Mesoderm Progenitor Cells of Common Origin Contribute to the Head Musculature and the Cardiac Outflow Tract

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et al.,1999;Tzahor2003). (Hacker andGuthrie,1998;MootoosamyDietrich,2002; Noden manipulation oftissuesandsignalingmoleculesinchickembryos Rudnicki etal.,1993;Tajbakhsh etal.,1997),aswellbythe transcription factors inmice(Kelly etal.,2004;Lu2002; and inthehead,asindicatedbygeneticlossofmyogenic controlskeletal muscleformationinthetrunk regulatory pathways and Trainor, 2005).Itappearsthatdifferent intrinsicandextrinsic pharynx thatwilleventually give risetothefacial structures(Noden the branchial(pharyngeal)arches,pairedthickenings around the segmentation. Together withcranialneuralcrestcells,CPMcellsfill paraxial mesoderminthetrunk,CPMlacksany overt signof mesoderm, CPM(Coulyetal.,1992;Noden,1983)].Unlike the mesoderm locatedanteriortothesomites[termedcranialparaxial 1998). et al.,1996;Reshef1998;Stern1995;Tajbakhsh etal., the trunk(Boryckietal.,2000;Munsterberg etal.,1995;Pourquie the lateralplatemesodermhave beenshown toblockmyogenesisin in thetrunk,whereasbonemorphogenicprotein with Sonichedgehog(Shh)fromthenotochord,inducemyogenesis signaling moleculessecretedfromthedorsalneuraltube,together et al.,2001;Buckingham,Pourquie,2001).Notably, Wnt skeletal musclefromsomiteshave beenintensively studied(Bailey the tissuesandsignalingmoleculesthatinduceformationof cartilage, endothelialanddermisprecursors.Duringthepastdecade, somites, whichgive risetotheskeletal musclelineage,aswellto and lateralcompartments.Paraxial mesoderminthetrunk formsthe ofthemesodermintoparaxial embryogenesis istheregionalization A key event intheestablishmentofbodyplanduringvertebrate INTRODUCTION KEY WORDS:Myogenesis,Secondaryheartfield,Cranialparaxialmesoderm,SplanchnicBmp4 as pathological,aspectsofheartandcraniofacialdevelopment. expectedtoprovideinsightsintothenormal,aswel understanding ofmesodermallineagespecificationinthevertebrateheadis play avitalroleincardiogenesis,asnewsourceofcardiacprogenitorsthatpopulatetheoutflowtractvivo differentiation ofskeletalmuscleprecursorsinthesecells.Ourresultsdemonstratethatcellswithinthecranialparaxialme head: applicationofBmp4,bothinvitroandvivo,inducescardiacdifferentiationthecranialparaxialmesodermbloc populations withinthecardiacoutflowtract.WefurthershowthatBmpsignalingaffectsspecificationofmesodermcellsin embryo, wedemonstratethatcellsfromthecranialparaxialmesodermcontributetobothmyocardialandendocardialcell skeletal muscles.Usingfate-mappingstudies,geneexpressionanalyses,andmanipulationofsignalingpathwaysinthechick vivo experimentalsystemsintheavianembryotoexplorehowmesodermprogenitorsheaddifferentiateintobothheartand (i.e. thelateralplatemesodermandparaxialmesoderm,respectively).Inpresentstudy,wehaveemployedbothinvitro During earlyembryogenesis,heartandskeletalmuscleprogenitorcellsarethoughttoderivefromdistinctregionsofthemesod Libbat Tirosh-Finkel, Hadas Elhanany, ArielRinonandEldadTzahor* the headmusculatureandcardiacoutflowtract Mesoderm progenitorcellsofcommonorigincontributeto Development 133,1943-1953(2006)doi:10.1242/dev.02365 Accepted 15March 2006 *Author forcorrespondence (e-mail:[email protected]) 76100, Israel. Department ofBiologicalRegulation,Weizmann InstituteofScience, Rehovot In vertebrates, theheadmusculatureisderived fromtheparaxial ( Bmp) signalsfrom both invitroand invivo, how headmesoderm cellsarespecifiedin established cellular andmolecularmodels aimedatexploring, we myocardial lineage.Accordingly, inthepresentstudy, discovered to considerwhethertheCPMcontributes inany way tothisrecently identification ofasecondaryheart fieldinvertebrate embryosledus cardiogenesis intheadjacentCPM (TzahorandLassar, 2001).The signals fromthedorsalneural tube (e.g.Wnt1andWnt3a)block remain elusive (Abu-Issa etal., 2004). secondary/anterior heartfieldanditsexact anatomicallocation 2002; OlsonandSchneider, 2003), thenatureof understanding ofthedevelopment oftheprimary heartfield(Kirby, [SHF (Buckinghametal.,2005)].Incontrasttoourrelatively broad termedthesecondheartfield now from mesodermprogenitors, arterial poleoftheheartdevelops bytheaddition ofcellsderived studiessuggestthatthe the primaryheartfield;however, these cardiacmyocytes arisefrom all tube. Itwas originallythoughtthat anterior poleoftheheartfollowing theformationoflinearheart formationofthe (albeit obscure)ofthemesodermcontribute tothe parts intheheadoriginatingvarious 2004) embryosthatcells al., et al.,2003;Dodou2004;Kelly etal.,2001;Zaffran et andmouse(Cai chick (Mjaatvedt etal.,2001;Waldo etal.,2001) embryo, beneaththefloorofpharynx.Ithasbeenshown inboth splanchnic mesoderm,SpM)islocatedontheventral sideofthe Mercola, 2001;TzahorandLassar, 2001). Foley andMercola,2005;Marvinetal.,2001;Schneider stages(BrottandSokol, 2005; cardiac differentiation duringthese contrast, membersofthecanonicalWntsignalingpathway canblock Sugi, 2000;Schlangeetal.,Schultheiss1997).By differentiation duringearlystagesofheartformation(Loughand and fibroblastgrowth factor (Fgf)actaspotent inducersofcardiac endocardium oftheheart.Inboth called theprimaryheartfield)thatlaterformsmyocardiumand cells withinthesplanchnicmesodermformcardiaccrescent(also mesoderm. Thistissuesplitsintosomaticandsplanchniclayers; In aprevious studyinchickembryos,wedemonstratedthat In thedeveloping head,thelateralmesoderm(alsotermed Cardiac progenitorcellsarederived fromthelateralplate Xenopus RESEARCH ARTICLE and chickembryos,Bmp . A deeper soderm ks the 1943 the in erm l

