aclCoumailleau Pascal *Author forcorrespondence (e-mail:[email protected]) Université P. etM.Curie,UMR S787,Paris75013,France. oiedrvdclsepesn h -E receptor, a somite-derived cellsexpressing theC-MET located inlimbmesenchymeisabletodirectthemigrationof the 1995; Kardon,1998).TheligandHGF(hepatocyte growth factor) 1977; Christetal.,Lance-Jones,1988;HayashiandOzawa, mesenchyme andnotbythemyogeniccells(Chevallier etal., positional informationformusclepatterningiscarriedbythelimb Robson etal.,1994). between E6/HH28andE8/HH32(ShellswellWolpert, 1977; forearm, thisprocesshasbeendescribedtooccurin48hours, muscles, aphenomenoncalledmusclesplitting.Inthechickwing separate intothevarious musclemassesproducingtheindividual (3) Lastly, thedorsalandventral musclemasseswillprogressively MYF5, MYOD andmyogenin,thendifferentiate intomyotubes. program bysuccessively expressing thebHLHtranscriptionfactors stage (E3/HH20),themuscleprecursorcellsactivate themyogenic dorsal andventral masses(SchrammandSolursh,1990). Atthis reached thelimbmesenchyme,muscleprecursorcellsorganize into (Chevallier etal.,1978;Solursh1987).(2)Oncethey have chick wingsasoccurringbetweenE2(HH15)andE3(HH20) differentiate. (1)Themigrationprocesshasbeendescribedinthe undergo threemainstepsofspatialorganization andconcomitantly Schienda etal.,2006).Somite-derived muscleprecursorcells part ofthesomites(Chevallier etal.,1977;Christ Myogenic cellsofvertebrate limbsoriginatefromthe ventrolateral during vertebrate limbdevelopment arenotfullyunderstood. The mechanismscontrollingtheorganization ofmusclesinspace INTRODUCTION Accepted 15May 2007 1 KEY WORDS:Chick,Limb,Muscle,Vessel, PDGF, VEGF, CollagenI,MyoD process. matrix andattractsconnectivetissuecellstothefuturesplittingsite,allowingseparationofmusclemassesduring s connective tissue-promotingfunctionsofbloodvessels.Finally, weproposethatPDGFBpromotestheproductionofextracellular secreted factorPDGFB(expressedinendothelialcells)asaputativemolecularcandidatemediatingthemuscle-inhibitingand correlation betweenbloodvesselsandmusclesuggeststhatareinvolvedinthesplittingprocess.We alsoidenti VEGFR1 leadstomusclefusion.Theendogenouslocationofendothelialcellsinthefuturecleavagezonesandinverse muscle formation,whilepromotingconnectivetissue.Conversely, localinhibitionofvesselformationusingasolubleform therefore perturbedvascularassemblyinchicklimbsbyoverexpressingVEGFA anddemonstratedthatectopicbloodvesselsinhibi endothelial cellsaredetectedinthefuturezonesofmusclecleavage,delineatingcleavagepatternmasses.We detected inlimbregionsbeforemusclecellsandcanorganizethemselvesspacetheabsenceofmuscles.Inchicklimbs, investigate therelationshipbetweenvasculatureandmusclesduringlimbbuddevelopment.We showthatendothelialcellsar Muscle formationandvascularassemblyduringembryonicdevelopmentareusuallyconsideredseparately. Inthispaper, we Samuel Tozer chick limbdevelopment Involvement ofvesselsandPDGFBinmusclesplittingduring (2007)doi:10.1242/dev.02867 Development 134,2579-2591 Saint-Bernard, Bât.C,6 Biologie duDéveloppement,CNRS,UMR 7622,UniversitéP. etM.Curie,9Quai Classical experiments inavian embryoshave shown that 1 , Marie-AngeBonnin e E, Case24,75252ParisCedex05,France. 1 and DelphineDuprez 1 , Frédéric Relaix 1, * 2 INSERM U787, 2 , SandrineDiSavino transcriptional target of Brohmann, 2000). Itisalsoworth noting thatthe classified asinvolved inmyoblastmigration (Birchmeierand 2000; Brohmannetal.,2000). However, thisgeneisusually muscles areunaffected (SchäferandBraun,1999;Grossetal., absence ofdorsalmusclesin forelimbs,whereastheventral muscle-patterning gene,because 1999). The display anoverall reductionofthemusclemasses (Mankoo etal., muscles inlimbs(Mankoo etal.,1999).However, thesemutants abnormal splittingofcertainmusclesoreliminationspecific Mox2 genes arecandidatesforinvolvement inlimbmusclepatterning. and formindividual musclesisstillpoorlyunderstood.Homeobox information isintegrated bythemusclemassesinordertosplit differentiation (Kardonetal.,2003).However, theway this a pre-patternthatwilldeterminethesiteofmyogenic Tcf4 et al.,1995;Chen1998).Recently, thetranscriptionfactor limb drives thedorsalmusclepattern(Riddle etal.,1995;Vogel respecification. this muscleposteriorisationisnotaconsequenceofcartilage directly toSHHsignaling(AhnandJoyner, 2004)suggeststhat al., 1999).Thegeneticproofthatlimbmusclecellscanrespond forearm musclesintowithaposterioridentity(Duprezet anteroposterior axisformation,isabletotransformanterior (Duprez, 2002).Sonichedgehog(SHH),whichisinvolved in in positioningthemusclesadditiontocartilage molecular pathways involved inlimbaxisformationareinvolved (Amthor etal.,1998;Bonafede2006).Moreover, the to restrictthepositionofpremusclemassesinchicklimbbuds 2005). Bonemorphogenicproteins(BMPs)have beenproposed and negatively, respectively (Swartz etal.,2001;Vasyutina etal., also controlthemigrationofmuscleprogenitorcellspositively 1999; Relaixetal.,2003).SDF1/CXCR4andephrin-A5/EPHA4 , locatedinlimbmesenchyme,hasbeenproposedtoestablish ( Meox2 Lbx1 ) -homozygousmutantmiceconsistentlydisplay Lmx1b homeobox genecanalsobe considered asa 1 , PilarGarcía-Villalba expressed inthedorsalmesenchymeof Pax3 Pax3 (Dietrich etal.,1999;Scaal Lbx1 RESEARCH ARTICLE mutant micedisplayan 1 , Hoxa11 plitting fy the 2579 and t e

DEVELOPMENT muscle. cells) promoteconnective tissueformationattheexpense of provide evidence thatvessels andPDGFB(locatedinendothelial and musclecellsoneachother duringlimbbud development. We of vessel muralcells(Hoch andSoriano,2003;Betsholtz,2004). PDGFB functionintherecruitment, proliferationandmigration knockout micedisplaysimilarphenotypes,consistent witha expressing itsreceptor, PDGFR signal fromendothelialcellstobloodvessel muralcells vessel formation(Betsholtz,2004).PDGFBactsasaparacrine derived growth factor B)isanothersecretedfactor importantfor 2003; Weinstein, 2005; Carmeliet,2005).ThePDGFB(platelet- attractive andrepulsive neuronalguidancefactors (Batesetal., highlight thatthepatterningofvascular systemisdirectedby endothelial network inthelimbs.However, recentstudies one ofthecuesinvolved inthecorrectorganization ofthe al., 2000;Pooleet2001;Cho2002),VEGFA isprobably various systemsandspecies(Cleaver andKrieg, 1998;Drake et