nerve, anddorsalobliqueEOM,innervated bythetrochlear rectus extraocular muscle(EOM),innervated bytheabducens mesoderm isthesourceoftwo oftheextrinsic eye muscles[lateral Noden, 1983a;Wachtler andJacob,1986).Moreover, this glossopharyngeal nerve) (Coulyetal.,1992;Jacob1984; laryngeal muscles(thirdbranchialarchmuscles,innervated bythe innervated bythefacial nerve) andthe anteriorpharyngealand opening andfacial muscles(secondorhyoidarch muscles, muscles (MAM),innervated bythetrigeminalnerve], thejaw including thejaw closuremuscles[firstormandibular arch mesoderm provides themusclesoffirstthreebranchial arches, (Gans andNorthcutt,1983).Theunsegmented paraxial head during vertebrate evolution andlargely develop like trunksomites tongue. They have beensecondarilyincorporatedintothehead posterior pharyngealandlaryngealmusclestheof Wachtler andJacob,1986).Theoccipitalsomitesdeliver the somites (Coulyetal.,1992;Jacob1984;Noden,1983a; otically, fromsegmented paraxialmesoderm,namelytheoccipital unsegmented para-andpre-oticheadmesoderm, post- a threattolife(Goodrich,1958). are crucialfunctions,andanimpairmentofcraniofacial musclesis gill breathing,andforthemovement oftheeyes. These,however, openings andfacial expression, forrespirationinanimalsrelyingon uptake, masticationandswallowing, forthecontrolofcranial purpose ofmusclesinthehead.Here,areusedforfood locomotion andthemaintenanceofbodyposture,thisisnot movements. However, whiletrunkmusclespredominantlyserve Vertebrate skeletal musclesarethebasisforallvoluntary INTRODUCTION arch, Jawmuscles, KEY WORDS:Chick,Quail,Embryo,Headmesoderm,Skeletalmuscles,EyeLateralrectus, Branchiomericmuscles,Mandibula signal, namelyBmp7,isanoverallnegativeregulatorofheadmyogenesis. function insupportingtheproliferativestateofmyoblasts,suppressingtheirdifferentiation,whileafurtherbranchialarch branchiomeric muscles.Moreover,weidentifiedFgf8asthebranchialarchderivedsignal.However,thismoleculehasanadditio known. Hereweshowthatantagonisticsignalsfromtheneuraltubeandbranchialarchesspecifyextraocularversus could beplacedintoamolecularnetwork,becausetheprogrammetocontroldevelopmentofmusclesinheadisnot Recent knockoutexperimentsinthemousegeneratedamazingcraniofacialskeletalmusclephenotypes.Yetnoneofgenes Gudrun vonScheven,LúciaE.Alvares*, RoyC.Mootoosamy specify eyeversusmandibulararchmuscles Neural tubederivedsignalsandFgf8actantagonisticallyto Development 133,2731-2745(2006)doi:10.1242/dev.02426 Accepted 3May2006 § † – PR,Brazil. Biológicas, DepartamentodeBiologiaCelular, 3oPiso,Sala201,81531-980 Curitiba das Américas,RuaCoronel FranciscoHoffman dosSantos100,SetordeCiências *Present address: UniversidadeFederaldoParaná–UFPR,Centro Politécnico,Jardim Tower, Guy’s Hospital,LondonBridge,SE19RT, UK. King’s CollegeLondon,DepartmentofCraniofacialDevelopment,Floor27Guy’s Wolfson Wing, HodgkinBuilding, Guy’sWolfson Wing, Campus,LondonSE11UL,UK. Author forcorrespondence (e-mail:[email protected]) Present address: King’s CollegeLondon,Wolfson Centre forAgeRelated Diseases, Muscles intheheaddevelop fromtwo sources:the Lbx1 , Paraxis , En2 , Myf5 , MyoR , Fgf8 , Bmp7 2000). Inzebrafishneuralcrestcells inmutantssuchas (Grammatopoulos etal.,2000;Noden,1983b;Pasqualetti etal., leads topatterningdefectsofbothboneandmuscle Moreover, they carrypositionalinformation,thealterationofwhich (Couly etal.,1993;KöntgesandLumsden,1996;Noden,1983a). tendons andmuscleattachmentpointsformusclesinthehead majority ofskullbones,andinadditiontheconnective tissue, head muscles(Noden,1983b).Neuralcrestcellsprovide the specify theindividual headmuscles,areknown. myogenic differentiation oftheheadmesoderm,norcascadesthat trunk muscle,defects.However, neitherthecascadesthattrigger 1999; Kitamuraetal.,1999),displaydistinctcraniofacial, but not et al.,2004),ormicelackingthehomeoboxgene Capsulin (Luetal.,2002),micelackingtheT-box gene mice forthetwo basichelix-loop-helixtranscriptionfactors MyoRand (Hadchouel etal.,2000;Summerbell2000),whileknockout promoter andenhancerelementsintheheadtrunk determining factor tobeexpressed intheembryo,employs distinct differentiation inthehead(Tzahoretal.,2003).Myf5,firstmuscle signals thatstimulatetrunkmuscledevelopment suppressmyogenic and Guthrie,1998;MootoosamyDietrich,2002).Moreover, Key regulators ofsomiticmyogenesis suchas distinct programmeofmyogenesis(MootoosamyandDietrich,2002). collectively refertoitasheadmesoderm. the samemyogenicpotential(thisstudy).We thuswill 1984; Wachtler andJacob,1986).Moreover, thismesenchymehas morphologically continuousstripofmesenchyme(Jacobetal., muscles oftheanteriorbranchialarchesdevelop froma mesoderm beforemuscledevelopment, suchthatEOMandthe Most oftheprechordalmesodermjoinsparaxialhead ventral obliqueEOM,allinnervated bytheoculomotornerve). head mesoderm(dorsalrectus,ventral rectus,medial rectus, nerve], whiletheremainingfourEOMoriginatefromprechordal gene forDiGeorge syndrome, (Kelly etal.,2004; Schillingetal.,1996).However, thecandidate in humanDiGeorge patients,headmuscleisseverely impaired muscle development directly (Kelly etal.,2004). Inaddition,in-vivo gene isexpressed inbranchial archmuscle,andhencemayinfluence indirectly (Vitelli etal.,2002a;Vitelli etal.,2002b).Moreover, the Classically, neuralcrestcellsarethoughttocontrolthedeveloping It was establishedonlyrecentlythattheheadmesodermemploys a † and SusanneDietrich § Tbx1 RESEARCH ARTICLE , affects neuralcrest cells Pax3 Pitx2 are absent(Hacker chinless (Gage etal., Tbx1 derived (Kelly 2731 r , and nal

DEVELOPMENT express additionalmarkers, with et al.,1992;Noden,1983a;Wachtler andJacob,1986).Here,muscles lateral rectusEOMismediallyadjacenttodeveloping MAM(Couly eye andbranchiomericmusclessitsidebyasthe anlage ofthe muscle innervation. and suppressesthespecification ofarchmuscles,inordertoaideye- This suggeststhattheneuraltube supportsthespecificationofEOM marker continued, themuscleanlage erroneously expressed theMAM unidentified cue.Initsabsence, whilemuscledifferentiation unrestricted, solubleandacts inconjuctionwithafurther, that specifiesthelateralrectusEOM.Thissignalisanteroposteriorly or musclespecification.However, theneuraltubeprovides asignal innervating nerves donotcontroltheonset of muscledifferentiation neural crestcells,theeye astheultimateEOMtarget, orthe to localised,extrinsic cues.Ablationexperiments thenrevealed that established thattheheadmesodermdevelops intomuscleaccording specification ofeye versus branchiomericmuscles. the toolstosimultaneouslyaddressdifferentiation and (Gardner andBarald,1992).Thus,focusingonthisterritory, wehave EOM (MootoosamyandDietrich,2002) before branchiomeric muscleprecursorsexpress resemble visceralmotoneurons(Jacobetal.,2001).Notably, 1992; Noden,1983a;Wachtler andJacob,1986).Theirmotoneurons mesoderm anddevelop withinthebranchialarches(Coulyetal., arise fromthemoreposteriorandlateralaspectofhead neurons inthetrunk(Jacobetal.,2001).Thebranchiomericmuscles Motor neuronsinnervating thesemusclesresemblesomaticmotor (Couly etal.,1992;Noden,1983a;Wachtler andJacob,1986). mesoderm andremainoutsidethebranchialarchesatalltimes EOM develop fromthemoreanteriorandmedialaspectofhead organised intotwo groups,theEOMandbranchiomericmuscles. development (Engle,2002). that innervation maynotberequiredfortheinitialstepsofmuscle Duane’s andFEOMtype2,muscle anlagenfirstdevelop, suggesting are absent(Nakanoetal.,2001).However, inanimalmodelsfor trochlear andoculomotor-innervated musclesfail, asthesenerves suffering fromfibrosisofextraocular muscles(FEOM)type2, the oculomotornerve (Engle,2002).Inasimilarvein, inpatients