotshown), andcell Similar tolimbtendons,wedidnotdetectcelldeathintailtendons get incorporatedintooneorasmallnumberoftendons(Fig. 5G). mutant embryosandtendoncellsfrommultiplesegments seemedto arrowhead). Consequently, tendonelongationwas very limitedin sparsely distributed tendoncellsinlaterstages(Fig. 5I,pink tendon progenitors(Fig.5G),andtherewas acontinuedpresence of phenotype emerged atE13.5withvery limitedcondensationof disrupted (Fig.5F, blackarrowheads). Asinthelimb,tendon separation ofthedorsalandventral progenitorpopulations werenot In conclusion,intailaswelllimbtendons,thestagethat is In Axial tendonprogenitorsareinducedinthesyndetome,at Scx –/– mutant embryos,theinductionofsyndetomeand Scx Scx –/– embryos containedonlyasmall –/– and images ofsectionswild-type and magnified Scx TEM imagesofwild-typeand mutant digitatE18.5.(B,C,F,G) section ofwild-type(WT)and and Eosin(H&E)stainingofa microfibrils. (A,E)Hematoxylin tenocytes; green arrowhead, cytoplasmic extensionsof bundles; red arrowhead, arrowhead, collagenfiber disrupted. Scx tendon inthedigitsof Fig. 6.TheECMoftheFDP E18.5 amplified profoundus (FDP)tendonsat embryos appearstobethe –/– –/– Scx ϫ flexor digitorum 38,000 (C,G).(B,H)TEM Development 134(14) embryos isalso –/– tail tendonsatE18.5 ϫ ( A-H 4000. ϫ ) Yellow 00(B,F) 7000 Scx .

DEVELOPMENT endotenon signalinthemutanttendon. in wild-type(E)andmutant(G,F)FDPtendon.Noticethecollapseof (green), andanti-tenascinCantibody(red) highlightingtheendotenon image ofacross sectionthrough theFDPtendon.(E-G) signal (green) andanti-EphA4antibody(red) are overlaidonaDIC arrowheads, tenocytes;red arrowheads, theendotenon. (C,D) second digitphalangeofwild-type(WT)and amplified E18.5. (A,B)TEMoftheflexordigitorumprofoundus (FDP)tendon littermate (Fig.4B,EandFig.6A,E).Thesizedifference might embryos was significantly smallercompared with awild-type matrix isdisruptedin Organization oftheendotenonandtendon Scleraxis isessentialfortendondifferentiation Fig. 7.In FDP tendon(Fig.4E,F).Theinthedigitof FDP tendon. a tendonsheathandare intermixedwiththetenocytesin tendon thatdoesformin aspects oftendonmorphogenesis,wefocusedonthemostrobust To investigate thepossibleinvolvement ofScxintheselater packed bundles ofcollagenfibers (BenjaminandRalphs,2000). structured extracellular componentsofthetendons, tightly tenocytes areproperlyorganized they producetheremarkably of tendondifferentiation. Inwild-typeembryos,oncethe We have sofar shown anessentialroleforScxintheearlystages Scx ϫ 550 toemphasizecellularaspectsoftheFDP. Green –/– TEM ( mutant embryos,theendotenoncellsfailtoform A , B ) andantibodystaining( Scx –/– Scx mutants –thedigitsegment ofthe –/– mutants Scx C-G –/– mouse embryosat ) ofsectionsatthe ScxGFP ScxGFP signal Scx –/– affected in absent in2-week-oldmutantmice. rudiments couldbeseeninmutantembryos,they were completely highly enlarged nuclei(Fig.6H).Indeed,althoughtendon seen undergoing apoptosisinthemutant,asevident bytheir 5E andFig.6D,H).Intailsections,thetenocytes could alsobe FDP ofwild-typeE18.5embryos,inSc endotenon cellswereorganized inasheathofstretchedcellsthe arrowhead) (BenjaminandRalphs,2000;Kannus,2000).Whereas tendons, ofasecondexternal layercalledepitenon(Fig. 7A,red making upatendonsheathcalledtheendotenonand,inmajor fibroblastic tenocytes, whicharesurrounded byflattenedcells limb tendons(Fig.7C).Inthemutant, that theephrinreceptorEphA4was expressed intheendotenonof observations inchickembryos(D’SouzaandPatel, 1999),wefound previous disrupted organization oftheendotenon.Inagreementwith (Fig. 7B).Stainingforendotenonmarkers furtherhighlightedthe cells appeareddisorganized andfailed togenerateacontinuouslayer Scx 6F,G). Anothercomponentofthetendonmatrixaffected inthe severely decreasedandappearedlesscomplex inthemutant(Fig. matrix components.Thesecytoplasmic extensions werealso collagen fibers inordertoregulate thelocalizeddepositionof extensions (Fig.6B-D,redarrowheads) thatengulfedbundles of the tenocytes developed acomplex network ofcytoplasmic but notinthewild-type,tendon.Moreover, inwild-typeembryos, (Fig. 6B,F),andunoccupiedspaceswerecommoninthemutant, detected