activate endothelialcellmigration,proliferationandsurvival in is lesswelldocumented.BasedonthecapacityofVEGFA to studied, itsparticularroleintheestablishmentoflimbvasculature of VEGFA in theformationofgeneralvasculature iswell physiological andpathological(Ferrara,2000).Althoughtherole development includingvasculogenesis andangiogenesis,both A) isakey moleculeinvolved invarious aspectsofbloodvessel secreted glycoproteinVEGFA (vascular endothelialgrowth factor Various signalingmoleculesareinvolved intheseprocesses.The recruitment andtheestablishmentofartero-venous identity. several processesincludingvascular remodeling,muralcell Vargesson, 2003).Subsequentvascular development involves Noden, 1991;Brand-Saberietal.,1995;Ambler2001; vessels isstillnotclear(Pardanaud etal.,1989;Feinberg and contribution ofeachprocessintheearlyassemblylimbblood endothelial progenitors(i.e.typeIIvasculogenesis). The angiogenesis) orbycoalescenceoffree(migrating)vascular vasculature canariseeitherfrompre-existing vessels (i.e.by network hasformedinthetrunkofembryo,limb the fact thatthelimbbuds appearaftertheprimitive vascular al., 1995;Pardanaud etal.,1996;Pouget2006). Owingto cardinal veins, intersomiticvessels, kidney andlimbs(Wilting et provide endothelialcellstoboththeroofandsidesof aorta,to transplantations fromquailtochickhave shown thatsomites vasculature inlimbmusclepatterning.Orthotopic somite of muscleseparation. provide evidence foralinkbetweenthevasculature andtheprocess 1995; MurrayandWilson, 1997).However, thesestudiesdidnot using inkinjection(SchroeterandTosney, 1991a;Flammeetal., investigated byhistologicalanalysis,afterhypervascularization or involvement ofbloodvessels inmusclesplittinghasalreadybeen Jones andLandmesser, 1980;Edom-Vovard etal.,2002).The in theabsenceofinnervation following neuraltubeablation(Lance- (Schroeter andTosney, 1991a),becausethemusclessplitnormally and Duprez,2004).Aninfluencefromnerves hasbeeneliminated involved inlimbmusclepatterning(Kardon,1998;Edom-Vovard muscle spatialorganization. Thetendonsaregoodcandidatestobe muscle patterningarenotclear(Yamamoto etal.,1998). individual musclesinchicklimbs,althoughtheir preciserolesin in restricteddomainsofthemusclemassesandspecific Hoxa13 2580 In thepresentpaper, weanalyzetheinfluenceofendothelial Analyzing new dataledustoreinvestigate theinfluenceof Other limbtissueshave beenstudiedascandidatesforinfluencing RESEARCH ARTICLE homeobox geneshave beendescribedasbeingexpressed ␤ . PDGFBandPDGFR ␤ library (Boardmanetal.,2002).Part ofthecodingsequenceforchick et al.,1997).Theprobeforcollagen IoriginatesfromtheUMISTEST 1993), hybridization. the expression oftheectopicgenewas systematicallychecked byinsitu infection. Owingtoacertainvariability inthevirusspreadamongembryos, 48 to72hours,graftinglimbsatHH22(E4)leadsmorelocalizedvirus stages oflimbdevelopment (HH14,E2)leadstoagenerallimbinfectionin progressively spread toalllimbtissues.Whereasgraftingembryosatearly control. Aftergrafting,theRCASvirus(andinsertedgene)will situ hybridizationtotissuesections.Theleftwingwas usedasaninternal various timesaftergrafting, embryoswereharvested andprocessedforin presumptive regions ofembryosatstagesHH14(E2)toHH22(E4).At approximately 50 described byEdom-Vovard etal.(Edom-Vovard etal.,2002).Pelletsof expressing cellsandRACS-expressing cellswerepreparedforgraftingas replication-competent retroviral vector RCASBP(A).VEGF/RCAS- Paris, France)was insertedinthesenseorientationinto The chick RCAS-expressing cells Production andgraftingofVEGF/RCAS-expressing orcontrol Embryos from Pax3 E6.5/HH29, E7/HH30,E7.5/HH31,E8/HH32. numbers andHHstagesareequivalent: E5/HH26,E5.5/HH27, E6/HH28, of daysincubationarereportedwiththeHHstages.Thefollowing day were stagedaccordingtodaysinovo. To facilitate comparisons,thenumbers (HH) (Hamburger andHamilton,1951),whereasoldembryos(E5.5-E10) Young embryos (E3-E5)werestagedaccordingtoHamburger andHamilton at 37°C.BeforeE2,embryoswerestagedaccordingtosomitenumber. Strasbourg) –andJapanesequaileggs (Chanteloup,France)wereincubated Sélection Animale(ISA),Lyon, France]andWhiteLeghorn (HAAS, Fertilized chickeggs fromcommercialsources–JA 57strain[Institutde Chick andquailembryos MATERIALS ANDMETHODS was isolated,usingprimer5 Hif2 (Marcelle andEichmann,1992), mouse labeled mRNA probeswereusedasdescribed: as previously described(Edom-Vovard etal.,2002).Thedigoxigenin- processed forinsituhybridizationtowhole-mountsandwax tissuesections Normal ormanipulatedembryoswerefixed in4%(v/v)formaldehydeand In situhybridizationtowhole-mountsandtissuesections then processedforwax sectioning. the circulationofembryos.Theembryoswerefixed 2hourslaterand Two daysaftergrafting,2 PDGFB orPBSbeadsweregraftedintotherightwingsatstageHH26 (E5). Bromodeoxyuridine (BrdU) labelinginovo in PBSandsoaked in500ng/ were obtainedfromRDSystems.Affigel Bluebeads(Biorad)werewashed The ratPDGFBandhumansolubleVEGFR1(sFLT1) recombinantproteins PDGFB andsFLT1 beadimplantation fertilization was consideredtotake placeat6am. al., 2005)micewerecollectedafternaturalovernight matings.For staging, primer3 sections. the vessels oftheallantoid,orprocessedforinsituhybridizationtotissue manipulatedwingswereeitherinjectedwithpureindianinkinto grafting, wings ofembryosataboutstageHH27/28(E5.5/E6).Two daysafter embryos ortotissuesections.sFLT1 orPBSbeadswere graftedintotheright grafting, embryoswereprocessedforinsituhybridizationtowhole-mount normal embryosatstageHH19/20(E3)toHH26(E5).2472hoursafter 4 hoursonice.PDGFBorPBSbeadsweregraftedintotherightwingsof ␣ mutant mice probe correspondstoapartofthemouse Hif2 MyoD Ј (5 Vegfa Ј ␣ -TTGCTCATGTCCATGTAGCC-3 (Favier etal.,1999), (Delfini andDuprez,2004),quail Pax3 coding sequence(provided byThierryJaffredo, CNRS, ␮ m indiameterweregraftedintotherightwingsorwing –/– mutant (Relaixetal.,2003)and ␮ l ofBrdU(Amersham)was directlyinjectedinto Ј ␮ (5 l PDGFBfor1hourorin Ј -CGTCATCTCCCTGATCATCC-3 Tcf4 Pdgfb (Kardon etal.,2003).Themouse (Horiuchi etal.,2002), Hif2 Ј ), fromanRT-PCR-derived Development 134(14) Vegfr2 Pax3 ␣ coding sequence(Ema Pax3 , MyoD (Eichmann etal., ␮ GFP/+ Cla g/ ␮ , I siteofthe l sFLT1 for Fgfr4 (Relaix et Pdgfr Pdgfr Ј ) and and ␣ ␤