EOM, whicheitherdegenerates orsurvives whenmisinnervated by syndrome, theabducensnerve fails toinnervate thelateralrectus muscle development inthehead.Inpatientssuffering fromDuane’s muscle development requiresthepresenceofmuscletargets. their eyes andeye muscles.Thus,itremainsopenwhetherhead have highlyspecialisedlifestylesandmayhave secondarilylost However, hagfishes,whichconstituteasistergrouptolampreys, musclesinvertebrates (http://tolweb.org/tree?group= Hyperotreti&contgroup=Craniata) (BrodalandRagnar, 1963). eye of remarkable exception totheotherwisestereotypearrangement 1981). Likewise, ineyeless hagfishes,noeye musclesarefound,a muscles areseverely reduced(Paterson andKaiserman-Abramof, in are alsosuspectedtocontrolcraniofacial myogenesis.For example, role ofthesecellsremainsunclear(Tzahoretal.,2003). requirement ofneuralcrestcellsformuscledifferentiation; thusthe and in-vitrostudiesledtoconflictingresultsregarding the 2732 At thelevel oftheanteriorhindbrain,precursorsforprospective Using heterotopictransplantationinthechickembryo,wefirst Conceptually, musclesthatdevelop fromheadmesodermcanbe Finally, theinnervating nerves have beenthoughttocontrol Besides neuralcrestcells,thestructurestargeted byheadmuscles ZRDCT-An Myf5 RESEARCH ARTICLE En2 . Moreover, theabducensnerve fellshortofitstarget. (von Scheven etal.,2006). anophthalmic mousemutantslackingeyes, eye Paraxis and En2 Lbx1 MyoR labelling theMAM characterising the and Capsulin MATERIALS ANDMETHODS differentiation. indicating thatFgf8controlsmuscleprecursorproliferationversus then harvested andfixed asoutlinedabove. from HH10donors.Aftersurgery, theeggs wereincubatedfor 48hours, and Dietrich,2005);themid-hindbrainneuraltubegraftswerederived levels wereperformedatHH16asdescribedbyLoursandDietrich(Lours 0.05 experiments using25- [Goodfellow; (Dietrichet al.,1998;Dietrichet1997)],filter experiments wereperformedusing7.5- emigration ofcranialneuralcrestcells(Lumsdenetal.,1991).Barrier Recombinant humanFgf4orFgf8(R&D)at500 Protein-loaded beads 1951). incubator. Embryoswerestagedaccordingto(Hamburger andHamilton, and PotterFarm (Woodhurst), andincubatedat38.5°Cinahumidified Fertilised hens’andquails’eggs wereobtainedfromWinter Farm (Royston) Chick andquailembryos undifferentiated archmuscleprecursors,andsuppressed addition, Fgf8upregulated signals andsupportsMAMagainstEOMdevelopment. In expression, indicatingthatitantagonisestheneuraltubederived function: itupregulated head muscledevelopment. Fgf8,however, displayedadual arches, wefoundthatBmp7hadageneralised,negative effect on beads loadedwithsignallingmoleculesexpressed inthebrachial stimulate MAMandsuppressEOMdevelopment. Implanting neural tubesuggestedthatbranchialarchderived signalsmight Bmp7 Probes andtheirexpression patternsaredetailedinthefollowing references: (Dietrich etal.,1998;Dietrich 1997;MootoosamyandDietrich,2002). Whole-mount in-situhybridisation was carriedoutaspreviously described In-situ hybridisation (Schubert andLumsden,2005). hindbrain levels andelectroporatedasdescribedinSchubertLumsden (Bell etal.,1999)was injectedintotheHH10neuraltubeatmidbrain- 2003) orthisvector harbouringtheopenreadingframeof mouse The pCA Electroporation mesoderm fragments,whichwerecarriedoutatHH8-9 neural crestablationsandtheheterotopictransplantationofhead development and innervation (Nodenetal.,1999).Theexceptions were Cranial operationswereperformedatHH10,beforeheadmuscle et al.,1998;Dietrich1997;MootoosamyandDietrich,2002). tungsten needlesasdescribedpreviously (Alvares etal.,2003;Dietrich In ovo microsurgery was carriedoutwithflame-sharpened100- Microsurgery described inChengetal.(Chengal.,2004). Celltracker Orange(MolecularProbes), aggregated andimplantedas Rat B1controlcellsandexpressing Wnt1wereraised,labelledwith Tissue culture cellimplants at 500 (Alvares etal.,2003).Recombinant humanBmp2,Bmp4orBmp7(R&D) loaded ontoHeparin-Acrylicbeads(Sigma)asdescribedbyAlvares etal. 1992); et al.,1998). Affi-Gel blueagarosebeads(Biorad)asdescribedinDietrichetal.(Dietrich 1997); (Saitoh etal.,1993); The upregulation of ␮ (Begbie etal.,1999); ␮ m [Costar;(Fan andTessier-Lavigne, 1994)].Operationsatflank Shh Fgf8 g/ml or100 ␤ -IRES-eGFP vector describedinAlvares etal.(Alvares etal., (Johnson etal.,1994). (Mahmood etal.,1995); MyoR ␮ g/ml andShh(R&D)at100 ␮ m-thick polycarbonatefilterswithaporesizeof En2 (von Scheven etal.,2006); En2 Dlx5 upon separationofheadmesodermand MyoR expression andsuppressed (Ferrari etal.,1995); Lbx1 , amarker forproliferative, (Dietrich etal.,1998); ␮ Development 133(14) m-thick tantalumfoil ␮ ␮ g/ml or100 g/ml was loadedonto Paraxis En2 – (Logan etal., , beforethe (Sosic etal., ␮ g/ml was Paraxis Hoxb1 Myf5 Myf5 ␮ m ,

DEVELOPMENT Dietrich, 2002). Whenthesametissuewas graftedanteriorlynext to rhombomere2,i.e.atthecorrect position(Mootoosamyand lateral rectusEOMmarkers was confinedtotheterritoryadjacent Wachtler andJacob,1986)( fate-mapping experiments (Coulyetal.,1992;Noden,1983a; hindbrain andwithinthemandibular arch,inlinewithclassical orthotopically, quail-derived tissuewas foundnext totheanterior (Mootoosamy andDietrich,2002). HH19, asallmarkers arerobustly expressed atthisstage staining). Theembryoswereanalysed48hourspost-surgery at performed beforeneuralcrestcellemigrationatHH8-9 donors andstage-matchedchicken hosts.Theoperationswere on therightsideofembryosasindicatedinFig.1A,usingquail intrinsic orextrinsic cues. localised formationofmuscleanlagenintheheaddependson (Trainor andKrumlauf,2000).We thusinvestigated whetherthe can superimposeitsidentityonindividuated neuralcrestcells 1983a; Wachtler andJacob,1986).Moreover, theheadmesoderm arches develop fromdistinctregions (Coulyetal.,1992;Noden, established thatEOMandthemusclesoffirstthreebranchial al., 2003).Fate mappingexperiments fortheheadmesodermhave programme formigratorymuscleprecursorformation(Alvares et possess positionalinformationthatpredisposesthemtowards the We recentlyshowed thatinthetrunk,limbmuscleprecursors Heterotopic graftingofheadmesoderm RESULTS Zeiss Axiophot,usingNomarskioptics. sagitally beforeanalysis.Embryosandsectionswerephotographedona cleared in80%glycerol/PBS.Whole-mountedembryosweresplitmid- After in-situhybridisationand/orimmunohistochemistry, embryoswere Photomicroscopy either 80%glycerol/PBS,orAquamount(BDH). Vibratome. Sectionswerecollectedongelatinisedslidesandmountedin up to2days,thenrinsedinPBSandsectioned50 cooled to4°C.Subsequently, blocksweretrimmedandfixed in4%PFA for Embryos wereembeddedin20%gelatin(Sigma)PBSat50°C,then Sectioning (Vector Laboratories). horseradish peroxidase(Dako; dilution1:100)andaperoxidasesubstratekit In allcases,signalsweredetectedusinganti-mouseIgGconjugatedwith protein usingananti-eGFPantibody(Molecularprobes;dilution1:2000). Studies HybridomaBank,University ofIowa, IA;dilution1:200),andeGFP dilution 1:200),thedeveloping eye usingaPax6 antibody(Developmental antibody (Developmental StudiesHybridomaBank,University ofIowa, IA; neurofilament subunit. QuailtissueswereidentifiedusingtheQCPN (Zymed; dilution1:3000),whichrecognisesthe155-kDaintermediate 1992). Axonalstainingwas performedusingtheRMO270antibody carried outaccordingtoGuthrieandLumsden(GuthrieLumsden, Upon in-situhybridisation,whole-mountimmunohistochemistrywas Immunohistochemistry Head musclespecification expression ofthemuscle-determiningfactor position ofallmuscleanlagenwas examined, assayingforthe staining) asmarkers; bothmarkers gave identicalresults.The investigated using H, brown staining).ThespecificationofthelateralrectusEOMwas grafted