inthemutant,amountofmatrixwas greatlyreduced arrowheads). Althoughbundles ofcollagenfibers couldbe the parallelcollagenfibers incrosssection(Fig.6B,F, yellow transmission electronmicroscope(TEM)images,whichshowed matrix contentwas apparentinintermediate-magnification that matrixdepositionisprobablyalsoaffected. Thereduced be morecompactinhistologicalstaining(Fig.6A,E),indicating methods). Moreover, thecellsinmutanttendonsappearedto cells foundinthewild-typeFDPtendons(seeMaterialsand mutant FDPtendonshadapproximately60%ofthenumber detected inthesetendons(Fig.4R-U),and,indeed,wefoundthat result fromlower cellnumberscausedbythe deaththatwe highly rudimentarytailtendonsof of cytoplasmic extensions was seeneven moredramaticallyinthe green arrowhead). Thedecreaseincollagendepositionandloss microfibrils inmutanttendonswerehighlydisorganized (Fig.6G, Normally foundindiscretebundles inthewild-typetendon, The cellularorganization ofthetendonsalsoappearedtobe –/– mutants werethemicrofibrils (Fig.6C,greenarrowhead). Scx –/– mutants. Inwildtype,tendonsarecomposedof collagen I(C,F)genes. the collagenXIV(A,D),tenomodulin(B,E)and wild-type (WT)littermate( an E16.5 hybridization onsectionsfrom theforelimb of Fig. 8.Genesregulated byScx. Scx –/– RESEARCH ARTICLE mutant embryo( Scx EphA4 x –/– –/– embryos, theendotenon mutants atE18.5(Fig. -expressing cellswere A-C ) usingprobes for D-F In situ ) anda 2705

DEVELOPMENT Molecular characterizationofthe earlier stagesoftheirdevelopment. Hand1 hybridization (datanotshown). Furthermore,neitherparaxisnor expression forany ofthesegeneswhenexamined byinsitu Fitch, 1997;Firulli,2003).We didnotdetectadistincttendon bHLH transcriptionfactors isalsohighlyrelatedtoScx(Atchley and more-distant subfamily members.ThesmallHandsubfamily of 1995), istheclosestrelative toScxandthe twistgenesrepresent known asTcf15–MouseGenomeInformatics)(Burgess etal., (Atchley andFitch,1997).Within thissubfamily, paraxis(also Twist subfamily ofthebHLHtranscriptionfactors the small redundancy withother, relatedtranscriptionfactors. Scxbelongsto We wanted totest whetherthiscouldbeexplained bypartial tendon progenitors,Scxisnotnecessaryfortenocyte specification. still forminthemutantssuggeststhat,despiteitsearlyexpression in loss ofScxactivity. sufficient toexplain thephenotypiceffects thatweobserve uponthe properties, itisunlikely thatthemodulationofthesethreegenesis organized intheendotenon(Fig.7E),but, in thematrixproteintenascinCwas exclusively digit flexors, regular organization oftheendotenon(Fig.7D).Similarly, inthe intermixed withthetenocytes, emphasizingadisruptionofthe 2706 expression in slight but reproducibledecreaseinthelevels ofcollagenIgene demonstrated inchick(Shukunamietal.,2006).We alsodetecteda tenomodulin geneexpression by mutants (Docheva etal.,2005).Interestingly, theinductionof proliferation defecthasbeendetectedintendonsoftenomodulin is asingletransmembraneprotein(Brandauetal.,2001),andmild collagen fibers (Gelseetal.,2003;Young etal.,2002).Tenomodulin associated collageninvolved intheregulation ofthediameter (Fig. 8A,B,D,Eanddatanotshown). CollagenXIVisafascicle- and of thelimbbud [e.g. described asbeingspecifically expressed intendons withinthecontext fibromodulin, lumicananddecorin(Ezuraetal.,2000)]orgenes van derRest,1987);tenascinC(ChiquetandFambrough, 1984); (Walchli etal.,1994;Young etal.,2000);collagenXII(Dubletand tenomodulin (Brandauetal.,2001);collagenIandXIV of genesknown tobeexpressed specifically inalltendons[e.g. function intendonformation,wethereforeexamined theexpression identify genesregulated byScxthatcouldberesponsiblefor formation, affecting bothtendondifferentiation andorganization. To We have foundthatScxhasmultipleessentialrolesintendon phenotype loss ofexpression in endotenon islostin 7F,G), againdemonstratingthatthestructuralintegrity ofthe tenascin Cproteinwas fullyintermixed withtheFDP tenocytes (Fig. determined, itisalsopossiblethatthesecellsmayexpress However, becausethelineageofendotenoncellshasnotbeen tenocytes, suggestingthattheeffect onthesheathisindirect. tendon formation, wecreatedatargeted