DEVELOPMENT enabled detectionof of 1995; Pardanaud etal.,1996;Kardon2002).Transplantation endothelial andmusclecellsoriginatefromsomites(Wilting etal., Classical surgical manipulationsinavian embryoshave shown that somatopleure before myogeniccells Angiogenic cellsare detectedinlimb RESULTS detected usingamonoclonalantibodyagainstBrdU(Amersham). the polyclonalantibodyanti-GFP(CellSignaling).Proliferatingcellswere limbs was carriedoutaccordingtoRelaixetal.(Relaixal.,2003)using Studies HybridomaBank).Fluorescentco-immunohistochemistryinmouse PECAM1(Developmental platelet/endothelial celladhesionmolecule1, embryos wererecognizedusingthePECAMantibody, whichrecognizesthe (Developmental StudiesHybridomaBank).Theendothelialcellsinmouse visualized inquailembryosusingtheQH1polyclonalantibody (Developmental StudiesHybridomaBank).Theendothelialcellswere antibody MF20thatrecognizessarcomericmyosinheavy chains Differentiated musclecellsweredetectedonsectionsusingthemonoclonal Immunohistochemistry 33342 (MolecularProbes)for15minutes. sections processedforinsituhybridizationwereincubatedwithHoechst cDNA librarymadefromE6-E8chickheart.To labelnuclei,adjacent Vasculature andmusclesplitting Vegfr2 at different stagesatthebrachiallevel (Fig.1A-F).We foundthat by insituhybridizationontransverse adjacentsections ofembryos distribution withthatof derived angioblasts(Shalabyetal.,1995)andcomparedits used thetyrosinekinasereceptor, endothelial progenitorcellsinthepresumptive wingregions. We therefore investigated thetemporal relationship betweenmuscleand there was noindicationofthetiminglimbcolonization.We developing limbbud (Huangetal.,2003).However, inthesestudies, indicating thatthesecelltypesmigratealongdifferent routesinthe between thedistribution ofquailendothelialandmusclecells, Single quailintochicksomitegraftsdonotleadtoastrictcorrelation cells alsooriginatefromsomites(BeddingtonandMartin,1989). morphology inearlylimbbuds, indicatingthatmouseendothelial lacZ -positive cellsareclearlydetectedinthepresumptive wing transgenic mousesomitesintowild-typeembryos lacZ Pax3 -positive cellswithanendothelial amarker ofmuscleprogenitorcells, , Vegfr2 as amarker ofthesomite- Hif2 andthebHLH-PAS transcriptionfactor gene PECAM1 markers, pattern intheabsenceofmusclecellsusingtwo mousevascular spatial organization ofthevasculature. We analyzedthebloodvessel mice hasalreadybeenreported(DeAngelisetal.,1999),but notthe (Relaix etal.,2003).Thepresenceofbloodvessels in deficient mice,inwhichnomyogeniccellisdetectedthelimbs vessel formation,wetookadvantage oftheexistence of In ordertoinvestigate theinvolvement ofmusclecellsinblood Vessel organizationintheabsenceofmuscle embryos. colonize thelimbsbeforemuscleprecursorcells,inchickandmouse migrate intothelimb(Fig.2A).Theseresultsshow thatangioblasts forelimb atE9.5,whereasPAX3-positive cellshave juststartedto isexpressed inthe (Baldwin etal.,1994).We foundthatPECAM1 recognized marker forearlyvascular precursorcellsinmice situation occursinmice.Thecelladhesionreceptor, PECAM1,isa somatopleure atthesametime.We alsoanalyzedwhetherasimilar progenitor cellsandangioblastsdonotcolonizethechickwing expression was notdetected(Fig.1G,H).Inconclusion, muscle somatopleure atthe22-and24-somitestages,whereas the sameresult:QH1-positive cellswereobserved inthewing QH1 antibodyonquailembryos(Pardanaud etal.,1987),wefound intermingled withthe positive cellshadstartedtheirmigrationtothechickwingbud and in thewingsomatopleure(Fig.1D,E).At30-somitestage, arrowheads) stages.Atthesestages,therewas no regions, at19-somite(Fig.1A,arrowheads) and25-somite(Fig.1B, the ventral musclemassthatgives risetoventral flexor muscles. wing (Sullivan, 1962;Robsonetal.,1994).We mainlyfocusedon Twelve mainmuscles canbeidentified intheforearmofchick The vasculature delineatesthecleavagepattern organizes itselfcorrectlyintheabsenceofmuscles. We concludethatthevasculature absent muscles(Fig.2C,E). appeared toorganize inasimilarpattern,delineatingtheareasof surround themuscles(Fig.2B,D).In control limbs,PECAM1-and ␣ ( Epas1 – MouseGenomeInformatics)(Pengetal.,2000).In Vegfr2 F) probes. ( hybridized with (B,E) and30(C,F)-somitestageswere embryos, atthewinglevel,19(A,D),25 ( progenitor cellsinavianembryos. wing somatopleure before muscle Fig. 1.Angiogeniccellsare detectedin So, somites. somites. NT, neuraltube;Nc,notochord; of expression of Arrows indicatetheoverlappingdomain probe (A,B)orQH1antibody(G,H). progenitor cellsvisualizedwith (brown). Arrowheads pointtoendothelial then incubatedwithQH1antibody hybridized with (H) and32(I)-somitestageswere embryos, atthewinglevel,22(G),24 A-F -positive cells(Fig.1C,F).Usingthe ) Adjacenttransversesectionsofchick Hif2 RESEARCH ARTICLE ␣ Pax3 G-I - positive cellswereseento ) Transverse sectionsofquail Vegfr2 Pax3 mutant limbs,thevessels Vegfr2 probe (blue)and (A-C) and (A) and Pax3 Pax3 expression Vegfr2 Pax3 Pax3 mutant Pax3 Pax3- 2581 (D) in Pax3 (D- -

DEVELOPMENT staining (blue)visualizesthenuclei.( vascular cells.GFP(green) reveals the embryos were incubatedwiththePECAMantibody(red) tovisualize visualized using a 3A,B,D,E). However, atthesestages,we detectedendothelialcells, (Fig. was noobvious sign ofseparationtheventral musclemass material). individual muscles inE10wings(seeFig.S1thesupplementary When thesplittingprocessis finished, thevessels surroundthe endothelial cellssurroundthedorsalandventral musclemasses. beginning ofthesplittingprocess,vessels composedof schematized (seeFig.S1inthesupplementarymaterial).Before the successive separationsoftheventral musclemasscanbe Based onserialcross-sectionsofwingsatdifferent stages,the corresponding totheabsentmusclesin ventral musclesincontrol limbs(B,D)andtheequivalentsites probe andthenincubatedwiththeMF20antibody. Theasterisks show mutant micewere cuttransversallyandhybridizedwiththe and muscle. Fig. 2.Thevascularnetworkorganizesitselfindependentlyof 2582 with thePECAMantibody. ( muscles andmuscle-lesssitesare surrounded bythe vasculature. At E5/HH26andE5.5/HH27,based onmyosinexpression, there Pax3 RESEARCH ARTICLE ( –/– A ) Transverse sectionsofE9.5forelimbs of mutant micewere cuttransversallyandthenincubated Vegfr2 probe, crossing the ventral musclemass at D , E ) E13.5hindlimbsof B Pax3 , C ) E13.5forelimbs from Pax3 -expressing