DEVELOPMENT available uponrequest). primers forcardiacandskeletal muscle markers (primersequencesare of randomprimers,followed byaPCRamplificationusingdifferent setsof treated totalRNA, usingaM-MLV reverse transcriptase-mediatedextension Versagene CellKit(GentraSystems).cDNA was synthesizedfromDNase- (R&D Systems)was addedtothemedium. RNA was harvested usinga To blockBmpsignaling,500ng/mlofrecombinantFc-Nogginprotein 293 cellsstablyexpressing humanBMP4-HA,wereaddedtotheexplants. well plates.ControlorBMP4-conditionedmedium,harvested fromHEK- medium, chickembryoextract 2.5%,penicillin/streptomycin0.5%)infour- were culturedinacollagendropcovered withmedium(10%FCSin including theendodermofpharynx,weredissectedseparately. Explants cells) plusendodermfromtheroofofpharynx,andSpMexplants, surface ectoderm (alongwithsomecranialneuralcrest explants comprising the tubular heartwas firstremoved fromstage10chickembryos.CPM In ordertodistinguishbetweentheCPMandsplanchnicmesoderm(SpM), Explant culture MATERIALS ANDMETHODS and arecontrolledbyBmpsignalinglevels. contribute totheheadmusculatureandOFTaretightlylinked, that thedevelopmental programsofmesodermprogenitorsthat propose progenitors duringvertebrate embryogenesis.We therefore This cellpopulationmayrepresentanadditionalsourceofcardiac myocardium andendocardiumofthecardiacoutflow tract(OFT). reveal, forthefirsttime,thatcellswithinCPMcontribute tothe mesoderm progenitors.Furthermore,ourfate-mapping studies and, atthesametime,blocksskeletal muscledifferentiation inhead cardiac andskeletal musclecellfates: Bmp4promotescardiogenesis the avian embryo.Ourfindingsshow thatBmpsignalsaffect both 1944 followed by Cy3-conjugatedanti-mouseIgG (1:100). incubation with the primaryantibody[MF20(1:10), QCPN(DSHB)], Sections wereblocked with5%goatserum,1%BSAinPBS,priorto Fixed embryoswereembedded inparaffin andsectionedas describedabove. Immunohistochemistry for sectioningandQCPNdetection. site. Theeggs wereincubated for24hours.Operatedembryoswereselected piece ofchickCPMwas removed, andthequailgraftwas implantedatthis were labeledwithDiIforvisualization),dissectedout.Acorresponding Quail graftsoftheCPM,atlevel ofrhombomere1-3(somewhich Quail-chick chimeras fluorescent imagesweretaken. and orleftCPM.Following fixation,brightfield injected intotheright ethanol, followed bydilutionintetraglycol(1:2).Thedyewas pressure- DiI (D282,C7001,MolecularProbes)at5or2.5mg/ml,respectively, in Micropipettes attachedtoamicromanipulatorwerefilledwithDiIor CM- Fate-mapping experiments wereperformed on stage8-10embryos. In-ovo dyeinjection situ hybridizationanalysiswas performed. incubated foranother24hours,embryoswerefixed with4%PFA, andin stage9embryos(Tzahoretal.,2003).Theeggs weresealedand into thenselectedforimplantation 24 hourstoformaggregates, whichwere HEK-293 orHEK-293-BMP4cellsweretransferredtoanagarplatefor Implantation ofcellaggregates Leica microtome. transferred toparaffin. Theembryosweresectionedat10-15 sectioning, fixed embryosweredehydrated(ethanol/xylene),washed, and For paraffin attached toadigitalcamera(DC300F, LeicaMicrosystems). request. ImageswereobtainedusingaLeicaMZ16FA stereomicroscope in situhybridizationprobesandadetailedprotocolareavailable upon labeled antisenseriboprobessynthesizedfromthecDNA. Afulllistofthe Whole-mount insituhybridizationwas performedusingdigoxigenin- In situhybridization RESEARCH ARTICLE ␮ m, usinga ␣ MEM Fgf8 players duringearlycardiacandskeletal muscledevelopment. Both not shown) signalingpathways, whichare known tobemajor the expression ofmemberstheBmp,Fgf(Fig.1C)and Wnt(data skeletal muscleandcardiacdifferentiation programs, weexamined (Fig. 1B). suggesting adegree ofplasticityinthesemesodermalcells invitro the SHF(Fig.2A), whereasthe developing heart,aswellinsurroundingtissuescorrespondingto 15-17 embryos(Fig.2). Guthrie, 1998;Nodenetal.,1999), wefocusedouranalysesonstage canbeobserved at stages14-15(Hacker and myogenesis intheBAs between embryonicstages10-22 (Waldo etal.,2001), andbecause markers (Fig.2A).BecausecellsfromtheSHFmigrateto theOFT demonstrate theexpression ofbothmyogenicandcardiogenic the OFT/secondBA, theheart/thirdBA andtheinflow tract,to were sectionedatfourdifferent levels, thefirstbranchialarch(BA), in whole-mountandsectionedchickembryos(Fig.2).Embryos initiated candidategeneexpression analysesbyinsituhybridization mesoderm precursorsintocardiacandskeletal musclelineages, we specific transcriptionfactors regulating thedifferentiation of To gaininvivo informationonthesignalingmoleculesandtissue- mesoderm specificationinvivo Candidate regulatory molecules forhead crucial roleinthespecificationoftheselineages. in SpMexplants. Moreover, Bmpsignalingpathways mayplaya observed inCPMexplants, whereascardiogenesiscouldbeobserved explant cultureresultsdemonstratethatmyogenesis couldbe correlates withcardiogenesis(Fig.1C).Taken together, these explants thanwithinculturedCPMexplants, afindingthatstrongly and were expressed inbothCPMandSpMexplants. Notably, both myosin heavychain expression of members oftheGatafamily ofzincfingertranscriptionfactors. The expressed thecardiacdifferentiation marker and methods).Atthetimeofdissection,stage10SpMexplants splanchnic mesoderm(SpM)populations(Fig.1,seealsoMaterials