null alleleofthe 2001; Tozer andDuprez,2005).To determinetheroleofScxin persistent marker known forthetendonlineage(Schweitzer etal., Scleraxis, abHLHtranscription factor, istheearliestandmost DISCUSSION The fact thattendonprogenitorsarepresentandmany tendons It isimportanttonotethat Fgf18 RESEARCH ARTICLE or Hand2 (Liu etal.,2002)].Ofthese,onlytwo showed acomplete Scx expression was upregulated in –/– Scx Six2 Scx embryos (Fig.8C,D).Basedontheirknown –/– –/– (Oliver etal.,1995), mutants. mutants: collagenXIVandtenomodulin Scx Scx is exclusively expressed in misexpression was recently Scx Eya1 Scx –/– Scx (Xu etal.,1997) –/– tendon –/– mutants. embryos, Scx Scx gene. at categories, inthemutants,mostofmuscles remainedattachedto between thesetwo tendon groups, itisimportanttonotethat,inboth Homozygous generated bymusclecontraction. function ismostlytoanchormusclesanddissipatetheforce was nodiscernibledefectinothercategories of tendonswhose musculoskeletal forceinthelimbs,tailandtrunk.Surprisingly, there tendons andoftheresponsiblefortransmitting in tendondifferentiation resultinginasevere lossofintermuscular tendons. Themoststrikingphenotypein incorporation oftendonprogenitorsintohistologicallydistinct phenotypes werefirst detectedatE13.5,concomitantwiththe affected in tendons, and,noticeably, allthetendonsinthesecategories are differentiation offorce-transmitting tendonsandofintermuscular categories. processes thatconferthecellularidentitiesofdifferent tendon the first directdemonstrationofdifferences inthemolecular rather isspecific toadistinctsubsetoftendontissues, thusproviding seem toberequiredforthatcoreidentityofthetendontissues, but et al.,2001).Itisthereforefascinating thatScxfunction doesnot well-defined cellandtissueidentity(Brentetal.,2003; Schweitzer tendon tissues,reinforcingthenotionthatthey allrepresentasingle maintaining theintegrity ofthemusclesinquestion. as themiddletendonofdiaphragmandprobablyplayarole in tendons, theintermuscularconnectmusclesegments such force generatedbythemuscles.Finally, adivergent groupof muscles orskeletal elements,reflectingtheneedtodissipate Anchoring tendonsfrequentlyexhibit broadcontactareas withthe muscles throughamonolayeroftendoncellsthatsurroundtheribs. trunk tendons,notablyincludingtheattachmentsofintercostal tendons includetheoriginoflimbmusclesandmany ofthe anchor musclestorespective skeletal attachments.Anchoring complemented bytendonswhosephysiologicalroleismostlyto considerable distances.However, theforce-transmittingtendonsare a remarkablespecializationtosupporttransmissionofforceover studies focusontherobust tendonsofthelimbsandtail,whichshow muscle toboneaccountsforahighlydiverse setoftissues.Most The definition oftendonsastheconnective tissuesthatconnect intermuscular tendons formation offorce-transmitting and Scx isrequired forthedifferentiation and model forthiscondition. arthrogryposis, suggestingthatmild reminiscent ofacongenitalhumanmalformationknown as the muscularskeletal system.Thesephenotypicfeaturesare physiological flexures andthecompletelossofsomefunctions of the limbswas disruptedinmutantmice,resultingnon- formation, thenormalbalancebetweenextensor andflexor forcesin interplay betweentenocytes andendotenoncells. cytoplasmic extensions thatsupportmatrixorganization, andinthe of structuralmatrixcomponents,intheproductionextended a roleinlateraspectsoftendonformation,includingthesecretion definition oftissuesastendons.Moreover, weshow thatScxplays highlights themolecularandstructuraldiversity withinthebroad distinct tendons.Thevariable effect ondifferent tendoncategories failure oftendonprogenitorstocondenseintomorphologically Tendon progenitorswereinducedin Our analysissuggeststhatScxfunction isrequiredforthenormal When wefirst cloned As aresultofdifferential severity inthedefectstendon Scx Scx –/– –/– mutants. Althoughthereisnoobvious similarity mice wereviablebut showed adramaticdefect Scx , wenoticedthatitwas expressed inall Scx Scx mutations mayserve asa Scx –/– Development 134(14) embryos andtendon –/– embryos was the