cells.DAPI –/– limbs (C,E).Both Pax3 Pax3 +/+ GFP/+ Hif2 and mouse Pax3 ␣ Pax3 +/+ –/– the expression of (Fig. 4B)ledtoadramaticincreaseofbloodvessels, visualized by fixed oncethesplittinghadfinished, atE8/HH32.EctopicVEGFA 4A), beforethemusclesplittingprocessstarted.Theembryoswere expressing cellsweregrafteddorsallyintoE4/HH22wing buds (Fig. RCAS retrovirus system(Fig.4).PelletsofVEGF/RCAS- Pacifici, 2001).We modified theexpression of et al.,1989;Flamme1995;Wilting etal.,1996;Yin and VEGFA induces angiogenesisinavariety ofinvivo models(Leung organization, wedecidedtomodifyvessel assembly. Ectopic In ordertoestablishwhetherthevasculature influencesmuscle Ectopic bloodvesselsinhibitmuscleformation between vessel assemblyandthesplittingprocess. effective separationofthemuscle masses.Thisresultsuggestsalink endothelial cellsdelineatesthecleavage sitesofmuscle,beforethe anterior masses(Fig.3L,arrow). Inconclusion, thelocationof delineating thefutureseparationofcentralandproximal positive cells(Fig.3L).We alsoobserved endothelialcellsanteriorly, containing numerous a daylater, atE6/HH28,thiscleavage zonehadbecomeobvious, extend over 300 crossing theventral musclemass,weestimatethisorganization to organization of and anteriormusclemasses(Fig.3C,F).Thisstereotyped cleavage zonewas visualizedatE6/HH28,separatingtheposterior the precisesiteoffuturecleavage zone(Fig.3A,B,D,E).This secondary effect toectopicvessels andnotadirectresponse of supplementary material),suggesting thatlatermusclealterationisa compared withthecontrol limb (seeFig.S3F-Kinthe (in termsofshapemusclemasses, densityofmyogeniccells) E5.5 VEGF-infectedlimbsdid notshow any musclemodification masses arepermissive tovessel progressionatthisstage.However, started toinvade musclemassesfromE5.5,indicatingthat muscle massesrestrictangiogenesisatthisstage.Ectopicvessels Vegfa masses werenever hypervascularized beforeE5,despiteectopic E2 VEGFgraftsatvarious stagesbeforeE6showed thatmuscle formation atvarious stagesafterVEGFmisexpression. Analysisof muscles byvessels was stage-specific, weanalyzedmuscle material). Inordertodeterminewhetherthisnegative effect on with thoseinthecontrollimb(seeFig.S3A-Esupplementary vessels. Theremainingmuscleswereseverely reducedcompared days aftergrafting,andtoanaccompanying increaseinblood regions ledtoageneralanddramaticectopicexpression of VEGF/RCAS earlier, atE2/HH14,into the presumptive wing thecorrespondingcontrolmuscles(Fig.4G-J).Grafting than show thattheremainingmusclescontainfewer myogeniccells Higher magnifications ofadorsalmuscle(arrowed inFig.4C-F) labels muscle-differentiated cells(Edom-Vovard etal.,2001). which labelsmuscleprogenitorcells,andmyosinexpression, which (Fig. 4E,F).Themuscleswerevisualizedby muscles comparedwiththenormalmusclepatternofleftwing we observed areductioninmusclesizeandeven alossofcertain 4C,D). Inthedorsalregions displayinganexcess ofbloodvessels, By countingthesectionsdisplayingendothelialmarker in theventral masswas observed over acertainlengthofthelimb. stereotyped locationofendothelialcellsatthefuturesitessplitting in quailembryos(seeFig.S2thesupplementarymaterial).This the futuresiteofseparationtwo partsoftheflexor carpiulnari E5/HH26 andE5.5/HH27.We alsoobserved QH1-positive cellsat mass was consistentlyobserved inthewingsofchick embryosat expression (datanotshown), suggestingthat repulsive cuesin ␮ Vegfr2 Hif2 m alongtheproximal-distalaxis(Fig.3G-K).Half Hif2 ␣ , ascomparedwiththeleftcontrolwing(Fig. -positive cellscrossingtheventral muscle ␣ - positive cellsandresidualsparseMF20- Development 134(14) Vegfa Fgfr4 using theavian expression, Vegfa Hif2 ␣ 5

DEVELOPMENT Hif2 vessels. We usedthesolubleform ofVEGFR1(FLT1), referredto splitting inordertoanalyzemuscle organization intheabsence of We next aimedtoblockvessel formation duringthetimeofmuscle fusion Local blockofvesselformation leadstomuscle inhibiting muscleformation. misexpression promoteconnective tissueECMproduction,while These resultsshow thatectopicvessels inducedbyVEGF misexpression, ascomparedwithcontrolmuscles(Fig.5C-H). was observed inhypervascularized musclesfollowing VEGF sites (Fig.5A,B).Moreover, anetincreaseincollagenIexpression masses anditspresencecanbecorrelatedwiththatofvessels atthese expression isalsoenhancedintherecentlycleaved sitesofmuscle collagenI muscles (Shellswelletal.,1980).We observed that collagen Iexpression isenhancedinmembranessurrounding In additiontobeinglocatedinallchicklimbconnective tissues, component oftheextra-cellular matrix(ECM)ofconnective tissues. connective tissuemarker collagen I.CollagenIisamajor connective tissueformation,weanalyzedtheexpression ofthe formation atthetimeofmusclesplitting. anarchic ectopicbloodvessels hasanegative influenceonmuscle have aneffect. Altogether, theseresultsshow thatthepresenceof possibility thatthevirusreachedthoseventral muscles toolateto displaying ectopicvessels (Fig.4C-F),isconsistent withthe of any shapemalformationoftheventral musclesatE8,despite muscle cellstoVEGF. Conversely, inE4-graftedwing,theabsence Vasculature andmusclesplitting elements andthenhybridizedwiththe of theventralmassesfrom C.( antibody (brown). (D,E)Highmagnificationofthefuture cleavagezoneoftheventralmassesfrom A,B.(F)Highmagnificationo E5/HH26 (A,D),E5.5/HH27(B,E)andE6/HH28(C,F)chickembryoswere hybridizedwiththe Fig. 3.Thelocationofendothelialcellsdelineatesthefuture cleavagezonesofmusclemasses. produce thecentralandproximal anteriormasses. arrow indicatesthe images runningfrom proximal todistal).