differentiation potentialsofthecranialparaxialmesoderm (CPM)and now appliedamoredefinedexplant culturesystemtostudythe as skeletal muscle,differentiation (Tzahoretal.,2003)invitro.We mesoderm cellsundergo cardiac(TzahorandLassar, 2001),aswell We previously observed usingexplant cultureassaysthathead myogenesis intheheadmesoderm,vitro Characterizing thenature ofcardiogenesis and RESULTS Gata6 both CPMandSpMexplants. Inaddition,theexpression of (Fig. 1B).Notably, muscle markers vitro, afindingconsistentwithSHFcells. cardiac lineage,andcanundergo furthercardiacdifferentiation in for threedays(Fig.1B).Thus,SpMexplants arecommittedtothe (Lu etal.,2002),was expressed inSpMexplants thatwerecultured transcription factor involved inheadmuscleformationthemouse levels graduallydecreasedinculture.Interestingly, identified SHFmarker culture (Fig.1B).TheSpMexplants alsoexpressed therecently In ordertoidentifycandidatesignalingmoleculesthatregulate Nkx2.5 In contrasttotheSpM,CPMexplants express theskeletal Bmp4 and and , anearlycardiogenicmarker, was broadlyexpressed inthe Fgf10 were expressed athigherlevels within culturedSpM Isl1 Gata4 , aswellthereceptorsforFgfandBmpligands, was detectedinbothCPMandSpMexplants, MyoD and thestructuralmyocardialmarker Myf5 ( vMHC Islet1 and expression was consistentlydetectedin Myogenin ) was augmentedafterthreedaysin [ Isl1 cardiac myosinheavy chain (Cai etal.,2003)],althoughits after threedaysinculture Development 133(10) Nkx2.5 Capsulin , aswell ventricular , abHLH ( cMHC Gata5 Bmp2 ) ,

DEVELOPMENT pharynx. mesoderm explantsunderwentcardiogenesis ( cardiac markersandsignalingmolecules.Althoughcranialparaxialmesodermexplantsunderwentmyogenesisinculture ( 10 embryos.RNAwasharvestedfrom theexplantseitherimmediately(0)orafter3daysinculture. RT-PCR analysiswasperforme specification of theSHF. Gata6 (Fig.2D,E,D3,E3).Unlike heart OFT(Fig.2D2,E2)and, to alesserextent, tothetubular the OFT(Fig.2C2), the embryonic heartatstage16(Fig.2C,C3)andlower levels in Although wefoundthat compare A4withB4). whereas bothgeneswereobserved intheinflow tract(Fig.2, endoderm, bothofwhichexpress mesoderm was clearlyseenasanepitheliallayerunderneath the (Fig. 2A1,A2).Atthelevel ofthethirdBA, thesplanchnic pharyngeal endoderm(thefloorofthepharynx),andinOFT (which isdirectlyconnectedtotheOFTatstage16),ventral region ofthefirstBA, theventral mesenchymeofthesecondBA removal ofthelinearhearttube;(A3)transversesectionembryo(dottedlineinA2).( paraxial mesoderm(CPM,purple)andsplanchnic(SpM,orange)ofastage10chickembryo:(A1)Ventral view;(A2)follo ada n kltlmusclecellfates Cardiac andskeletal expression analysessuggest thatinchickembryos, the inflow poleof theheart(Fig.2C4,D4,E4).Thus,ourgene bedetectedat pharynx (Fig.2C3,D3,E3).All threegenescould BAs was detectedinthesplanchnicmesodermbeneath and Nkx2.5 (Fig. 2B3).Inthemostposteriorareaofembryonicheart, (Fig. 2B). was expressed onlyinmyocardialcellswithintheembryonicheart Fig. 1.Invitro differentiation potentialof cranial paraxialmesodermandsplanchnicinchickembryos. Nkx2.5 Gata6 , but not , but not Nkx2.5 skont soit ihmmeso h aafamily. is known toassociatewithmembersoftheGata (as wellas cMHC was expressed inthemesodermalcoreatdistal Gata4 Gata5 Nkx2.5 , was expressed inthedorsalmesocardium, , mayplayimportantroles inthe Gata4 and ) atthelevel ofthesecondandthird Nkx2.5 Gata4 Gata6 a expressed throughoutthe was , theexpression of (Fig. 2A3)but not eerestrictedmoreto were n =20/20). T, totalchickRNA;da,dorsalaorta;nc,notochord; nt,neuraltube;oft,outflowtract;ph, Gata5 cMHC Gata5 and h adoei ieg;yet,similarto the cardiogeniclineage; Theexpression of et al.,2005). ‘stem cellcharacteristics’oncardiacmyocytes afterbirth (Laugwitz (Cai etal.,2003).Itwas recentlyshown that this genemightconfer in thespecificationofSHFlineageduringmouseembryogenesis Nkx2.5 third BA (Fig.2F3),andinthedorsalmesocardium2F4).Unlike endoderm (Fig.2F2),inthesplanchnicmesodermatlevel ofthe expressed inthemesenchymeofsecondBA, andinthepharyngeal Isl1 myocardium (Fig.2F2,F4)inamannersimilartotheexpression of 2003; Yuan andSchoenwolf, 2000). within theheadmesoderm,consistentwithprevious reports(Caietal., chick embryosrepresentsapoolofundifferentiated cardiogeniccells expression of together withourdatafromexplant culturesdemonstratingthe the OFT(seeFig.S1insupplementary material).Thesefindings, addition, weobserved the expression of the splanchnicmesodermsurroundinggut(Fig.2G4). In mesodermal coreofthefirstandsecondBAs (Fig.2G1,G2),andin myogenic andcardiogenic lineages. paraxial andsplanchnicmesoderm cellsthatcontribute toboth to proposethat The LIMhomeodomaintranscriptionfactor The skeletal musclemarker in themouse(Caietal.,2003).Thus, , Gata5 Capsulin B and , C Capsulin CPM andSpMexplantswere dissectedfrom stage ) Gata6 in differentiating SpM cells(Fig.1),leadus , , like Isl1 expression was notseenintheheart Isl1 Isl1 Capsulin tsae1 a notrestrictedto at stage16was RESEARCH ARTICLE , isspecificallyexpressed in Capsulin Isl1 n Nkx2.5 =17/20), splanchnic was expressed inthe expression instage16 ( Isl1 A ) Dissectedcranial at thedistalendof d fortheindicated is akey molecule , thisgenewas wing the 1945