DEVELOPMENT on Scxfunction,noristheexpression of induction andmaintenanceofthetendoncellfate arenotdependent molecular processesinvolved intendondifferentiation. Second,the identity andfunctionofScxtargets willthereforehelpunravel the tendon progenitorsinmaturetendons.Futurediscovery ofthe is essentialfortendondifferentiation andfortheincorporationof pivotal observations regarding thetendontranscriptome.First,Scx addressed previously. Thisstudyprovides forthefirst timetwo The transcriptionalregulation ofthetendoncellfate hasnotbeen Transcriptional regulation oftendoncellfate them maysecondarilydisrupttheother. structure aremutuallydependentsothataprimaryeffect ononeof both directlyregulated byScxorwhethertheseaspects oftendon the organization ofthematrixandoncellularmorphologyare be interestingtodetermineinfuturestudieswhethertheeffects on regulate thelocalsecretionandsynthesisofcollagenfibers. Itwill network ofcytoplasmic processesthatenablethetenocytes to decrease inthenumberoftenocytes presentandapartial lossofthe fibers andintheirorganization. Concomitantly, weobserved a matrix, manifestinginadramaticdecreasethenumberofcollagen displayed avariety ofdefectsinitsabilitytoproducethetendon Ameye, L.andYoung, M.F. References http://dev.biologists.org/cgi/content/full/134/14/2697/DC1 Supplementary materialforthisarticle isavailableat Supplementary material NIH grantPO1DK56246toC.J.T. part bystartupfundsfrom the ShrinersHospitalsforChildren toR.S.,andby Riordan andSpencerWatson fortechnicalhelp. Theresearch wassupportedin The authorsthankAva Brent forcriticalreading ofthemanuscriptandTim robust tendonin genesis (BenjaminandRalphs,2000;Kannus,2000).Themost secretion andorganization ofthesematrixstructuresduringtendon molecular processesthatenablethetenocytes tocoordinatethe the musclesandskeleton. Little,however, isknown aboutthe organized bundles ofcollagenfibers thattransmittheforcesbetween The functionalcomponentsofthematuretendonarehighly extracellular matrixorganizationintendons Scx affects bothcellulardifferentiation and smaller rudimentarytendons. progenitor condensationandpartialresultingin might representthedifference betweenthecomplete failure of discrete tendons.Consequently, thedifference inphenotypicseverity likely toberelatedtheincorporationoftendonprogenitorsinto overtly distincttendons,suggestingthatthescleraxisfunctionis detected inthetransitionfromtendonprogenitorstocondensedand transmitting tendonsinmutants,allcases,thephenotypewas first considerable variability observed inthephenotypesofforce- of tendonstomediatetissueattachments.Moreover, despitethe generation ofthecomplex tendonstructures,but notforthecapacity their targets, emphasizingthatScxfunctioniscrucialforthe Scleraxis isessentialfortendondifferentiation important andoftenneglected tissue. point forfuturestudiesofthetranscriptionalregulation ofthis and thetoolsthatitallowed ustodevelop provide apowerful starting differentiation phenotype.Moreover, theelucidationofScxfunction therefore representkey playersintendonformation. of expression ofthe proteoglycans: novelinvivomodelsforosteoporosis, osteoarthritis,Ehlers- This studyrepresentsthefirst demonstrationof atendon Scx –/– mutants. Transcriptional regulators of the ScxGFP Scx –/– (2002). Micedeficient insmallleucine-rich mutants, thedigitsegment oftheFDP, reporter was maintainedintendoncells Scx itself, because Scx gene may Brent, A.E.andTabin, C.J. Brandau, O.,Meindl,A.,Fassler, R.andAszodi,A. Burgess, R.,Cserjesi,P., Ligon,K.L.andOlson,E.N. Brown, D.,Wagner, D.,Li,X.,Richardson, J.A.andOlson, E.N. Brent, A.E.,Braun,T. andTabin, C.J. Brent, A.E.,Schweitzer, R.andTabin, C.J. Firulli, A.B. Benjamin, M.andRalphs,J.R. Chiquet, M.andFambrough, D.M. Farley, F. W., Soriano,P., Steffen, L.S.andDymecki,M. Atchley, W. R.andFitch,W. M. Docheva, D.,Hunziker, E.B.,Fassler, R.andBrandau,O. D’Souza, D.andPatel,K. Cserjesi, P., Brown, D.,Ligon,K.L.,Lyons, G.E.,Copeland,N.G.,Gilbert,D. Liu, Z., Xu, J., Colvin, J. S. and Ornitz, D.M. Liu, Z.,Xu,J.,Colvin,J.S.andOrnitz, Kannus, P. Gregory, K.E.,Keene,D.R.,Tufa, S.F., Lunstrum,G.P. andMorris,N.P. Gelse, K.,Poschl,E.andAigner, T. 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