orthotopically. control, headmesodermatthelevel ofthe anteriorhindbrainwas patterning cuesforthedeveloping muscles(Noden,1983b).Asa crest cellshave beensuggestedtoprovide positionalinformationand When headmesodermfromrhombomere2levels was grafted Heterotopic graftingofheadmesodermfragmentswas carriedout The graftedtissuewas tracedusingtheQCPNantibody(Fig. 1B- Paraxis (not shown) and n =4, Fig.1B).Theexpression of Myf5 Lbx1 ␮ m onaPelco1000 (Fig. 1G,H,blue (Fig. 1B-F, blue – , asneural lateral rectus EOMwith Quail tissueswere detected withtheQCPNantibody(brown), the to thetop,dorsalleft,ventral(branchialarches) totheright. Lateralviewofthe headsofoperatedembryosatHH19;anterior (B-H) otic levels/rhombomere 5-6wasgraftednexttorhombomere 2. rectus EOM. neural tube;ov, oticvesicle;r, rhombomere; som,somite;vr, ventral hyoid arch; lr, lateralrectus EOM;m,midbrain;ma,mandibulararch; nt, Scale bar:200 Note thatthemolecularmarkersshowed normalexpression patterns. the neuraltube),andmuscleanlagen ingeneralwith levels wasgraftednexttorhombomere 2;in levels/rhombomere 5-6( anteriorly nexttotheanteriormidbrain( rhombomere 2levelswasgraftedorthotopicallyascontrol ( of embryos,anteriortothetop.Quail-derivedheadmesodermfrom local environment. Fig. 1.Headmesodermdevelopsintomuscleaccording toits ␮ m. do,dorsalobliqueEOM;dr, dorsal rectus EOM;hy, ( A ) SchemeofoperationsatHH8-9 Lbx1 D ). In (B-F; blue;note:additionalexpression in E , G , headmesodermfrom midbrain RESEARCH ARTICLE C ) orposteriorlynexttootic F , H , headmesodermfrom Myf5 – ; dorsalview (G,H, blue). B ), 2733

DEVELOPMENT ( also thecasewhenheadmesodermfromanteriormidbrainlevels developing lateralrectus,next torhombomere2(arrows). Thiswas graft. However, normalexpression was seenattheusualsiteof ( to theanteriormidbrain( 2734 n n =2; Fig.1D),thelateralrectusmarkers werenotexpressed inthe =6; Fig.1E)orfromoticlevels ( RESEARCH ARTICLE n =4; Fig.1C)orposteriorlytooticlevels n =6; Fig.1F)was rhombomere 2levels was confirmedby grafting ofanteriororposteriorheadmesodermfragmentsto normal development ofmuscleanlagenupontheheterotopic heterotopically graftedtoanteriorhindbrainlevels (arrows). The identical (notshown). Thissuggeststhatheadmuscles develop in Moreover, operatedandunoperatedsidesoftheembryoslooked (R-U) neural crest ablatedembryos(P,Q). and express and ventralretcus EOMare differentiating lateral rectus, dorsaloblique,rectus mandibular andhyoidarch musclesandthe wild type(N,O),muscleanlagenforthe (L,M) embryo.(N-Q) both theunoperated(J,K)andoperated expressed inthemandibulararch musclesof embryos, (B-E) antibody totracethenervoussystem(brown). Embryos are doublestainedwiththeRM0270 anterior istothetop,dorsalleft. D,H,L,P,T. Embryosare atHH19;lateralviews, lateral rectus/MAM region asindicatedin ␮ unsegregated (smallarrows). Scalebars:400 expression domainsremained proximally the absenceofneuralcrest cells,the (R,S) andoperated(T,U) embryo.Notethatin muscle anlagenispresent inthewildtype associated withtheventral/branchiomeric rectus EOM.(J-M) successful. (F-I) indicates thatthesurgicalprocedure was trigeminal ganglionare separate.This and themandibulomaxillaryaspectof (open arrowheads). Moreover, theophthalmic neural-crest-associated expression isabsent and mandibularectodermremained, the otic placodesandexpression inthemaxillary expression inthederivativesofnasaland arches. (D,E)Operatedembryo.While the neuralcrest cellsfillingthebranchial (H,I), wild type(F,G) andintheoperatedembryo ventral rectus EOM. vesicle; r, rhombomere; VII,facial nerve;vr, aspect ofthetrigeminal(Vth)nerve; ov, otic aspect ofthetrigeminal;oph,ophthalmic mandibular arch; mmx,mandibulomaxillary dorsal rectus EOM;hy, hyoidarch; ma, diencephalon; do,dorsalobliqueEOM;dr, ␮ embryos ( boxed area inB,F,J,N,R. ( lateral rectus/MAM region corresponding to ( ( top, ablatedarea inturquoise. neural crest cells.Dorsalview, anteriortothe tube/neural foldablationatHH8toremove specification. for earlyheadmuscledifferentiation and Fig. 2.Neuralcrest cellsare dispensable C B m inB,F,J,N,R; 200 m inD,H,L,P,T; 200 , , G F , , J K Dlx5 Paraxis , MyoR N , O , R , Dlx5 E S Headsofunoperatedembryos; ) expression. (B,C)Inwild-type , ) highermagnificationofthe I expression. Myf5 , labels thedevelopinglateral M is predominantly expressed in ( , Paraxis A Q Myf5 ) Schemeofdorsalneural . Thisisalsothecasefor , U En2 ) highermagnificationof Myf5 ␮ ␮ Development 133(14) m inC,G,K,O,S;400 m inE,I,M,Q,U.d, expression. Bothinthe expression. MyoR staining (Fig.1G,H). D , expression. Inthe H , L expression , P , T ) Operated En2 MyoR Dlx5 is

DEVELOPMENT lateral rectus anlagen.(D,E) Also in operatedembryostheeyeismissing; onlytherest oftheopticstalkremained (C,E,open arrowhead). (B,C) sides (C,E).(B-E)Brown staining: Pax6antibodystainingdetectingstrong expression inthetelencephalon(t),eye,neuraltub ventral rectus EOM. unsegmented headmesoderm;hy, hyoid arch; lr, lateralrectus EOM;ma,mandibulararch; nt,neuraltube;ov, oticvesicle;t,t mesoderm. ( view, anteriortothetop.Theoptic placodeandtheregion oftheforebrain delivering theopticcupwere removed. Pink:unseg ( markers wereappropriatelyexpressed atthelevel ofrhombomere2 their neuronalphenotype.We foundthatthelateralrectusEOM judging theneural-crestablatedstateofembryosonbasis investigated themuscularphenotypes oftheoperatedembryos, al., 1999;Veitch etal.,1999)(Fig.2D,E,oph,mmx).Next, we the mandibulomaxillary aspectofthetrigeminalganglion(Begbie et crest-deficient embryos,namelyaseparationoftheophthalmicand RMO antibody, wefoundthe typical neuronalphenotypeofneural- open arrowheads). Moreover, tracingthenervous systemwiththe embryos lacked neural-crest-associated neural crest(Ferrarietal.,1995)(Fig.2B-E).Indeed,operated were analysedfortheexpression of et al.,1999).To confirmthatthiswas thecase,embryosatHH19 crest regeneration (Begbie etal.,1999;Lumsden1991;Veitch This operationeliminatesthesourceofneuralcrestcellsandprevents diencephalic tooticlevels atHH8-9 dorsal thirdoftheneuralplatewereablatedbilaterallyfrom specification. To investigate theirroleinvivo, theneuralfoldsand signals positively regulating headmuscledifferentiation and (Noden, 1983b),they werethefirstcandidatesinourquesttoidentify As neuralcrestcellshave beenimplicatedinheadmyogenesis Ablation ofneuralcrest cells muscles. suggests thatextrinsic cuesgovern thedevelopment ofhead tune withtheirlocalenvironment (ortsgemäss).Thisfurthermore Head musclespecification Fig. 3.Thepresence ofthe eyeisnotrequired foreyemuscledifferentiation andspecification. muscle precursors(von Scheven etal.,2006)was present,although Finally, wild typewerealsopresentintheoperatedembryos(Fig.2N-Q). arches. Importantly, alldifferentiating muscleanlagenpresentinthe the headmesodermoccupiedallavailable spaceinthebranchial expression patternindicatedthatintheabsenceofneural crestcells, labelled theMAM(GardnerandBarald,1992)(Fig.2J-M).The Paraxis : Fig.2F-I, MyoR B-E , amarker forproliferative, undifferentiated arch ) LateralviewsofHH19heads;anterior tothetop,dorsalrightforunoperatedsides(B,D), totheleftforoper Lbx1 not shown). Likewise, Myf5 Dlx5 – expression (blue)iswildtype. Scalebar:200 ( n =34), asindicatedinFig.2A. Dlx5 , amarker forbranchialarch expression (Fig.2D,E, En2 correctly Myf5 expression ofthelateralrectusmarkers and,using a After reincubationfor48hours,theembryoswereassayed the partofforebrainproviding theopticcup(Fig.3A; specification, atHH10weunilaterallyablatedtheopticplacodeand whether theeye isresponsibleforEOMdifferentiation and reduced (Paterson