DEVELOPMENT 1946 tract; mn,motoneuron; nt,neuraltube;oft, outflowtract;ov, oticvesicle;ph,pharynx; spm,splanchnicmesoderm. tract. ba1,branchialarch 1; ba2,branchialarch 2;da,dorsalaorta;dm,mesocardium; end,endoderm;V, trigerminalga Firstbranchialarch. (A2-J2) Outflowtract/secondbranchialarch. (A3-J3)Epithelial splanchnicmesoderm/third branchial (A1-J1) hybridization fortheindicatedgenes instage16chickembryos.(A1-J4)Transverse sectionsatfourlevels (indicatedbythed Fig. 2.Geneexpression analysis ofcandidateregulatory moleculesthatspecify theheadmesoderm. RESEARCH ARTICLE ( A-J ) Whole-mountinsitu Development 133(10) otted linesinA). nglion; ift,inflow arch. (A4-J4)Inflow

DEVELOPMENT (Fig. 2H1,H2). BAs, aswellinthepharyngealendodermandoticvesicle strongly expressed inthemesodermalcoreofﬁrstandsecond 2004) andheadmuscleformation(Kelly et al., 2004),was transcription factor known toinﬂuenceSHFformation(Xuetal., vertebrate heart(Plageman,JrandYutzey, 2005) shown toplayafundamentalroleinpatterningthe developing musclecellfates Cardiac andskeletal transcription factors (i.e. material). either theBA orOFTregions (seeFig.S2Ainthesupplementary expression patternsof lineage inchickembryos. mice, recently shown tobeinvolved inthisnew myocardiallineagein Although both role inthedevelopment oftheSHFlineageinchickembryo. that, basedontheirspatiotemporalexpression, arelikely toplaya not detectedintheOFT. regulator thatwas shown tonegatively affect theFgf8 supplementary material).Furthermore, and paraxialmesoderm(Fig.2J2,J3;seealsoFig.S2Cinthe stage 16chickembryos,attheboundarybetweensplanchnic detectedinboththeendoderm andectodermof expression was SHF lineageinmice(Brown etal.,2004;Kelly etal.,2001). Members oftheT-box family ofproteinswerepreviously Taken together, ourgeneexpression analysesrevealed agroupof Fgf10 Gata5 and , Gata6 Fgf8 Tbx1 Tbx1 were alsoshown toplayacrucialroleinthe (Xu etal.,2004)and and expression attheseaxiallevels resembledthe Isl1 Capsulin Gata5 Tbx5 , Capsulin , incontrast , Gata6 represent new candidatesforthis and , Isl1 Isl1 Nkx2.5 Mkp3 , (Cai etal.,2003)were was notexpressed in , Tbx1 , aMAPkinase , althoughitwas Tbx1 . and Capsulin , aT-box Fgf8 ) expression of sac andthedorsalmesocardium(Fig.2I1-I4,Fig.6).Notably, the BAs, thepharyngealendoderm,splanchnicmesoderm,aortic and BA region (Fig.2I). Bmp7 material). expressed inasimilarmanner(seeFig.S2Bthesupplementary pathway inthechicklimb(Kawakami etal.,2003),is signaling abolished inSpMexplants following BMP4administration.Time- same explants afterthreedaysinculture. genes three days(Fig.3).BMP4inducedtheexpression of the cardiac fortwo or to bothCPMandSpMexplants thathadbeencultured fates. To testthispossibility, BMP4-conditionedmediumwas added major roleinthedeterminationofcardiacandskeletal musclecell in vivo (Figs1,2)suggeststhatthissignalingpathway mayplaya The expression ofBmpfamily membersbothinSpM explants and in vitro andinvivo skeletal muscledifferentiation inthehead,both Bmp4 inducescardiac geneexpression, andblocks regulation of F4 withI3andI4),suggestingthat mesoderm andinthedorsalmesocardium(Fig.2,compareF3 muscle markers CPM explants. Inparallel,Bmp4blocked expression oftheskeletal We next looked attheexpression of ; seeFig.S1D,Einthesupplementarymaterial)heart Nkx2.5 Isl1 , Bmp4 Gata4 MyoD expression. overlapped withthatof , in culture ( in splanchnicmesodermexplantsafter3days ( CPM explantsafter2and3daysinculture and blockedskeletalmuscledifferentiation in Noggin protein. BMP4inducedcardiogenesis days intheabsence(–)orpresence (+)of ( RT-PCR analysiswasperformedafter3days. or presence (+)ofBMP4-conditionedmedium. cultured intheabsence(–) explants. Explants vitro. cardiogenesis andblocksmyogenesisin Fig. 3.Bmp4signalingpromotes , Gata5 n B Myf5 Bmp4 =15/15), whileNoggininducedmyogenesis TPRaayi fepat cultured for3 ) RT-PCR analysisofexplants ( A , and was expressed intheectodermof Gata6 RT-PCR analysisofCPMandSpM ) n =7/8). Myogenin RESEARCH ARTICLE Bmp4 , vMHC Bmp4 Myf5 may beinvolved inthe that appearedinthese Isl1 (as wellas and expression was also in thesplanchnic Capsulin Bmp2 in the 1947 and

DEVELOPMENT we didnotdetectsignificantinductionof the impactofBMP4on (Fig. 4C1)andsectionedembryos4C2,C3).We furthertested the headmesodermandectoderm,asevidenced inwhole-mount BMP4 stronglyinduced the rightsideofCPMstage9-10chickembryos(Fig.4A,B). (Fig. 4).Pelletsofcellsoverexpressing BMP4wereimplantedinto tested whetherectopicBmp4couldinducecardiogenesisinvivo application ofBMP4toCPMcellspromotescardiogenesis,wenext blocks skeletal muscleformationinCPMexplants. promotes acardiaccellfate inbothCPMandSpMexplants, and Together, ourexplant culturedatademonstratethat,invitro,BMP4 levels remainlow. ifBMP differentiate intomyogenicprogenitors cardiogenic lineage,but notfullydifferentiated, couldtrans- sufficiently low. Alternatively, SpMcellsthatarecommittedtothe levels are progenitors thatcanundergo myogenesisifBMP SpM explants, suggestthatSpMcellsmaycontainafew myogenic These results,alongwiththeexpression of level ofexpression ofendogenous Noggininthesecells(Fig.1C). Noggin werenotsignificantlyaffected, inlinewiththesignificant expressed Noggin(datanotshown). CPMexplants treatedwith elevated (Fig.3B).Similarresultswereobtainedusingvirally increased theexpression of 4E3). We thereforesuggest thatBMP4actsupstreamof the BA mesenchyme,aswellintheectodermandendoderm(Fig. 4E1,E2). Bycontrast,BMP4inducedtheexpression ofthisgenein neuronal expression of implanted intherightsideofCPM(Fig.4E)blocked the that BMP4mightregulate region (Fig.4D1,D2,D3). (data notshown), the skeletal musclemarkers (but notothercardiacmarkers) was reduced,whereasthelevels of SpM explants treatedwithNoggin,theexpression level of Bmp antagonistNoggintoourexplant culturesystem(Fig.3B).In from thesplanchnicmesoderm. levels controlthedifferentiation andfunctionofmyocytes derived of theSpM-derived cardiomyocytes, suggestingthatinvitro administration ofBMP4(orBmp2,datanotshown) inducedbeating culture (Fig.3A).Importantly, inallcaseswenotedthat endogenous express in theabsenceofectopicBMP4(Fig.1;theseexplants also Although SpMexplants expressed cardiacdifferentiation markers and anupregulation ofmyogenicdifferentiation atdaythree. during thefirstday, followed byadownregulation ofthesemarkers (data notshown) revealed the expression ofcardiogenicmarkers course RT-PCR andreal-timePCRanalysesofCPMcellsinculture 1948 Furthermore, BMP4 efficientlyFurthermore, BMP4 blocked Capsulin mayactasarepressor ofheadmuscledifferentiation. role inskeletal muscledifferentiation, theseresultssuggestthat signals areknown toplayaninhibitory 3A). ConsideringthatBMP its effect on expression inthemesenchymeofBA (Fig.4F1,F2,F3),similarto induced lineage specificationinthehead(Figs1,2).BMP4 suggesting itspossibleroleinbothcardiacandskeletal muscle progenitors (Mootoosamy andDietrich,2002)was alsoinhibitedin BA region. Theexpression of 4G1,G2,G3), andreduced Because datafromourinvitroassaysdemonstratedthat The expression patternsof nodrt lc h noeosBPsignals,weappliedthe In ordertoblocktheendogenousBMP Capsulin RESEARCH ARTICLE Isl1 is expressed inbothparaxialandsplanchnicmesoderm, Bmp2 and consistentwiththeeffects invitro(Fig. ofBMP4 Gata5 and expression was inducedbyBMP4intheBA Gata Nkx2.5 Isl1 Bmp4 Tbx1 Isl1 Bmp4 in thecranialnerve gangliaV(Fig. Gata4 gene expression (Fig.4D).Although MyoD expression (Fig.4H1,H2,H3) inthe gene expression ontherightsideof ), ectopicapplicationofBMP4 Myf5 expression. Bmp4cellsthatwere and and and at thesiteof Isl1 Gata4 Gata5 Myogenin Myf5 genes (Fig.2)indicated Myf5 or after two daysin Gata6 expression (Fig. and lateral rectus were slightly Capsulin Isl1 expression Capsulin in vivo. vMHC , BMP in for was comparedwithidentical sectionsofembryosthatwerestained OFT (Fig.6C2).Thesignalresulting fromthelabeledCPMcells of thefirstBA (Fig.6C),theaorticsac/secondBA (Fig.6C1) andthe embryos werefixed and sectionedtransversely (Fig.6A)atthelevel DiI was injectedintothe CPMofstage10embryos.Stage16 and molecularanalysesofsectionedchickembryos(Fig.6).CM- route totheOFT, wecomparedourresultsfromthefate mapping lineages ofthecardiacOFT. second BAs contribute toboththeendocardialandmyocardial demonstrate thatcellsfromtheCPMmigratetofirst and OFT (Fig.5O,P).Collectively, theseDiIlabelingexperiments were foundinboththemyocardialandendocardiallayersof the located intheCPM(Fig.5N).Notably, DiI-labeledCPMcells of thefluorescencesignal.Asexpected, theinjectedDiIwas were immediatelysectionedtransversely tovisualizethelocation than intothesplanchnicmesoderm,someofinjectedembryos (data notshown). from theCPMinOFTcranialneuralcrest-ablatedembryos migrate totheOFT. Furthermore,weobserved DiI-labeledcells second BA (seeFig.S3inthesupplementary material),CPMcells indicating thatatthelevel ofthefirstBA, and,toalesserextent, the embryos, DiI-labeledcellsweredetectedintheOFT(Fig.5K,L), to theonsetofcranialneuralcrestmigration(Fig.5I-L).Inthese we injectedtheDiIatanearlierstageofdevelopment (stage8),prior were derived fromtheCPMandnotcranialneuralcrestcells, al., 1983)].InordertoconfirmthattheDiI-labeledcellsinOFT heart development [thustermedcardiacneuralcrestcells(Kirbyet hindbrain correspondingtorhombomers6-8arerequiredfornormal colleagues demonstratedthatneuralcrestcellsoriginatingfromthe vivo. that cellsfromthecranialparaxialmesodermmigratetoOFTin Table S1inthesupplementarymaterial).Thisnew findingshows detected intheoutercurvature oftheOFT(Fig.5H;summarizedin curvature oftheOFT(Fig.5D),DiIinjectedintorightsidewas from theleftsideofembryoscouldbedetectedininner the aorticsac(Fig.5C,D,G,H).AlthoughDiI-labeledCPMcells DiI-labeled cellsfromtheCPMweredetectedinbothOFTand studies (Hacker andGuthrie,1998).Strikingly, inmostembryos, migrated intothefirstBA (Fig.5B,F)inaccordancewithprevious was subsequentlyfollowed atstages17-22.MostoftheCPMcells rhombomers 1-4(Fig.5A,E).ThelocationoftheincorporatedDiI of theCPMstage9-11embryosinovo attheaxiallevel of was injectedintotheleftorrightsides chick embryo(Figs5,6).DiI to thedeveloping heart,weemployed fate-mapping analysesinthe stages. To test,invivo, ourhypothesisthatCPMcellsarerecruited contribution ofthesecellstothedeveloping heartatthelooping mesoderm invitroandvivo, promptedustoinvestigate the The fact thatBmpsignalinginducescardiogenesisincranialparaxial cardiac outflowtract both themyocardial andendocardial layersofthe Cranial paraxialmesodermcellscontributeto marker genes,andblocked myogenicdifferentiation. proteininducedtheexpressionapplication ofBMP4 ofcardiac crucial roleindeterminingthefate oftheheadmesoderm:ectopic and invivo experimental playsa systemsdemonstratethatBMP4 application(Fig.4G1,G2,G3).Thus,bothinvitro response toBMP4 expected, labeledCPMcellswerelocalized inthemyogeniccoreof To gaininsightsintothe molecularmake-up oftheCPMcellsen In ordertoconfirmthatDiIwas injectedintotheCPMrather Previous fate-mapping studiesinthechick byKirbyand Nkx2.5 , Gata5 , Myf5 , Bmp4 and themyocyte marker MF20.As Development 133(10)