( In ordertodeterminewhetherectopicvessels couldinfluence ␣ - positive cellsatthefuture splittingsiteoftheventralmusclemasswasestimatedtoextendover300 Hif2 ␣ pstv el eietn uuecevg oei h neirpr ftecnrlmsl as–cleavagethatwill -positive cellsdelineatingafuture cleavagezoneintheanteriorpartofcentralmusclemass– G-K ) Wings from E5.5/HH27 chickembryoswere) Wings cuttransverselyfrom proximal todistalareas alongtheforearm Hif2 L ) AtE6/HH28,theventralmusclemasswaswellseparatedintoposteriorandanteriormasses.The ␣ probe (blue)followedbyimmunohistochemistryusingtheMF20antibody(brown). Thepresence of is expressed invascular smooth musclecells(Lindahlet al., 1997). as beinglocatedinendothelialcells, whereasitsreceptorPDGFR candidate. Duringmousedevelopment, PDGFBhasbeendescribed muscle. PDGFB,secretedby endothelialcells,isaputative vascular network couldaccountforthisnegative effect ofvessels on We next triedtodeterminewhichmolecularfactors locatedinthe muscle andconnectivetissue PDGFB reproduces theeffect ofbloodvesselson blood vessels inlimbmusclesplitting. together withthevessel experiments suggest an involvement of endogenous locationofvessels inthefuturemusclecleavage zones whereas localhypovascularization leads to musclefusion.The vessels andmuscle.Hypervascularization inhibits muscleformation, splitting. experiment shows thatthelocalabsenceof vessels prevents muscle of two muscles(seeFig.S4inthesupplementarymaterial). This fusion betweenthetwo muscleswas observed alongtheentirelength PBS beadsdidnotaltermuscleorganization (datanotshown). The the dorsalregions ofthesFLT1 graftedwings(Fig.6E-H),whereas Analysis of organization (Fig.6A-D; days aftergrafting,whereasPBSbeadsdidnotaffect vessel chick wingsledtoconsistentlocalinhibitionofvessel formation 2 2003). ApplicationofsFLT1 beadstothedorsalaspectofHH28/E6 and toreduceangiogenesisinvivo (Drake etal.,2000;Bates as sFLT1, whichhasbeenshown tobindVEGFwithhighaffinity The vessel experiments highlightaninverse correlationbetween MyoD Vegfr2 expression showed reproduciblemusclefusionin probe (blue)andthenincubatedwiththeMF20 ( n A-F =22 sFLT1 beads; ␮ m inlength(shownare example ) Transverse sectionsofwingsfrom RESEARCH ARTICLE n f thecleavagezone =14 PBSbeads). 2583 ␤

DEVELOPMENT and leftisposterior. u,ulna;r, radius. corresponding control muscles(I).Forallsections,thetopisdorsal hypervascularized musclescontain fewermyogeniccells(J)thanthe regions ofthecontrol andmanipulatedlimbs,showingthatthe (Anconeus) muscle(arrowed inC-F)locateddorsalandposterior in theVEGF-treated limbs(F).(G-J)HighmagnificationsoftheANC (E). Asteriskslabelthecontrol muscles(E)andtheirputativelocations compared oftheleftcontrol withthenormalmusclepattern wing reduction ofmusclesizeandevenalosscertainmuscles(F),as dorsal regions displayinganexcessofbloodvessels,weobserveda with thenormalvascularnetworkofcontrol wing(C).Inthe Consecutive sectionsofthemanipulatedwing( E4/HH22 chickwingsandtheembryoswere fixedatE8/HH32. Hif2 order toallowcomparison.Sectionswere hybridized with control leftwing( ( Fig. 4.Ectopicbloodvesselsinhibitmuscleformation. 2584 blood vessels,visualizedbytheexpression of wings. EctopicVEGFA leadstoadramaticincrease inthe densityof transcripts showtheextentofinfectionindorsalregions ofthe with theMF20antibody(brown) thatrecognizes myosins. (B) A ) VEGF/RCAS-expressing cellswere graftedtothedorsalaspectof ␣ (C,D,G,H) and RESEARCH ARTICLE C , E Fgfr4 , G , I ) were cutatthesameproximodistal levelin (E,F,I,J) probes (blue),andthenincubated Hif2 B , ␣ D , (D), compared F , H , J ) andofthe Vegfa Vegfa (B), also inhibitedtheexpression ofitsreceptor, the PDGFBeffect is stage-specific. ApplicationofPDGFBbeads control leftwings(F-H)were hybridizedwiththe E8/HH32. ConsecutivesectionsofVEGF-infectedrightlimbs(C-E)and to thedorsalaspectofE4/HH22wingsandembryoswere fixedat ventral musclemasses.( are detectedwithendothelialcellsintherecently cleavedsitesofthe immunohistochemistry withtheMF20antibody. collagenItranscripts We alsoobserved anadditionalandunexpected siteof smooth musclecells(datanotshown), similartothemousesituation. ahrn( cadherin wings from E6.5/HH29chickembryoswere hybridizedwiththeVE- vasculature inlimbmuscles. Fig. 5.CollagenIisassociatedwithnormalandectopic on musclemarkers beforeE5( PDGFB beadapplicationinthechicklimbdidnothave any effect PBS beadsdidnotimpair MyoD Ectopic PDGFBinhibitedtheexpression ofthemusclemarker, buds atE5andanalyzedtheconsequencesformuscle development. cleavage, weappliedbeadssoaked inrecombinantPDGFBtolimb splitting. Inordertoinvestigate apossibleroleforPDGFBinmuscle muscle cellscouldalsorespondtoPDGFBsignalingduring expression inchicklimbmusclemasses(Fig.7B,C),indicatingthat transcripts inendothelialcells(Fig.7A)and During chicklimbdevelopment, weindeedobserved muscles (H). increase incollagenIexpression inmuscles(E)compared withcontrol same level.Ectopichypervascularization(C,D)leadstoanobvious muscle inexperimentalwings(C-E)andcontrol wings(F-H)atthe antibody (brown). Highmagnificationsare shownofthesamedorsal and collagenI(E,H)probes (blue)thenincubatedwiththeMF20 akr collagenIaroundthebeads, intheregion negative for marker, upregulation oftheexpression ofthemuscleconnective tissue effect onconnective tissue.PDGFBapplicationledtoa clear masses (Fig.7G-I). We next determinedwhetherPDGFBcouldmimic thevessel (Fig. 7D,E),aroundthebeads,2daysaftergrafting,whereas A ) andcollagenI( C-H MyoD ) VEGF/RCAS-expressing cellswere grafted B ) probes followedby Transverse andconsecutivesectionsof n =20; datanotshown), showing that expression (Fig.7F).Interestingly, Development 134(14) Vegf Pdgfr Pdgfr (C,F), ␤ transcripts in ␤ Hif2 in muscle Pdgfr ␣ Pdgfb (D,G) ␤

DEVELOPMENT limb musclepatterning(Kardonetal.,2003)(Fig.8C). the Wntsignalingpathway, provides apre-patternforvertebrate bead implantation(Fig.8C-F). expression 8E). PDGFBapplicationalso ledtoanupregulation ofthe of musclecleavage beforetheeffective separationofmuscles(Fig. shown). I, ingeneralandmuscleconnective tissues,inchicklimbs(datanot collagen transcripts displayanexpression patternsimilartothatof connective tissue(Ataliotis,2000).We observed that transcripts have beendescribedasbeinglocatedinchicklimb other connective tissuemarkers, (Fig.8A,B).We alsoanalyzedtheexpression oftwo muscle markers Vasculature andmusclesplitting digitorum communis;u,ulna;r, radius. treated wings.EMU,extensormetacarpiulnans;EDC,extensor, muscles, are cleavedincontrol wings,whereas theyare fusedinsFLT1- magnification ofE,F, respectively. TheEMUandEDC,twodorsal probe inorder tovisualizemuscleorganization.(G,H)High wings (F,H) andcontrol leftwings(E,G)were hybridizedwith the the control leftwing(C).