andKaiserman-Abramof,1981).To investigate anophthalmic mousemutantslackingeyes, EOMarestrongly The eye isthetarget ofthedeveloping EOM,andin Ablation oftheeye muscle differentiation orEOM-MAMspecification. spacing andseparationofmuscleanlagen,but notintheinitiationof small arrows). Thus,neuralcrestcellsseemtoplay aroleinthe expression domainsremainedproximallyunsegregated (Fig.2R-U, rectus EOMusingaprobefor was tracedwiththeRMOantibody(brown staining), andthelateral and 6atHH10( innervation was prohibitedbysurgically removing rhombomeres5 cognate nerves (Engle,2002).To testthishypothesis,lateralrectus EOM formationhasbeensuggestedtodependonthepresenceof innervation Prevention oflateralrectus eyemuscle and specificationdoesnotrelyonthepresenceofmuscles’target. staining) werecorrectlyexpressed. Thus,earlymuscledifferentiation shown), was lacking(Fig.3C,E,openarrowheads). Significantly, side onlyasmallremnantoftheopticstalkwas present,whiletheeye were photographedandcompared.We foundthatontheoperated staining, theembryoswerebisectedmid-sagittally, andbothhalves using the the presenceofheadmusclesingeneral.Theablationwas controlled from theinside.Onunoperatedside,rootletsofabducens staining). Theembryoswerethenmid-sagittallybisectedandviewed ␮ m. do,dorsalobliqueEOM;dr, dorsal rectus EOM;hm, Lbx1 Pax6 (Fig. 3C,bluestaining)and n antibody (Fig.3B-E,brown colouring).Upon =10; Fig.4B).AtHH19-20,thenervous system ( A ) SchemeofeyeablationatHH10; dorsal Lbx1 Lbx1 RESEARCH ARTICLE (blue staining)revealing normal (not shown) or e (nt)/hindbrain.Note, Myf5 elencephalon; vr, mented head Myf5 (Fig. 3E,blue Paraxis Paraxis ZRDCT-An probe, for ated n =11). 2735 (blue (not

DEVELOPMENT notochord/floor plateseparationexperiments, inwhichthe was tracedwiththeRMO antibodyexcept inthecaseof for muscledifferentiation markers. Moreover, thenervous system expression oflateralrectus EOMmarkers, forMAMmarkers and of rhombomere3( inserting foilperpendiculartothe longitudinalbodyaxisatthelevel rhombomere 1( perpendicular tothelongitudinalbodyaxisatlevel of ( located tissuesinthemandibular archbyinserting foillaterally with thetrigeminalganglion);(4)separationfromlaterally inserting foilunderneath( ectoderm interface, peelingtheectodermbackaidedbydispase, then mesoderm andsurface ectoderm,makingacutattheneural tube- lateral tofloorplate/notochord( mesoderm withfoilintroducedthroughtheroofplate,theninserted ( impermeable barrieroftantalumfoilatthelevel ofrhombomere2 separation ofneuraltubeandheadmesodermbyinsertingan tissues. SixtypesofoperationswerecarriedoutatHH10:(1) therefore systematicallyinvestigated therolesofneighbouring of themuscleanlagenmayaccountfortheirdevelopment. We cognate nerve. Thissuggeststhattissuesintheimmediate vicinity not, ornotexclusively, provided byneuralcrestcells,target organ or muscle differentiation andspecification.However, thesecuesare Our studyshowed thatextrinsic cuesarerequiredforearlyhead mesoderm from surrounding tissues Separation oftheeye-muscle-deliveringhead that fortheonsetofmuscledevelopment thenerve was dispensable. Paraxis the operatedside,abducensnerve was missing.However, innervating thelateralrectusanlage(arrow) (Wahl etal.,1994).On and 6(Fig.4A,arrowheads), withaxonsextending anteriorly, nerve wereclearlyvisibleintheventral region ofrhombomeres5 (open arrowhead), but Rhombomeres 5/6ablatedside.Theabducensnerveismissing (B) (arrowheads) andaxonshavingentered the nerve (cranialVI)withrootlets inrhombomeres 5and6 RMO270 inbrown. (A)Unoperatedcontrol side.Notetheabducens stained for anterior tothetop,dorsalleftin and specification. Fig. 4.Innervationdoesnotcontrol eyemuscledifferentiation 2736 r, rhombomere; VI,abducensnerve. n n =18); (5)separationfromanteriortissuesbyinsertingfoil =59); (2)separationofnotochordandfloorplatefromthehead The embryoswereanalysed48 hoursaftertheoperationfor RESEARCH ARTICLE expression was identicaltotheunoperatedside,indicating Paraxis expression (blue);nervoussystemtracedwith n Internal viewsofHH20bisectedchickheads; Internal n =6); (6)separationfromposteriortissuesby =8). Paraxis n =19) (thisoperationalsoprevents contact is expressed normally. Scalebar:200 n =23); (3)separationofhead A and rightin Paraxis domain (arrow). B . Embryo ␮ m. well establishedonthecontrolside,expressing shown inFig.5A,Ai,then48hourslater, thelateralrectusEOMwas When neuraltubeandheadmesodermwereseparatedatHH10,as gene expression Separation ofheadmesodermandneuraltube-marker the markers andareshown here(Fig.5). and headmesodermledtosignificantchangesingeneexpression of performed. However, onlytheexperiments separatingneuraltube sectioning atthelevel ofrhombomere2andthemandibular archwas examine marker geneexpression indetail,transverse vibratome notochord andfloorplatewerestainedfortheexpression of its specification maybeachieved eitherviaasolublesignal or neural tubebeforebecomingengaged withthedeveloping eye. Thus, The lateralrectuseye muscle anlageresidesincloseproximitytothe Transmission oftheneuraltubederived signal rectus EOM Properties ofthesignalspecifyinglateral specified lateralrectusanlageandtheabducensnerve was defective. (arrowhead). Thissuggeststhatthecommunicationbetweenre- axons notreadilyfindingatarget (Tosney andLandmesser, 1985) Paraxis the abducensnerve (arrow). Ontheright, operated sidethatlacked Paraxis (Fig. 6B,C; innervation patternsontheunoperated(Fig.6A)andoperatedsides Paraxis as before,cultivated theembryosto HH19-20, doublestainedfor blocked theneuraltubederived signal forthelateralrectusmuscle target recognitionoftheabducens nerve arelinked. We therefore It thusseemsconceivable thatlateralrectusspecificationandthe the moreanteriorlylocatedremainingfive EOM(Wahl etal.,1994). rectus muscleanlage,disregarding thelaterallyadjacentMAMand of theneuraltubeinananteriordirectionandtoidentifylateral have tosampletheirenvironment toextend alongtheventral surface (Hernandez etal.,2004;Moens1998).However, theaxons information andpatterningcueswithinrhombomeres56 The specificationofabducensmotoneuronsdependsonpositional innervation phenotype Separation ofheadmesodermandneuraltube- arrows) and arrows). Ontheoperatedsidehowever, the moremediallylocatedanlageoflateralrectusEOM(Fig.5N, closure musclesdeveloping inthemandibular arch,stayingclearof unoperated side, was normal,asevidenced bythewild-typeexpression of Likewise, thebalancebetweenmuscleproliferationanddifferentiation on theunoperated,aswelloperated,side(Fig.5H-J,arrows). was notduetothelossofmuscleanlage,as 5C,D,F,G, openarrowheads). ThislossofEOM-marker expression however, both markers werestronglyreducedorabsent(Fig. expresses markers forthemandibular archmuscles. differentiates, but lacksthelateralrectusmarkers and,instead, tube derived signal,muscleinthepositionoflateralrectusanlage muscle anlage(Fig.5O,P, arrowheads). Thus,inabsenceoftheneural adjacent MAM,westainedtheembryosfor occupied bythelateralrectusmayhave adopted thefate ofthelaterally but thespecificationaslateralrectusEOMhadfailed. 5K-M, arrows). Thus,muscledifferentiation commencedcorrectly, To investigate whetherthemuscleinpositionnormally -RMO andmid-sagittallyopenedtheembryostoreveal the , thenerve showed adefasciculated appearance typicalfor -expressing muscleanlage(arrowhead) was innervated by n Paraxis =17). We foundthaton theleft,unoperatedside, En2 signals wereconfinedtotheprospective jaw (Fig. 5E,G,arrows). Ontheoperatedside En2 staining encompassedthis En2 Development 133(14) Myf5 expression. Onthe Lbx1 was expressed (Fig. 5B,D, MyoR Shh (Fig. . To