DEVELOPMENT ada n kltlmusclecellfates Cardiac andskeletal expression of effect wasobservedfor tube. ﬁrst, secondbranchialarches; lr, lateralrectus; nt,neural ectoderm oftheﬁrstBA( expression ofthisgenewasfoundinthemesenchymeand the skeletalmarkers ( markers Ectopically-applied Bmp4inducedexpression ofthecardiac muscle markersfollowingBMP4applicationisindicated. (open arrowhead) oftheexpression ofcardiac andskeletal Induction(whitearrowhead) orreduction Bmp4 treated). of theembryosare shown(leftpanel,control; rightpanel, the indicatedgenes.Whole-mountandtransversesections the transfectedcells.( analysis ofHA-taggedBMP4intheconditionedmedium expressing HA-BMP4(dashedcircle). ( ( blocks skeletalmuscledifferentiation invivo. Fig. 4.BMP4inducescardiac geneexpression and n A =5/6). Bycontrast,BMP4cellsblockedtheexpression of Dorsalviewofastage9embryo,implantedwithcells ) Nkx2.5 Isl1 ( n in thetrigeminalganglion(V),anelevated =7/7), Myf5 C-H Isl1 Gata5 n ) Insituhybridizationanalysesfor ( RESEARCH ARTICLE =7/7). as,aorticsac;ba1,ba2, n : althoughBmp4abolishedthe 44 and =4/4) ( n =7/14) and B Tbx1 ) Westernblot ( n Capsulin =3/4). Adual 1949

DEVELOPMENT myocardium; nt,neuraltube;ov, oticvesicle;ph,pharynx. markers. aaa1,aorticarch artery 1;aaa2,aorticarch artery2;as,aorticsac;ba1,ba2, first, secondbranchialarches; end, Myosin heavychainusingMF20antibody. (F-I2)Sectionsthrough embryossubjectedtowhole-mount insituhybridizationwiththe chimera assay. ( field andfluorescence images) (D 1950 view), followingremoval of theheart.Dottedlinesrepresent thethree section levelsdisplayedinthelowerpanels.( Fig. 6.Cellularandmolecularanalysesofcranialparaxialmesoderm enroute totheheart. levels. (D-D2)Transverse sectionsthrough thequail-chickchimera,followedbyimmunostaining withthequail-specificantibody (middle line).( RESEARCH ARTICLE C2-I2 C-I ) Transverse sectionsatthefirstbranchial arch (ba)level(upperdottedlineinA).( ) Transverse sectionsattheoutflow tract(OFT)level(lowerline).(C-C2)SectionsofCM-DiI-labeledcells attheaforemention Ј -D2 Ј ) ϫ 20 fluorescence imagesof the markedarea showninthe ϫ C1-I1 ( A 10 images(D-D2).(E-E2)Immunostaining for iga ofastage16embryo(ventral ) Diagram ) Transverse sectionsattheaorticsac level ta, truncusarteriosus. vesicle; ph,pharynx;rv, rightventricle; curvature; oft,outflowtract;ov, otic notochord; nt,neuraltube;oc,outer curvature; myo,myocardium; nc, aorta; end,endocardium; ic,inner arch; cc,conuscordis; da,dorsal branchial arch; ba2,secondbranchial the paraxialmesoderm(N).ba1,first (M) toverifythelocationofdyein dissected shortlyaftertheDiIinjection section (P)oftheOFT. (M,N)Embryo cross section(O)andtransverse the myocardium andendocardium: (O,P) TheCPMcellscontributetoboth contribute totheoutercurvature (H,L). (D), whereas cellsfrom therightside the innercurvature ofthecardiac OFT the leftsideofCPMcontributeto of dissectedhearts.Cellslabeledon images. (D,H,L)Highermagnification of brightfieldandfluorescence stage 17-18are shownasanoverlay transverse (C,G,K)viewsofembryosat chick embryos.Lateral(B,F,J) or (E,I,M) CPMofstage10(or8,I) injections intotheleft(A)orright of CPMcellsbyDillabelling.DiI outflow tractinvivo. and endocardium ofthecardiac cells contributetothemyocardium Fig. 5.Cranialparaxialmesoderm B ) Diagramofquail-chick endocardium; myo, Development 133(10) (QCPN, indicated ( A-P ϫ ) Tracking 10 bright ed

DEVELOPMENT presumably adoptamyocardialcellidentity( Fig. 6Cwith6H).Cellsthatmigratedthroughtheaorticsac molecular characteristicsofskeletal musclecells( results suggestthatCPMcellsmigrateintotheBAs adopted Gata5 the level oftheaorticsacweredetectedinaregion where between thefirstandsecondBAs (Fig.6A).CM-DiI-labeledcellsat which connectstheaorticarcharteriestoOFT, isconnected caudally (Waldo etal.,2001),suchthatatstage16-18theaorticsac, detected atthelevel ofthefirstBA (Fig.6F,G, Fig.2). ( and invivo (Figs1,2)revealed agroupoftranscriptionfactors specific transcriptionfactors andsignalingmoleculesbothinvitro muscles. Ourdetailedgeneexpression analysesofcandidate tissue- progenitors inthehead,differentiate intobothheartandcraniofacial experimental systemsintheavian embryoto explore how mesoderm In thepresentstudy, weemployed bothinvitroandvivo DISCUSSION cardiac OFT. contribute tothemyocardialandendocardialcomponentsof confirm theobservation thatcranialparaxialmesodermcells and retroviral infection together, ourdyemarkingtechnique,quailchickgraftingprocedure neural crestcontribute totheOFTduringloopingstages.Taken This experiment furtherindicatesthatCPMcellsrather thancranial which alsoexpress myocardium (Fig.6C2,E2;myocardialcellsareMF20positive), DiI-labeled cellscouldbedetectedintheendocardiumand myogenic tocardiogenicprogenitors.Atthelevel oftheOFT, CM- BA (Fig.6I.1)iscorrelatedwiththechangeincellfate from intheaorticsac/second the firstBA level (Fig.6I)anditsexpression compare Fig.6C1,F1andG1).Thelackofexpression ofBmp4at the cardiogenicgenesmentionedabove, only endocardium andmyocardiumofthecardiacOFT(Fig.6D2,D2 6D,D specific antibody)withinthemesenchymeoffirstBA (Fig. existence ofquail-derived CPMcells (recognizedbyaQCPN- embryos (Fig.6B).Sectionsoftheoperatedrevealed the embryos weretransplantedintotheCPMofstage-matchedchick material). CPMgrafts(withthesurface ectoderm)ofstage8quail labeling results(Fig.6B;seealsoTable S2inthesupplementary the firstBA, asindicatedby