( bead implantationlocallyinhibitsvesselformation(D)compared with assembly (B)ascompared withthenon-graftedleftwing(A).sFLT1 organization. PBSbeadimplantationatE6doesnotmodifyvessel undergone inkinjectionbefore fixationinorder tovisualizevessel ( Fig. 6.Blockingvesselformationleadstomusclefusion. the tendonmarker, scleraxis, 2daysaftergrafting(data notshown), Application ofPDGFBbeadsdid notinduceectopicexpression of A-D Dorsal viewsofwingsfrom E8chickembryos,whichhave ) Pdgfr Tcf4 ␣ expression was also enhancedinthefutureregions and Pdgfr E-H ) Transverse sectionsofsFLT1-treated right ␣ Tcf4 Tcf4 around thebeads(Fig.8C-F). , atranscriptionfactor linked to and Pdgfr ␣ , following PDGFB Pdgfr Pdgfr MyoD ␣ ␣ express PDGFreceptors,PDGFR Both celltypesareabletorespondPDGFsignalbecausethey embryological celltypes:connective tissueandmyogeniccells. The beadexperiments showed thatPGDFBactsontwo unrelated acts onmyogeniccells PDGFB actsonconnectivetissuecellsbefore it on muscleandconnective tissue. makes PDGFBanobvious candidateformediating thevessel effect vessels. Thefact thattheonlysourceofPDGFBis endothelialcells inhibiting andconnective tissue-promotingfunctionofblood to preventing muscleformation.Thus,PDGFBmimicsthemuscle- expression ofgenesspecific tomuscleconnective tissue,inaddition does notcontainsmoothmusclecells(datashown). that PDGF-inducedconnective tissueisnothighlyvascularized and cadherin 5)]orthatofsmoothmusclecellmarker (SMA),indicating of endothelialmarkers [HIF2 beads. ApplicationofPDGFBbeadsdidnotmodifytheexpression excluding atendonidentityforthetissuesurroundingPDGFB after grafting,whereasthedownregulation of hours PDGFB activated theexpression ofcollagenIassoon9.5 various timesafterPDGFBbeadimplantation,weobserved that define onwhichcelltypePDGFBactsfirst. Byfixing embryosat attracted connective tissuecellsaroundthebeads.We next triedto beads (Fig.7,Fig.8,9D,G),weconcludedthatPDGFB expression (andtheabsenceofmusclemarker) aroundthePDGFB attracting cells.Given thenetincreaseofconnective tissuemarker This impliesthatPDGFBincreasedcelldensityaroundthebeadby beads, 24(datanotshown) and48hoursaftergrafting(Fig.9E-G). PDGFB applicationdidnotmodifythecellproliferationaround (Fig. 9A-D).However, analysisofBrdUincorporationshowed that cell densitywas clearlyenhanced2daysafterbeadimplantation PDGFR organizes properlyintheabsence ofmusclein of musclecells.Moreover, the fact thatthevasculature ispresentand indicating thattheangioblastscolonizelimbbud independently before muscleprogenitorcellsinpresumptive limbbud regions, In thethispaper, weshow thatangiogeniccellsareclearlydetected myogenic cellsinchickandmouselimbs Angiogenic cellsbehaveindependentlyof DISCUSSION was observed beforethecellaccumulationaroundPDGFBbeads. (upregulation ofcollagenIanddownregulation of modifying celldensity. Themodification ofgenetranscription However, wecannotexclude theexistence ofcellmovement without cell accumulation48hoursafterbeadimplantation(Fig.9A-D). (Fig. 10G,H)afterPDGFBimplantation,comparedwiththeobvious density aroundthebeads9.5,12,16(datanotshown) and24hours implantation. We didnotobserve any obvious signofincreaseincell Hoechst-labeled nucleibehavior atvarious timesafterPDGFBbead contribution ofgenetranscriptionandcellmigration,weanalyzed PDGFB actsonconnective tissuecellsfirst. Inordertoestimatethe only observed 24hoursaftergrafting(Fig.10A-F).This shows that somite (lombar somite) atthe36-somitestage(Kardon etal.,2002). demonstrated thatthecommon progenitorexists inthethirtyfirst al., 2002).Lineagetracing experiments inchickhindlimbs originate fromacommonprogenitor (DeAngelis,1999;Kardonet experiments clearlyindicate thatendothelialandmyogeniccells to musclecells,inthedeveloping limbs.Different sets of confirms thecomplete independenceofvascular cellswithrespect Altogether, theseresultsshow thatPDGFBpromotesthe ␤ (muscle). AnalysisofHoechst-labelednucleishowed that ␣ , VE-cadherin(alsoknown as RESEARCH ARTICLE ␣ (connective tissue)and MyoD Pax3 Pax3 MyoD mutant embryos expression was expression) 2585

DEVELOPMENT Interestingly, there isevidence in chicklimbsindicatingthatblood of ECM-richconnective tissuebetweentwo dividing muscles. Thus, weproposethatvessels areinvolved insetting upaboundary which isalsoincreasedatthesplitting sitesindeveloping embryos. expression, we have shown thatectopicvessels promotecollagenI potential roleforthevascular network inmusclesplitting.Moreover, vessel formationleadsto musclefusion.Theseresultshighlighta vessels leadstoaninhibitionofmuscleformation,whereasblocking an inverse correlationbetweenvessels andmuscles.Anincreaseof in theventral musclemass.Inaddition,thevessel experiments show have shown thatthevasculature delineatesthefuturecleavage zones additional role:directingadevelopmental patterningprocess.We 2001). Hereweprovide evidence thatthe vascular systemplaysan to bloodvessel function(Matsumotoetal.,2001;Lammert endothelial cellspromoteliver andpancreatic organogenesis prior (Caplan andKoutroupas, 1973).Ithasalsobeenreportedthat nutrients andoxygentodifferentiating tissue,includingmuscle The vasculature isusuallyseenasasupplierofessentialmetabolic splitting process The vascularnetworkinfluencesthemuscle microscopy studies(Solurshetal.,1987),remainstobedetermined. myogenic cellsinthelimb,assuggestedbyprevious electron Whether angiogeniccellshave aninfluenceon themigrationof precursor cellsoutsidethesomitesinchickandmouselimbs. analysis shows thatangioblastsbehave differently tomuscle brachial epithelialsomites(Fig.1A,D,arrowed). Ourmarker Pax3 Consistent withthis,thereisanoverlapping expression domainof 2586 and RESEARCH ARTICLE Vegfr2 in thedorsolateralcompartmentofchick in theembryo(Bates etal.,2003;Weinstein, 2005;Carmeliet,2005). Eph receptors,have beenshown tocontrolvascular morphogenesis axon outgrowth, such assemaphorinsorephrinsandtheirassociated network (Weinstein, 1999).Recently, guidanceproteinsinvolved in interactions, leadingtotheestablishment oftheembryonicvascular adhesive interactions withtheECM,probablythroughintegrin accepted thatthepositionofendothelial cellsisregulated bytheir vasculature intheembryoniclimbarelargely unknown. Itis mechanisms directingthestereotypedorganization ofthe does notresolve theproblemofmusclepatterning,as connective tissueisanimportantissuetobe addressed. in musclepatterning.Theconnectionbetweentendons,vessels and 2004) andconnective tissue(Kardonetal., 2003),arealsoinvolved tissues, suchastendons(Kardon,1998;Edom-Vovard