DEVELOPMENT Head musclespecification markers neural tubefrom theheadmesoderm,lateralrectus of thebarriersare indicated.Notethatuponseparationofthe the top,operatedsidestoright,markersandposition sections atanteriorhindbrain/mandibulararch levels;dorsalto I,facialnerve. VII, ov, oticvesicle;r, rhombomere; V, trigeminalganglion; arch; ncc,neuralcrest cells;not,notochord; nt,neuraltube; tube; ht,heart;hy, hyoidarch; m,midbrain;ma,mandibular ectoderm; fge,foregut endoderm;fp,floorplateoftheneural bar: 200 specification, butnotdifferentiation, wasperturbed.Scale expressing thelateralrectus markers.Thus,muscle anlage medially, encompassingthemusclenormally expression (N-P)spread formthemandibulararch muscle Myf5 sides; anteriortothetop,dorsalleft.( top, dorsaltotheright.( ( impermeable barrier(tantalumfoil)isindicated. HH10; headmesoderminpink,thepositionof (dorsal tothetop)illustratingsurgicalprocedures at muscle. Fig. 5.Theneuraltubespecifiesthelateralrectus eye B , E , H (H-J) and , K Lbx1 , ( ␮ N A m. d,diencephalon;da,dorsalaorta;ect,surface Lateralviewsofunoperatedsides;anteriortothe ) ) Schematicdorsalviewand(Ai)cross section (B-D) and MyoR (K-M) expression continued. Paraxis C , F , I RESEARCH ARTICLE , L , (E-G) failedtobeexpressed. O ) Lateralviewsofoperated D , G , J , En2 M , P ) Cross 2737

DEVELOPMENT development atanectopiclocation. specific, andfurthermore,notsufficient totriggerlateralrectusEOM indicating thattheneuraltubederived signalisnotrhombomere2- lateral rectusmarker scheme oftransplantationsisshown inFig.7A.Inallcases,the hindbrain (Fig.7F, diencephalon-anterior mesencephalon(Fig.7E, oticlevels (Fig.7D, or midbrain toanteriormidbrain-diencephaliclevels (Fig.7C, rhombomere 2plustheanteriorlyadjacentr1,isthmusandposterior orthotopically ascontrol(Fig.7B, 2000). Therefore,wetransplantedatHH10rhombomere2 positional values unlesstheisthmusisincluded(IrvingandMason, Rhombomeres, whengraftedtoaposteriorposition,losetheir Heterotopic neuraltubegrafting of rhombomere2via heterotopic graftingofneuraltissues(Fig.7);and(2)transformation 2-specific. For this,two setsofexperiments werecarriedout:(1) required forthedevelopment ofthismusclewould berhombomere rhombomere 2,weasked whethertheneural-tube-derived signal and Mason,2000).With thelateralrectusEOMdeveloping next to Rhombomeres ofthehindbraineachcarryadistinctidentity(Irving Spatial restriction oftheneuraltubederivedsignal abducens nerve was restored(Fig.6E,F, arrows). Paraxis for theirinnervation phenotype.Notably, ontheoperatedside,both the expression of Lavigne, 1994)( cellular extensions but admitssolublefactors (Fan andTessier- the anteriorhindbrainandheadmesoderm.Thisfilterexcludes inserting a25-mm-thickfilterwithporesizeof0.05 strategy asfortheearlierbarrierexperiments; thistime,however, through cell-cellcontact.To investigate this,weusedthesame 2738 used. We expected tofindan upregulation ofthelateral rectus and RatB1fibroblastcarryingthe emptycloningvector ( controls, beadsloadedwithbovine serumalbumin (BSA)( mesoderm atthelevel ofthefuturelateralrectusEOM(Fig.9A).As Fan etal.,1997;Münsterberg etal.,1995)( inserted beadsloadedwith100 neural tubefitthisdescription(BrentandTabin, 2002).We therefore Shh derived fromthefloorplateandWnt1 derived fromthedorsal signal fromtheneuraltubeactsinlateralrectusEOMspecification. Our analysissuggestedthatasoluble,anteroposteriorlyunrestricted Test forneuraltubederivedsignalling molecules EOM marker (shown schematicallyinFig.8A).However, neitherthelateralrectus established bySchubertandLumsden(SchubertLumsden,2005) the posteriormidbrain-anteriorhindbrain,usingconditions D, the pCA rhombomere 4(Belletal.,1999).We electroporatedatHH10either in rhombomere2,homeoticallytransforms2into Hoxb1 Hoxb1 misexpression ( is notconfinedtorhombomere2. experiments, thesignalsupportinglateralrectusEOMdevelopment 8D,G) wereaffected. Thus,consistentwithourheterotopicgrafting n =6) orRatB1cellsengineeredto releaseWnt1(Chengetal.,2004; n =5) orthisvector expressing mouse controls theidentityofrhombomere4and,whenmisexpressed RESEARCH ARTICLE ␤ expression andinnervation ofthelateralrectusby -IRES-eGPF controlvector (Alvares etal.,2003);(Fig.8B- Paraxis n Paraxis =16, Fig.6D-F).Theembryoswereanalysedfor n Hoxb1 =7) tomidbrain-rhombomere2levels. The (Fig. 8C,F)northemusclemarker Lbx1 and doublestainedwiththeRMOantibody n misexpression (Fig.8). =5), andwegraftedtheposterior was expressed atthenormalsite, ␮ n g/ml recombinantShhprotein =3), but heterotopicallygrafted Hoxb1 (Fig. 8E-G, n n =13) intothehead =4) ortheposterior ␮ m between Myf5 n n =2) were =11) into n n =12) (Fig. =3) side theabducensnerveiswellfasciculatedandinnervated with tantalumfoil,animpermeablebarrier. Notethatonthecontrol filter isindicated.(A-C)Separationofneuraltubeandheadmesoderm dorsal totheleftinA,D;rightB,C,E,F. Positionofthebarrieror and after(C,F)removal ofthebarrierorfilter. Anterioristothetop, Paraxis the abducensnervenavigatedaround thefilterandinnervated factors. Notethat and headmesodermwithapolycarbonatefilterthatadmitssoluble express nerve isdefasciculatedanddidnotreach thearea thatnormallywould expressing lateralrectus anlage(A).Ontheoperatedside(B,C), pharyngeal pouches (endodermplusectoderm) andtheoral 2006) (Fig.9Ai,iii).Subsequently, elevated signalsarefoundinthe at HH10,thetime Fgf andBmpmoleculesareexpressed inthepharyngealendoderm mesoderm anddeveloping muscles(S.D.,unpublished).Moreover, Importantly, receptorsforthesemoleculesareexpressed inthehead development areFgfandBmpmolecules(Helmsetal.,2005). of-function experiments. arches andleadtofatal bleeding,weperformedcandidategene/gain- because they disturbarchpatterningasawhole,injuretheaortic development. Asablationexperiments inthisareaarenotfeasible from thebranchialarchesmightsuppressEOMandsupportMAM markers fortheneighbouringMAM.We thusasked whethersignals simultaneously specifiesthelateralrectusEOMandsuppresses Our experiments indicatedthatthesignalfromneuraltube Signals forthebranchialarch muscles described in(Tzahoretal.,2003). material), inlinewiththeirrepressive functionforheadmyogenesis I abducensnerve. VI, views of Fig. 6.Thesignalfrom theneuraltubeisasolublefactor. strongly suppressed markers inresponsetoShhorWnt1.Instead,Wnt1slightly, Shh ( A , Signalling moleculesknown toinfluencebranchialarch D Unoperatedcontrol sides,( ) -expressing lateralrectus. Scalebar:200 Paraxis Paraxis next torhombomere 2.(D,E)Separationofneuraltube -RMO270 stained,bisectedembryoatHH20. Paraxis MyoR Paraxis is expressed ontheoperatedside.Moreover, expression commences (von Scheven etal., (see Fig.S1inthesupplementary B , C , E , F ) operatedsidesbefore (B,E) Development 133(14) ␮ m. r, rhombomere; Internal Paraxis -