musclecellfates Cardiac andskeletal al., 2000;Schultheiss etal.,1997;Shi2000). Similarly, Dpp, vertebrate embryos(Jiao etal.,2003;Liu2004;Schlange Bmp signalingmoleculesarerequired forearlyheartformationin head myogenesisduring heartloopingstages Bmp signalinginducescardiogenesis andrepresses vivo (Figs 5,6). cardiogenic progenitorsthatpopulatethecardiacoutflow tractin that cranialparaxialmesodermcellsrepresentasource of both fate-mapping experiments andmolecularanalysesdemonstrate progenitors inthecranialparaxialmesoderm(Figs3,4).Datafrom specifying thecellfates ofbothmyogenicandcardiogenic mesoderm. We alsoprovide evidence thatBmp4playsakey rolein between cardiacandskeletal muscleprecursorsin the head analysesfurtherrevealed aconsiderabledegree ofplasticity These development ofthesecondary/anteriorheart field(SHF)inthechick. spatiotemporal expression, arelikely toplayaroleinthe Gata5 During heartloopingstagesinchickembryos,theOFTshifts We next usedthe quail-chickgraftingtechniquetoverify ourdye Ј , but not ), theaorticsac/secondBA (Fig.6D1,D1 , Gata6 , Myf5 Isl1 Nkx2.5 , wereexpressed (Fig.6F1,G1,H1).These ( see Fig.S4inthesupplementarymaterial) and and Myf5 Capsulin Gata5 in situhybridization(Fig.6H).Of (Fig. 6F2,G2). ) that,basedontheir Nkx2.5 Ј Nkx2.5 ) andwithinthe Myf5 and Nkx2.5 , compare could be Gata5 and Ј ). , a commonprogenitor population. the OFTderive frommultipleoriginswithintheembryo,ratherthan suggestthatendocardial andmyocardialprecursorswithin studies myocardial andendocardialcell populations.Collectively, these 1991). Ourdatademonstratethat CPMcellscanbefoundinboth to angioblaststhatpopulatethe endocardiumoftheOFT(Noden, progenitor cellsoriginatinginthe cephalicmesodermcangive rise Results fromquail-chicktransplantationassaysfurthersuggest that developing myocardium(Lietal.,2003; SatoandYost, 2003). have shown thatcardiacneural crestcellscontribute tothe vasculature (HutsonandKirby, 2003).Studiesinzebrafishembryos cells provide yetanothersourceforsmoothmusclecellsoftheblood base ofthegreatarteries(Waldo etal.,2005).Cardiacneuralcrest these mesodermalcellsprovide smoothmusclecellsthatformthe contribute tothemyocardiumofOFT(Waldo etal.,2001);later, addressed inthisstudy. neural crestonthemigrationofCPMcellsintoOFTwas not cranial neuralcrestcells(Figs5,6),theinfluenceof within theOFTwerederived fromthemesodermandnot myocardial cellfate. AlthoughweconfirmedthatDiI-labeledcells mechanism bywhichcranialparaxialmesodermcellsadopta vertebrate embryos.Taken together, ourdataidentifyanovel heart looping,acriticalstepduringmorphogenesisin as they move toward theOFTcouldaffect theprocessofrightward differences inoriginandthemigratorypathstaken byCPMcells curvature oftheOFT(Fig.5).Thesefindingssuggestthat OFT, whereas thosefromtherightsideweredetectedatouter the leftsideofCPMwerefoundininnercurvature ofthe Figs S3,S4inthesupplementarymaterial).DiI-labeledcellsfrom myocardial andendocardiallayersoftheOFT(Figs5,6;seealso cells thatmigrateprimarilytothefirstBA contribute toboth transplantation experiments, demonstratethatparaxial mesoderm Our fate-mapping experiments usingvitaldyesandourquailchick cardiogenesis The involvementofcranialparaxialmesodermin between apositive feedbackloop neuronal tissue.Thus,thereappearstobe induces Isl1 Bmp4 (aswellasotherBmpandFgffamily members)isatarget of Bmp signalingintheterminaldifferentiation ofcardiac progenitors. beating ofcardiomyocytes invitro,suggestingapossiblerolefor application ofBMP2andBMP4toSpMcellsinducedarhythmic al.,1996;Reshefet1998;Tzahor2003).Ectopic et and invivo (Figs3,4),consistentwithprevious studies(Pourquie CPM, BMP4blocked skeletal myogenesisinthistissue,bothvitro (Fig. 3).Concomitantwiththeinductionofcardiogenesisin blocked cardiogenesis,andinducedmyogenesisinSpMexplants signalsbyNogginpartially (Fig. 4),whereasinhibitionoftheBMP Gata5 BMP4 duringthesestagesinducedrobust expression of with cardiacgeneexpression (Figs1,2).Ectopicapplicationof during heartloopingandtheearlystagesofmyogenesisiscorrelated vitro (Waldo etal.,2001). SHF/OFT myocardium,affects theproliferation of thesecellsin suggestedthatBmp2,whichisexpressed inthe was further dorsal vessel, theequivalent of theheartinflies(Frasch,1995).It the It was previously shown thatSHF cellsinthechickembryo oso-ucinsuisof Loss-of-function studies Here, wedemonstratethattheexpression ofBmpfamily members Drosophila in theSHF(Caietal.,2003).We now demonstratethatBMP4 , Isl1 Isl1 Isl1 and expression intheSHF, whileblockingitsexpression in and ortholog ofBmp,isrequiredfortheformation Capsulin Bmp4 . in theCPMvitro(Fig.3)andvivo Isl1 in micehadpreviously shown that RESEARCH ARTICLE Nkx2.5 1951 ,