andDuprez, between muscles,secondarilytomusclefiber orientation. Otherlimb mesenchymal cellstomyogenicthatwould setupboundaries cleavage. Thevasculature would bearelaysystemfromlimb organization ofthevasculature, whichinturninfluencesmuscle hypothesize thatsignalsinthelimbmesenchymedirectspatial before any splittingevent occurs (Kardon, 1998).We therefore Kardon, 1998).Moreover, musclefibers know theirorientation cells (Chevallier etal.,1977;ChristLance-Jones,1988; positional informationformusclepatterningresidesinnon-somitic chondrogenesis (Yin andPacifici, 2001). initiation ofcorrectmesenchymalcondensationandsubsequent limb presumptive cartilageregions isarequiredconditionforthe vessels alsodictatecartilagepatterning,asthevascular regression in However, theinvolvement ofthevasculature inmusclesplitting From classicalembryologicalexperiments, weknow thatthe chick embryoswere hybridizedwiththe consecutive sectionsofwingsfrom E5.5/HH27 with theMF20antibody. ( Pdgfb E5.5/HH27 chickembryoswere hybridizedwiththe posterior; u,ulna;r, radius. For allthesections(A-C,E-I),topisdorsalandleft only observedinproximal region oftheforearm. muscles visualizedinthecontrol PBSlimbs(H)are between limbs.Thetwoanteriorandventral the difference inventralmuscles ofmusclepattern proximal levelthanthePDGFBbead(I),explaining expression. ThePBSbead(H)isshownatamore with preparations ofPDGFB-implantedwingshybridized later, atE7/HH30orE7.5/HH31.(D)Whole-mount wings andtheembryosfixed2daysor2.5 were implantedintothedorsalregions ofE5/HH26 and markers. Fig. 7.PDGFBinhibitstheexpression ofmuscle expression of have anyeffect on around PDGFBbeads,whereas PBSbeadsdidnot the PBS (F,G,H) -treated wingswere hybridizedwith around thebead.SectionsofPDGFB(E,I)or MyoD MyoD MyoD probe followedbyimmunohistochemistry ( (E-G) orthe A probe (C).( show inhibitionof ) Transverse sectionsofwingsfrom MyoD MyoD (E) and D-I Pdgfr ) PDGFBorPBSbeads (F,G) or B Development 134(14) , C Pdgfr ␤ ) Transverse and (H,I) probes. The MyoD ␤ Pdgfr (I) wasinhibited expression ␤ Pdgfr (H) ␤ (B)

DEVELOPMENT section ofanE6wingshowingtheendogenousexpression of muscle connectivetissue. Tcf4 (A,B) and probes forthemuscleconnectivetissuemarkerscollagenI of thecorresponding control leftwings( the PDGFreceptors, mediating thevessel effect onmuscleandconnective tissue.Since muscle splitting,weproposethat thePDGFBisacandidatefor expression inthelimbis restrictedtoendothelialcellsatthetimeof on muscleandconnective tissue.Since PDGFBendogenous We observed thatPDGFBbeadapplicationmimicsthevessel effect Role ofPDGFsignalinginmusclesplitting endothelial cells. limb musclesplittingbyinducingthecorrectpositioning of for limbmusclecells(Kardonetal.,2003)couldalsobeinvolved in connection remainstobeestablished,TCF4providing apre-pattern transcriptional activation ofIL8(Levy etal.,2002).Althoughthe TCF4 hasbeenshown toinduceendothelialcellmigrationviathe signaling invessel development (GoodwinandD’Amore,2002). determined. Interestingly, observations pointtoaroleforWnt rules govern thisorganization, althoughthey remaintobe limbs andreproducibleamongembryos,suggestingthatspecific organization oftheearlyvasculature isvery stereotypedinavian is nosuchevidence inlimbs.Ourresultsindicatethatthe differentiation intheskin(Mukouyama etal., 2002).However, there influence vascular remodelinganddeterminethepatternofarterial There arealsoarguments indicatingthatthesensorynerves Vasculature andmusclesplitting dorsal musclemass(brown) inafuture siteofcleavage.( E7/HH30. ConsecutivesectionsofthePDGFB-treated wings( of E5/HH26chickwingsandtheembryoswere fixed2dayslater, at markers. Fig. 8.PDGFBactivatestheexpression ofconnectivetissue could beaconsequence ofthecombinedresponses ofconnective respectively, theinhibitoryeffect ofPDGFBonmuscleformation suitable timeinmusclemasses andmuscleconnective tissue, treated wingswere hybridizedwiththe (C,D) andthenincubatedwiththeMF20antibody. ( PDGFB orPBSbeadswere implantedintothedorsalregions Pdgfr Pdgfr ␤ ␣ and transcripts (blue)are detectedinthe Pdgfr Pdgfr A , C ␣ ) were hybridizedwith arebothexpressed ata , ␣ probe. E F ) PDGFB- ) Transverse B , Pdgfr D ) and ␣ in myoblasts from various musclecell lines(Jinetal.,1990;Yablonka- muscle massesandmuscles;(2) PDGFactivity inundifferentiated PDGF signaling:(1)thepresence of arguments indicatingthatmyogeniccellscandirectlyrespond to around thebeadsandexcluding myogeniccells.Thereareseveral MyoD whether PDGFBactsdirectlyon musclecells,possiblybyinhibiting in collagenIexpression (Fig.10).Oneinterestingquestionis (Li etal.,2004). shown todrive dermalfibroblast migrationontypeIcollagenmatrix cells toward thesourceofPDGFB.Interestingly, PDGFBhasbeen ECM (collagenI)allows themigrationofmuscleconnective tissue application inchicklimb(Figs9,10)suggeststhattheincrease of situ hybridization)versus celldensity(Hoechst)afterPDGFB 2004). Theanalysisofthetimingtranscriptionmodification (in human skinandrattendonmodels(Nesbitetal.,2001;Wang etal., Levels ofcollagenIhave beenshown tobemodified byPDGFBin increase inexpression oftheconnective tissuemarker collagenI results show thatthefirst event afterPDGFBbeadapplicationisan tissue (throughPDGFR incubated withtheMF20antibody. section tothatshowninF, hybridizedwiththe does notmodifycellproliferation around thebead.(G)Theadjacent incubated withtheanti-BrdU antibodyshowthatapplicationofPDGFB with the chick wings.( visualized withHoechst-labelednucleiatE7,2daysaftergraftinginto proliferation. Fig. 9.PDGFBincreases celldensitywithoutmodifying (A-D). Transverse sectionsfrom PBS( higher inmusclescompared withlimbconnectivetissueatthisstage modify muscleorganization(C).Itshouldbenotedthatcelldensityis whereas PBSbeadsdonotinduceanycellaccumulation(A)and around PDGFBbeads(B),where The PDGFeffect on expression, orindirectly byrecruitingconnective tissuecells MyoD C , Cell densityaround PBS( D probe. There isanincrease incelldensityhomogenously daetscin othoseshowninA,B,hybridized to ) Adjacentsections MyoD ␣ ) andmuscle(throughPDGFR MyoD expression occursaftertheincrease E ) orPDGFB( RESEARCH ARTICLE expression isinhibited(D), A Pdgfr ) andPDGFB( MyoD ␤ F transcripts inchick , G ) -treated wings probe andthen B ) beadswere ␤ ). Our 2587 .