DEVELOPMENT rhombomere. rectus EOM;m,midbrain; ma, mandibulararch; ov, oticvesicle;r, arrow). Scalebar:200 of anectopicoticvesicle,andalso permitted otic placodetomidbrain-rhombomere 2levels,ledtothedevelopment arrow). (F)Graftingoftheposteriorhindbrain,includingparts expressed atthenormallocationdorsaltomandibulararch (green tissues from anteriormidbraintodiencephaliclevels, the posteriormidbraintorhombomere 2wasreplaced withneural transplanted inplaceofrhombomeres 4-6(openarrowhead). (E)When Similarly, noectopic (D) 2 didnottriggerectopic place ofmidbrainandposteriordiencephalon,theectopicrhombomere tube from posteriormidbraintorhombomere 2levels wasgraftedin expressed atthenormallocation(green arrow). (C)Whentheneural indicated. (B)Orthotopiccontrol graftingofrhombomere2; dorsal totheleft.Theanteroposterior orientationofthegraftsis in blueandthequailcellmarkerQCPNbrown; anteriortothetop, operated embryosatHH19,stainedforthelateralrectus marker grafting atHH10;dorsalview, anteriortothetop.( Head musclespecification lateral rectus development. Fig. 7.Thesignalfrom theneuraltubeisnotsufficient totrigger ␮ Lbx1 m. d,diencephalon;hy, hyoidarch; lr, lateral Lbx1 expression occurred whenthegraftwas expression (openarrowhead). ( A ) Schemeofquail-chickneuraltube Lbx1 expression (green B-F Lbx1 ) Lateralviewsof was Lbx1 Lbx1 is neighbouring EOM,neither state. time kept thecellsspecifiedasMAMinanimmature,proliferative Thus, Fgf8supportedtheMAMover theEOMfate, but atthesame control ofearly muscledifferentiation andspecificationofhead still amystery. Inthisstudy, weaimedatsheddinglightontothe (Goodrich, 1958).Despitetheir importance,theirdevelopment is Head musclesfulfilversatile yetvitalfunctionsinthebody DISCUSSION progress ofdifferentiation. has ageneralisedeffect onthedeveloping headmuscles,haltingtheir slightly downregulated (Fig.9T,U, openarrowheads). Thus,Bmp7 arrowhead), while however, neural tube-headmesodermseparationexperiments. Unlike those, arrowhead), thusevoking aphenotypesimilartothatobtainedbythe upregulated theexpression oftheMAMmarker markers (shown for The moleculedownregulated theexpression oflateralrectusEOM strong effect onthecraniofacial musclemarkers (Fig.9H-O, monitored by the operationdidnotaffect positionalvalues intheneuraltube,as tube, therebyindirectlyinfluencingmuscledevelopment. However, implanted intotheheadmesodermmightalsore-patternneural experiments deemedthisunlikely, wetestedwhetherFgf8beads heterotopic neuraltubegraftingand pattern thehindbrain(IrvingandMason,2000).Althoughour on theisthmicorganiser, suppress Fgf8 beads,whenimplantedintotheneuraltube,mimicfunction Effect ofFgf8 side intact( phenotype obtainedwithBmp7at100 we observed strongbilateraleffects. Thus,wefocushereonthe Bmp moleculesledtothesamephenotypes.However, at500 ␮ Fgf8, Bmp2,Bmp4orBmp7proteinwas loadedontobeadsat500 ectoderm (shown forHH19,Fig.9ii,iv). Thus,recombinantFgf4, mandibular arch(Fig.9R,S,arrowheads). eliminated, bothintheareaoflateralrectusand the (Fig. 9T,U, openarrowheads). Moreover, reduced markers (shown for Bmp7 beads(Fig.9P-W, Effect ofBmp7 carrying later, wefoundthattheFgf8beadshadinducedectopiclimbs Lours andDietrich(LoursDietrich,2005).Forty-eight hours anterior hindbrain( 1995). Fgf8-loadedbeads( mesoderm asawell-establishedFgf8responseassay(Cohnetal., mesoderm, employing limbinductionfromtheflanklateral therefore testedtheabilityofisthmustosignaladjacent experiments at100 with Fgfmoleculesgave identicalresults;hereonlytheFgf8 control, BSA-soaked beadswereused(Fig.9B-E).Beadsloaded the headmesodermatlevel ofrhombomere2(Fig.9A).Asa Fig. S2inthesupplementarymaterial). isthmus, thoughmaintainingitsown g/ml or100 The isthmusisaprominentsourceofFgf8,but inthe En2 Fgf8 Myf5 n =18, Fig.9P-W). expression inthedistalareaofmandibular arch ␮ Hoxa2 -expressing apicalectodermalridges,whilethe g/ml. Subsequently, thebeads wereimplantedinto expression was downregulated (Fig.9J,K,open MyoR n ␮ Paraxis =6) wereimplantedatHH16asdescribedby expression (Fig.9F,G, g/ml areshown ( Paraxis was upregulated (Fig.9N,O,arrowhead). n En2 =23) downregulated thelateralrectus , Fig.9P,Q, openarrowhead), but also n , Fig.9H,I,openarrowhead) and =3) orHH10posteriormidbrain- nor Hoxa2 MyoR RESEARCH ARTICLE Fgf8 ␮ n g/ml, whichleftthecontrol =30, Fig.9F-O).Alsoall and anteroposteriorlyre- expression arefound.We expression, hadnot(see Hoxb1 MyoR Myf5 n =7). Yet Fgf8hada En2 expression was expression was misxepression (Fig. 9L,M, ␮ n =39). 2739 g/ml,

DEVELOPMENT arch; ov, oticvesicle;r, rhombomere; t,telencephalon. r2*). However, (C,D,F,G) of electroporated embryosatHH19;anteriortothetop,dorsalleft.(B,E)Thetargetedarea revealed bymeansofeGFPfl Dietrich, 2002). Moreover, thevarious headmusclesdevelop at follow aspecifichead programmeofmyogenesis(Mootoosamyand We recentlyshowed thattheheadmesoderm ispredisposedto from thelocalenvironment Head muscledevelopment dependsonsignals (data summarisedinTable 1). muscle differentiation, whichwas distinctfromtheeffect ofBmp7 signal. However, Fgf8hadanadditionalfunctioninregulating specification, thusservingasantagonisttotheneuraltubederived positive regulator forMAMandnegative regulator ofEOM its innervation. Furthermore,weidentifiedarch-basedFgf8asa with afurtherfactor, specifiesthelateral rectusEOM,possiblytoaid regulates theMAMmarkers. Thissignalissolubleand,together regulates theexpression oftheEOMmarkers andnegatively However, weidentifiedasignalfromtheneuraltubethatpositively dispensable forearlymuscledifferentiation andspecification. – namelyneuralcrestcells,target organ andinnervating nerve –are extrinsic cues.Yet tissueslongsuspectedtocontrolheadmyogenesis simultaneously investigate muscle differentiation andspecification. 2006). Thus,weareinthefortunatepositionofbeingableto proliferative, undifferentiated MAMprecursors(von Scheven etal., Fig. 8.Thesignalfrom theneuraltubeisnotrhombomere 2-specific. 2740 MAM (GardnerandBarald,1992) rectus EOM(MootoosamyandDietrich,2002), express asetofmarkers with differentiation (Nodenetal.,1999),thesemusclesdifferentially Myf5, amuscle-determiningfactor indicatingtheonsetofmuscle within themandibular archresidesidebyside.Moreover, besides forming outsidethearchesandjaw closuremuscles developing on theregion oftheanteriorhindbrain,becausehereeye muscles muscles derived fromthenon-somiticheadmesoderm.We focused Paraxis respectively. (B-D)Control electroporation withthepCA Our studyshows thatheadmuscledevelopment iscontrolledby and RESEARCH ARTICLE Paraxis Myf5 Paraxis (C,F) or . (E-G)Misexpression of and Myf5 Myf5 (D,G) expression inblueandstainingwiththeanti-GFPantibodybrown. SameembryoshowninB,CandE,F, Lbx1 expression are notperturbed.Scalebar:250 and Hoxb1 Paraxis in theanteriorhindbraintransformsidentityofrhombomeres intothatofrhombomere 4(r1*, MyoR labelling thelateral En2 ␤ -IRES-eGFP vectorlackingtheopenreading framefor labelling the labelling the commenced correctly. Thus,neuralcrestcells, target tissuesand experiments thenmuscle differentiation andspecification mesoderm was separated fromthesetissuesthroughablation and innervating nerves (Engle,2002).However, whenthehead al., 2000),target tissues(Paterson andKaiserman-Abramof, 1981) cells (Grammatopoulosetal.,2000; Noden,1983b;Pasqualetti et Classically, headmyogenesiswas thoughttodependon neuralcrest dispensable forearlyheadmuscledevelopment Neural crest cells,target tissuesandnervesare environment. specification ofindividual musclesrelyoninstructive cuesfromthe programmes inthetrunk,onsetofthisprogrammeand the bound toemploy amyogenicprogrammedistinctfrom markers werenotexpressed. Thus,whiletheheadmesodermis proceeded inaccordancewiththenew positionandthelateralrectus anterior midbrainortooticlevels, then muscledevelopment head mesodermnormallyproviding thismusclewas moved tothe endogenous muscleanlageonthecontrolside.Conversely, when expressed thelateralrectusmarkers inthesamefashion asthe grafted tothelevel oftheanteriorhindbraininitiatedmyogenesisand local environment. Headmesodermfrommidbrainoroticlevels differentiation andspecificationiscontrolledbysignalsfromthe heterotopic locationsshowed, however, thatheadmuscle extrinsic cues.Graftingfragmentsofheadmesoderm into equally possiblethatheadmyogenesisiscontrolledbyintrinsicor stray neuralcrestcells(Trainor andKrumlauf, 2000).Thus,itwas possesses positionalinformation,whichitcansuperimposeonto Wachtler andJacob,1986).Inaddition,theheadmesoderm and Barald,1992;MootoosamyDietrich,2002;Noden,1983a; and theMAMexpress distinctmarkers (Coulyetal.,1992;Gardner defined, stereotypepositions,andeventually, thelateralrectusEOM ( A ␮ ) Schemeofneuraltubeelectroporation atHH10.( m. d,diencephalon;hy, hyoidarch; m,midbrain;ma,mandibular Hoxb1 allows normalexpression of Development 133(14) uorescence. B-G ) Lateralviews