DEVELOPMENT programs. paraxial mesoderm, andblockstheskeletalmuscle differentiation Bmp4 bothinvitro andinvivo promotes cardiogenesis inthecranial spatiotemporal expression of Bmp4.Moreover, ectopicapplicationof shift from askeletalmuscle toacardiac cellfateiscorrelated withthe cardiac markers expression of the connects thebranchialarches totheOFT, initiatecardiogenesis (note furthertowards theaorticsac,which upper box).Thosecellsmigrating skeletal musclemarkers (e.g. myoblast precursors,asevidenced bytheexpression ofvarious aortic sac,andintothecardiacOFT. Initially, thesecellsrepresent can migratealongthemyogeniccoreoffirstBA, throughthe demonstrates thatinchickembryos,DiI-labeledcellsfromtheCPM Our resultsaresummarizedinaschematicmodel(Fig.7),which paraxial mesoderminresponse toBmpsignals A modelforthespecificationofcranial development, andfromvarious locations(Abu-Issa etal.,2004). (or heartfields)arerecruitedintotheatdifferent stagesof hypothesis thatcardiacprogenitorpopulationsfrommultiplesources nature ofheartmorphogenesis,ourdataisconsistentwiththe (Waldo etal.,2005;Waldo etal.,2001).Inview ofthedynamic splanchnic mesodermintotheOFT, ashasbeenpreviously shown results donotruleoutacontribution ofcardiacprogenitorsfromthe are consistentwiththesedescriptionsoftheanteriorheartfield.Our mesodermal coreofthepharyngealarches)contribute totheOFT, levels ofthefirstandsecondBAs (whicheventually formthe et al.,2001).Ourresults,whichdemonstratethatCPMcellsatthe OFT, aswellthemesodermalcoreofpharyngealarches(Kelly In mice,theanteriorheartfieldincludesrightventricle andthe mesodermal cellssurroundingtheaorticsac(Mjaatvedt etal.,2001). whereas theanteriorheartfieldisrestrictedtocephalic of thepharynx,thatliescaudaltoOFT(Waldo etal.,2001), SHF includestheepithelialsplanchnicmesoderm,beneathfloor those originatinginthecranialparaxialmesoderm?Inchick, populations thatconstitutetheSHF(Buckinghametal.,2005)and 1952 markers are expressed (e.g. myocardium andendocardium oftheOFT, where different cardiac Nkx2.5 adopt amyogeniclineage( migrate through thefirstbranchialarch (markedbyorange line),first chick embryo. Fig. 7.Amodelforcranialparaxialmesodermspecificationin the What, then,istherelationshipbetweencardiacprogenitor ; middlebox).Asmallerportionof thesecellsmayreach the RESEARCH ARTICLE Our modelproposes thatcellswithintheCPM Myf5 Gata4 Myf5 , Capsulin Gata5, Gata6 and , Capsulin cMHC and ; lowerbox).Thegradual Tbx1 , and Capsulin ; highlightedinthe Tbx1 , ; highlighted Isl1 and Brown, C. B.,Wenning, J.M.,Lu, M. M.,Epstein,D.J.,Meyers,E.N.and Brott, B.K.andSokol,S.Y. with cardiogeniccellsexpressing middle box).CPM-derived cellswithintheOFTareco-localized adopt myocardialcharacteristics( in theupperbox).CPMcellsthatsurroundaorticsacpresumably Borycki, A.,Brown, A.M.andEmerson,C.P., Jr Bailey, P., Holowacz, T. andLassar, A.B. Abu-Issa, R.,Waldo, K.andKirby, M.L. References http://dev.biologists.org/cgi/content/full/133/10/1943/DC1 Supplementary materialforthisarticleisavailableat Supplementary material their helpandsupport. critical reading ofthemanuscript, andmembersofourlaboratoryteamfor thank Andrew Lassar, ElazarZelzer, HerveKempfandStephaneZaffran for incumbent oftheGertrudeandPhilipNollmanCareer DevelopmentChair. We Foundation forBiomedicalResearch, andfrom Ruth&AllenZiegler. E.T. isthe This workwassupportedbyresearch grantstoE.T. from theEstelleFunk the notionofasingle and theanteriorpoleofheartaretightlylinked, consistentwith progenitor populationsthatcontribute toboththeheadmusculature respectively). We proposethatthedevelopmental programsof mesoderm (i.e.thelateralplateandparaxialmesoderm, cells wereoriginallythoughttoderive fromdistinctregions ofthe and Lassar, 2001). Wnt1 andWnt3a)fromtheneuraltube(Tzahoretal.,2003;Tzahor the differentiation oftheCPMcellsisrepressedbysignals(e.g. skeletogenic lineagestake placeatsitesadjacenttotheaxialtissues, differentiation andpatterningofthesomitesintobothmyogenic al., 2003;TzahorandLassar, 2001).Althoughtheinitial et range oflineagesthandoesthetrunkparaxialmesoderm(Tzahor cells intheheadpossessabilitytodifferentiate intoabroader as onthoseofprevious studies,weproposethatparaxial mesoderm unique cardiogenicproperties.Basedonourcurrentresults,aswell 1991), thepresentstudydemonstratesthatthesecellsfurtheradopt angiogenic differentiation (Coulyetal.,1993;Noden, 1983;Noden, mesoderm (Fig.7). progenitorcellpopulationsoriginatinginthehead myogenesis in signaling pathways thatpromotecardiogenesisandsuppress Thus, weproposethatBmp4standsattheapex ofaconsortium cells, andreducesthedifferentiation ofskeletal muscleprecursors. ectopic applicationofBmp4inducescardiacdifferentiation inCPM in theheadregion isconsistentwithitspositive roleincardiogenesis: the dorsoventral axis,incorrelationwithBmpsignalinglevels. mesoderm cellssuggestthatthesearegraduallyspecifiedalong box). Ourcellularandmolecularanalysesofthecranialparaxial major focusofourfutureresearchefforts. impact andfunctionofthesecellswithintheOFTwillconstitutea awaits detailedmolecularandcellularanalyses.Inaddition,the lineages, orwhetherthiscellpopulationisheterogeneousinnature mesoderm thatdifferentiates intobothcardiacandskeletal muscle Whether thereisacommonprogenitorcellinthecranialparaxial this fieldmayleadtobothcardiacandcraniofacial abnormalities. (Hutson andKirby, 2003).Accordingly, insultstoany componentof Dev. Biol. Dev. Cell regulator Dbf4isaninhibitorofWntsignalingrequired forheartdevelopment. 127 pathways convergetocontrol Gligeneactivationinaviansomites. stem cellsintheembryoandadult. During earlyembryogenesis,heartandskeletal muscleprogenitor CPMcellscanundergo myogenic,skeletogenic or Although The expression ofBmpfamily members–inparticular, Bmp4– , 2075-2087. 8 272 , 703-715. , 281-285. (2005). Avertebratehomologofthecell cycle cardiocraniofacial Nkx2.5 Nkx2.5 Curr. Opin.CellBiol. (2004). Heartfields:one,twoormore? (2001). Theoriginofskeletalmuscle , , Gata5 (2000). ShhandWntsignaling Gata4 Development 133(10) morphogenetic field , Isl1 and 13 , 679-689. and cMHC Development Capsulin (lower ;

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