DEVELOPMENT obvious effect on expression wasobserved(B),whereas no an increase incollagenI the MyoD MyoD implantation. ( visualized withHoechst-labelednuclei, 24hoursafterPDGFBbead observed 24hoursaftergrafting. (E). ( Seifert, 1993;Fiaschietal.,2003).Moreover, abnormalitiesin et al.,1990;Jin1991;1993;Yablonka-Reuveni and various skeletal musclecelllines(Yablonka-Reuveni etal.,1990;Jin been shown tospecifically inhibitmuscleterminaldifferentiation in by previous invitrostudies,whichPDGFB(and notPDGF-A)has muscle marker expression inchicklimbisnevertheless supported on muscleisdirectorindirect.Anegative effect ofPDGFBon signaling itisnotclearwhether, inourexperiments, PDGFaction consistent withthenotionthatmyogeniccellscanrespondtoPDGF mutagenesis (Chenetal.,2004).Althoughthesearguments are PDGFB transcriptionaltargets bymicroarray-coupledgene-trap 1998); and(4)theidentification ofvarious musclemarkers as ( after grafting.ConsecutivesectionsofthePDGFB-treated wings,9.5 of E5/HH26chickwingsandtheembryoswere fixedatvarioustimes myogenic cells. Fig. 10.PDGFBactsonconnectivetissuecellsbefore itactson phenotype observed in Reuveni etal.,1990;Fiaschi2003);(3)theskeletal muscle 2588 A , B MyoD ), 12( expression (lossof probe. RESEARCH ARTICLE C (A,C,E) andcollagenI(B,D,F)probes. Assoonas9.5hours, , D ) and24( H ) AnadjacentsectiontoGwashybridized withthe PDGFB beadswere implantedintothedorsalregions MyoD E expression wasobserved(A). Aneffect on , MyoD F Pdgfr ) hoursaftergraftingwere hybridizedwith expression around the bead)was ␤ mouse chimaeras(Crosbyetal., G ) Analysisofcelldensity, PDGFR tissue and/ordirectly byPDGFB actingonmusclecells,whichexpress inhibited asaconsequenceofthe accumulationofmuscleconnective muscle massseparation.Concomitantly, muscledifferentiation willbe tissue cellsandtheformationofa newmusclemembrane,allowing by producing collagenI.This promotes theaccumulationofconnective However, analysisofskeletal musclesinE13.5andE14.5 cells (expressing PDGFR cells, whichexpress PDGFR endothelial cellsactsinaparacrinemanneronmuscleconnectivetissue ( muscle massesare separated. (red) andmuscleconnectivetissuecells(green). ( cleavage siteinthemusclemass,whichiscomposedofmyogeniccells separation. theeffect ofthevasculature onmuscle mass Fig. 11.Modelfor Pdgfr patterning, includingfusionofmyotomes,have beenobserved in andKazlauskas,2004).Interestingly, defectsinmyotome Tallquist eye, lungandskin,inmousetransgenicmodels(Betsholtz,2004; endothelial cells.PDGFBcanalsoactivate PDGFR connective tissuecellsarealsoresponsive toPDGFBproducedby mesenchymal interactions,wefoundthatchicklimbmuscle signaling ismainlyassociatedwithPDGF-Ainepithelial- with anotherPDGF(Bergsten etal.,2001).AlthoughPDGFR muscle pattern(datanotshown); indicatingapossible redundancy mutant micedidnotshow consistentmodification ofthelimb Pdgfb skeletal muscleshave beennoted,althoughnotcharacterized,in cleavage. We cannotexclude anadditionalanddirect effect of splitting sitescouldexclude myogeniccellsandthusallow muscle progressive accumulationofconnective tissuecellsatthefuture migration andaccumulationtothefuturesiteofcleavage. The ECM (collagenI),whichinturnwillpromoteconnective tissuecell cells crossingthemusclemasses)willincreaseproductionof 2000). One attractive hypothesisisthatPDGFB(producedbyendothelial ␣ –/– ␤ . mutant mouseembryos(Soriano,1997;Tallquist etal., mutant mice(Lindahletal.,1997;Betsholtz2001). ( A ) Theendothelialcells(blue)delineatethefuture ␣ ) increase thesecretion ofextracellularmatrix ␣ Inresponse toPDGFB,connective tissue . C ) ThePDGFBsecreted bythe Development 134(14) B ) Atalaterstage,the ␣ signaling in Pdgfb –/– ␣

DEVELOPMENT Ataliotis, P. http://dev.biologists.org/cgi/content/full/134/14/2579/DC1 Supplementary materialforthisarticleisavailableat Supplementary material supported byINSERM. S.T. issupportedbytheFrench MinistryofResearch andbytheAFM.F.R. is (CNRS) andtheEU6thPCRDTthrough theMYORESNetworkofExcellence. Recherche Médicale(FRM),theCentre Nationaldela Recherche Scientifique theFondationpourla the Ministère delaRecherche (ACIjeuneschercheurs), Myopathies (AFM),theAssociationpourlaRecherche contre leCancer(ARC), Pdgfb Birchmeier, C.and Brohmann, H. Betsholtz, C.,Karlsson,L.andLindahl,P. Betsholtz, C. Bergsten, E.,Uutela,M.,Li,X.,Pietras,K.,Ostman,A.,Heldin,C.H.,Alitalo, Bates, D.,Taylor, G.I.,Minichiello,J.,Farlie,P., Cichowitz,A.,Watson, N., Baldwin, H.S.,Shen,M.,Yan, H.C.,DeLisser, H.M.,Chung,A.,Mickanin, Amthor, H.,Christ,B.,Weil, M.andPatel,K. Ambler, C.A.,Nowicki,J.L.,Burke,A.andBautch,V. L. Ahn, S.andJoyner, A.L. References mutant mice;JudithFavierforchick previous versionofthemanuscript;ChristerBetsholtzforproviding laboratory; LucPardanaud forhelpfuldiscussionsandcriticalreading ofa for herencouragementsandtheuseofunpublishedreagents from her We forhelpwiththeillustration;Margaret thankSophieGournet Buckingham molecular mechanismunderlyingmusclesplitting. endothelial andmusclecells,viaconnective tissuecells,provides a PDGFB/PDGFRsignalinginthecommunicationbetween of for vessels inthecleavage ofmusclemasses.Theinvolvement mass separation(Fig.11). tissue locallyandexclude musclecells,leadingultimatelyto PDGFB producedbythevessels canincreasemuscleconnective PDGFB experiments provide amolecularmechanismwhereby increase inapoptosisatthesiteofcleavage (datanotshown). Our and Tosney, 1991b).However, wedidnotdetectany significant removed byphagocytic cellsviaanunknown mechanism(Schroeter myotubes inthecleavage zonesarethenthoughttobeselectively electron microscopy (SchroeterandTosney, 1991b).Theresidual the myogeniccellsincleavage zonehasbeenobserved using PDGFB on Vasculature andmusclesplitting Beddington, R.S.andMartin,P. Boardman, P. E.,Sanz-Ezquerro, J.,Overton,I.M.,Burt,D.W., Bosch,E., Bonafede, A.,Kohler, T., Rodriguez-Niedenfuhr, M.andBrand-Saberi, B. Brand-Saberi, B.,Seifert, R.,Grim,M.,Wilting,J.,Kuhlewein, M.andChrist, platelet-derived growth factors. genetic studiesinmice. chondrogenesis bothinvivoandvitro. differentiation duringlimbmuscledevelopment. the PDGFbeta-receptor. K. andEriksson,U. 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