DEVELOPMENT tube specifiesthe lateralrectuseye muscle versus theMAM. for MAM(GardnerandBarald, 1992).Thissuggeststhattheneural anlage normallyproviding thelateralrectusexpressed position, signal, whileallmuscleanlagen differentiated attheappropriate lateral rectusEOMmarkers. Intheabsenceofneuraltubederived neural tubeprovided a signal thatpromotedtheexpression ofthe the effect was masked throughsignalredundancy). However, the head muscleformation,wefoundthatmosttissueshadnoeffect (or Searching fortheextrinsic cuesthatmightcontroltheearlyevents in the lateralrectus versus mandibulararch muscles A solublesignalfrom the neuraltubespecifies molecular cascades. tissues servingthesimilarfunctionsemploy relatedor different al., 2001).Itthuswillbecrucialtoinvestigate whetherthedistinct are suppliedbyneuronsresemblingvisceralmotoneurons(Jacob et somatic motoneurons,whileinthehead,branchialarchmuscles 1983a). Moreover, inthetrunk skeletal muscleisinnervated by head itstemsfromneuralcrestcells(Coulyetal.,1993;Noden, In thetrunk,connective tissueismesoderm-derived, whileinthe distinct programmes,eventually theseprogrammesmayconverge. although theonsetofmyogenesisinheadandtrunkiscontrolledby al., 2004;Kardon,1998;Punet2002).Thus,itispossiblethat initial phase,thesetissuesbecomeinterdependent(Hippenmeyer et development independently. However, afterthecompletionofthis trunk, muscle,connective tissuesandnerves begin their Remarkably, studiesonsomiticmyogenesisestablishedthatinthe development aftertheonsetofdifferentiation andspecification. reported roleofneuralcrestscellsandnerves affects headmuscle dispensable forearlyheadmuscledevelopment, thisimpliesthatthe for cardiacdevelopment. crest cellswererequiredtooverride thesignalsrecruitingmesoderm it ispossiblethatunderthechosencultureconditions,neural expressed anumberofcardiacmarkers (Tzahoretal.,2003).Thus, fulfilling asimilarfunction.However, invitrotheheadmesoderm role oftheneuralcrestcellsmaybecomemasked byothertissues 1997). Ifsignalredundancy alsooccursinthehead,then thein-vivo surface ectodermshow adegree ofredundancy (Dietrichetal., various surroundingtissues,ofwhichthedorsalneuraltubeand however, thatinthetrunk,somiticmyogenesisiscontrolledby muscle differentiation (Tzahoretal.,2003).Ithas tobenoted, experiments showing thatcranialneuralcrestcellspromotehead invivo. Ourfindingscontrastwithrecentin-vitro development innervating nerves arenotnecessaryforearlyheadmuscle Head musclespecification g8basDwrglto peuainDwrglto Upregulation Milddownregulation Downregulation Downregulation Normal Proximal:normal Upregulation Downregulation Normal Downregulation muscle development. suppression oflateralrectus andsupportofmandibular arch muscleidentity, (2)supportofmuscleprecursor proliferation ove muscle: Signal from neuraltube:specifieslateralrectus EOM, suppresses mandibulararch muscleidentity;noeffect ontheprogress of Upregulation Bmp7 beads Fgf8 beads Downregulation Separation neuraltube– Treatment Table 1.Summaryofmanipulationsaffecting headmusclespecificationanddifferentiation head mesoderm As ourwork suggeststhatneuralcrestcellsandinnervation are Paraxis and Lbx1 expression was lost. Instead,themuscle etsetaclr adblrac Dfeetaigmsl muscleprecursors Differentiatingmuscle Mandibulararch rectus extraocular Lateral Paraxis , Lbx1 uceptenMuscledifferentiation Muscle pattern Distal: downregulation En2 muscles: , amarker En2 tube re-patterning,as expressed. Thiseffect was notanindirectconsequenceofneural mesoderm harbours Fgfreceptors(S.D.,unpublished), andinFgf8 lateral rectus,theEOMmarkers werelostand Fgf8 beadswereimplantedinto themesodermdestinedtoform not Bmp7,hadaspecificeffect onEOMandMAMmarkers. When Performing gain-of-functionexperiments, wefoundthatFgf8,but in epithelial-mesenchymalinteractions(Helmsetal.,2005). pharyngeal pouches(endo-andectoderm)oralectoderm act and development; they areexpressed inthepharyngealendoderm, might specifyMAMbut suppressEOMdevelopment. expressed, weasked whethersignalsfromthebranchialarches the absenceofneuraltube,MAMmarkers wereectopically epaxial andhypaxialmuscleprecursors(Parkyn etal.,2002).Asin the neuraltubeandlateralmesodermactantagonisticallytospecify It isestablishedthatduringtrunkmuscledevelopment, signalsfrom tube signalandspecifiesMAM Branchial-arch-based Fgf8 antagonisestheneural (Tzahor etal.,2003). repressive roleforShhandWntsignalsincraniofacial myogenesis observation isinlinewithourearliercollaborative studyshowing a expression ofthelateralrectusmarkers, suppressedthem.This function experiments, thesemolecules,insteadofpromoting the of theelusive neuraltubederived signal.However, ingain-of- and Tabin, 2002);they hencefitthephenomenologicaldescription are solublesignallingmoleculesthatoperateatallaxiallevels (Brent fashion. derived signalmightactinapermissive, ratherthaninstructive, neighbours thelateralrectus.Thissuggeststhatneural-tube- Moreover, thesignalisnotconfinedtorhombomere2,which is notsufficient totriggerectopiclateralrectusdevelopment. EOM. Theseexperiments showed thattheneuraltubederived signal changed theidentityofneuraltubenext tothelateral rectus signal, weperformedheterotopicgraftingofneuraltissuesand soluble factor. markers was restored.Thissuggeststhattheneuraltubereleasesa and Tessier-Lavigne, 1994),thentheexpression ofthelateralrectus preventing cell-cellcontactbut admittingsolublemolecules(Fan was insertedbetweenneuraltubeandheadmesoderm,thereby cell-cell contact.However, whenafilterwithporesizeof0.05 conceivable thattheneuraltubemighttransmititssignalthrough Markers for Fgf8 andBmp7areknown regulators ofbranchial archpattern Shh provided bythefloorplateandWnt1dorsal neural tube To make inroadsintotheidentificationofneuraltubederived Given theproximityofneuraltubetolateralrectus,itwas precursors: Myf5 Hoxa2 r differentiation. Bmp7:suppression ofskeletal differentiation. Fgf8:dualfunction(1) expression was unaltered. Thehead Proliferative, undifferentiated RESEARCH ARTICLE (branchial arches): En2 was ectopically MyoR 2741 ␮ m

DEVELOPMENT 2742 Fig. 9. See nextpagefor legend. RESEARCH ARTICLE Development 133(14)

DEVELOPMENT the isthmusarerequiredtoupregulate further factors foundinthemandibular archenvironment but notat available totheheadmesoderm.Itcannotbeexcluded, however, that neighbouring mesodermislimited.Thus,isthmicFgf8maynot be response assay, wefoundthattheabilityofisthmustosignal Head musclespecification not express and Mason,2000).However, themesodermnext totheisthmusdoes signalling inaplanarfashion toorganise mid-andhindbrain(Irving neural tube/EOMsystem. supporting branchialarchmuscledevelopment andopposingthe Fgf8 signalstotheheadmesodermdirectly, simultaneously signal have beenobserved (Franketal.,2002).Thissuggeststhat responses oftheheadmesodermtolossepithelialFgf hypomorph mousemutantsthatsucceedingastrulationspecific orientation asinFig.3A.(Ai,ii) Fgf8 andBmp7. Fig. 9.Differential effect ofthemandibular-arch-derived signals the top).Notepersistentexpression of anterior tothetop)andHH19(Aii,lateralviewofrightside, marker for proliferative, undifferentiated MAMmuscleprecursors patterning rolealsoinfluenced muscledifferentiation: differentiation remained oncourse.However, Fgf8,inadditiontoits When headmesodermandneural tubewereseparated,muscle muscle differentiation Role ofFgf8andBmp7in thecontrol ofhead lateral rectus andthemandibulararch muscles(R,S). upregulated (N,O;compare withFig.5).Bmp7dowregulated Myf5 (L,M). Unlikeneuraltube-mesodermseparationexperiments,however, rectus marker(H,I)andupregulated themandibulararch musclemarker expression of the hindbrainorneuralcrest cells,asevidencedbytheunperturbed placed intotheheadmesodermdidnotinfluencepositionalvaluesof position ofthebeadsismarkedbyasterisks.NotethatFgf8 by openarrowheads, upregulated expression byfilledarrowheads. The expression domainsare markedbyarrows, downregulated expression as inFig.3B-E.Theembryoswere doublelabelledasindicated.Normal sides oftheembryosonleft,treated sidesontheright,orientation ( ( the lateralrectustrigeminalganglion. EOMunderneath outflow tractoftheheart. pharyngeal pouches,theoralectoderm(arrows), andthein- endoderm andheart;atHH19expression isupregulated inthe mandibular arch). (Aii,iv) pouch (Aii,arrows pointtowards expression associatedwiththe and intheendodermplusoverlyingectodermofeachpharyngeal endoderm andatHH19,upregulated expression intheoralectoderm proximally closetothebead(T,U). was downregulated inthedistalpartofmandibulararch butnot the region ofthelateralrectus EOM(P,Q) and the top).Notepersistentexpression of anterior tothetop)andHH19(Aii,lateralviewofrightside, prominently inthedistalregion ofthearch (V,W). Scalebars:250 ganglion; VII,facialnerve. ov, oticvesicle;pchp,prechordal plate;r, rhombomere; V, trigeminal ectoderm; hy, hyoidarch; i,isthmus;m,midbrain;ma,mandibulararch; endoderm; fn,frontonasal Fgf8signalinventralforebrain andsurface Ai,Aiii; 100 F-O B-E Besides thearches,Fgf8isstronglyexpressed bytheisthmus, Lateralviewsofembryostreated withBSA-loadedcontrol beads, ) ) Fgf8-loadedbeadsand( expression wasalsodownregulated (J,K),while ␮ En2 m inAii,Aiv;200 Hoxa2 . Employing limbinductionaswell-establishedFgf8 ( A ) SchemeofbeadimplantationatHH10, (F,G). However, Fgf8downregulated thelateral Bmp7 Fgf8 P-W ␮ Fgf8 expression atHH10(Ai,ventralview, and m inB-U.di,diencephalon;endo, MyoR ) Bmp7-loadedbeads;untreated expression atHH10(Ai,dorsalview, Bmp7 Fgf8 was alsodownregulated, most En2 Bmp7 are absentfrom thearea of in thepharyngeal . Myf5 in theHH10 in theregion ofthe En2 MyoR expression was Paraxis MyoR ␮ m in , a in of muscleprecursorintheheadandmayunderpingrowth. trunk (Itohetal.,1996;Kahane2001),mayexpand thepool downregulated. ThussuggeststhatFgf8,similartoitsroleinthe more mildly, (von Scheven etal.,2006)was upregulated byFgf8while expressing leading toerroneousexpression of When theheadmesodermwas separatedfromtheneuraltube, the targetrecognition ofthecognatenerves Eye musclespecificationmaybeaprerequisite for mesoderm, diverting itfromaskeletal muscletoacardiacfate. 2003). HenceitispossiblethatBmp7actedonthebranchialarch molecules areimportantregulators ofheartdevelopment (Brand, subsequently totheoutflow tractoftheheart(Kelly, 2005).Bmp contributes toskeletal musclebut alsototheanteriorheartfieldand into account,however, thatbranchialarchmesodermnotonly muscle precursorsintoany myogenicprogrammes.Ithastobetaken specifies theneighbouringmuscle anlageas signal fromtheneuraltube(green), togetherwithafurtherfactor, Summarising ourresults(Fig. 10),weproposethatatasoluble Model development. antagonism isaglobalregulator ofeye versus branchiomericmuscle Helms etal.,2005).Thus,itislikely thattheneuraltube-Fgf8/arch observed (Abu-Issa etal.,2002;Frank2002)(reviewed in mouse mutants,severe pharyngealandaortic archdefectswere MAM isexpressed inallbranchialarches,andhypomorphFgf8 development inthisregion. However, Fgf8assignaltospecify and marker geneexpression serve aunique functioninheadmuscle 2004; Wahl etal.,1994). Thus,isitpossiblethatsignallingevents projecting laterallyintothemandibular arch(Chilton andGuthrie, anterior regions andthemaxillo-mandibular branch ofthetrigeminal arriving fromposteriorregions, theoculomotornerve arriving from is traversed byanumberofcranialnerves, withtheabducensnerve Noden, 1983a;Wachtler andJacob,1986).Furthermore,thisregion branchiomeric musclesdevelop sideby(Coulyetal.,1992; Moreover, itisonlyatrhombomere2levels thateye and muscle develops next torhombomere 2(Wahl etal.,1994). abducens nerve isborninrhombomeres5and6;thelateral rectus nerve andmusclenotoriginatingfromthesameaxial level: the surface orsolubleaxonguidancecues. specification ofthelateralrectusEOMleadstoproductioncell all transcriptionfactors, wecanexpect thatultimately, the muscle toaiditsinnervation. However, as of phenotype was linked tothepresenceof hindered bytheobstacleinitspath.Rather, theinnervation progress ofaxonaloutgrowth andtarget recognitionwas not innervation ofthelateralrectuswas restored. Thisindicatesthatthe barrier, thefilterwas used,thenmarker geneexpression and and Landmesser, 1985).Wheninsteadoftheimpermeablefoil target andhencewereactively samplingtheenvironment (Tosney defasciculated, indicatingthattheaxonsstruggledtoidentifytheir this musclefellshortofitstarget. Moreover, thenerve nerve. Within thebranchial arches,however, thepresenceof Fgf8 of thelateralrectusthenfacilitates theinnervation bytheabducens the expression oftheMAM marker lateral rectusEOM(yellow). Simultaneouslythesesignalsprevent In contrasttoFgf8,Bmp7stronglydownregulated The abducens-lateralrectuspairisoneoftherareexamples of En2 or both.Thissuggeststhattheneuraltubespecifieseye Paraxis MyoR and . Thus,Bmp7mayinhibittheentryofhead Lbx1 , thentheabducensnerve destinedfor RESEARCH ARTICLE En2 En2 Paraxis . Thecorrectspecification Paraxis in themusclenormally Paraxis/Lbx1 / Lbx1 , Lbx1 or theabsence and Myf5 Myf5 -positive En2 2743 , and was are

DEVELOPMENT Brand, T. lateral rectus extraocularmuscle,MAM,mandibulararch muscles. MAM markersare repressed inthisarea. MAM(magenta)are specifiednexttothemandibularFgf8signalsthatsuppress EOMmarker by afurther, unidentifiedsignal.Asaresult ofthesignallingevents( tube (green) andFgf8from thebranchialarches (blue)signaltotheneighbouringheadmesoderm.Thefrom theneuraltub at theonsetofEOMandMAM-specificmarkergeneexpression; thenon-somiticheadmesodermisdepictedingrey. ( Brent, A.E.andTabin, C.J. Bell, E.,Wingate,R.J.andLumsden,A. Dietrich, S.,Schubert,F. R.andLumsden, A. Cohn, M.J.,Izpisua-Belmonte,J.C.,Abud,H.,Heath,K.andTickle,C. Chilton, J.K.andGuthrie,S. Cheng, L.,Alvares, L.E.,Ahmed, M.U.,El-Hanfy, A.S.andDietrich, Brodal, A.andRagnar, F. Begbie, J.,Brunet,J.F., Rubenstein,J.L.andGraham,A. Alvares, L.E.,Schubert,F. R.,Thorpe,C.,Mootoosamy, R.C.,Cheng,L., http://dev.biologists.org/cgi/content/full/133/14/2731/DC1 Supplementary materialforthisarticleisavailableat Supplementary material EU MarieCurieEarlyStageFellow. and theEuropean NetworkofExcellence,Myores. Gudrun vonSchevenisan Charitable FoundationofGuy’s Hospital,theHuman Frontier ScienceProgram Patterson forkindlyproviding in-situprobes. Thework wassupportedbythe I.Mason,E.OlsonandB. We alsothankA.Graham,T. Lints,C.Logan, helpful commentsandtoFrankSchubertforcriticallyreading themanuscript. We are mostgratefultoFrankSchubert,Lynda ErskineandSarahGuthriefor the cellsinaproliferative state. an additionalroleinpreventing MAMdifferentiation, maintaining Fgf8 actantagonisticallyinheadmusclespecification.Yet, Fgf8has (magenta) aresupported.Thus,theneuraltubederived signaland (blue) ensuresthatEOMmarkers arerepressedwhileMAMmarkers Fig. 10.Modelforthespecificationoflateralrectus eyeversusmandibulararch muscles. 2744 Abu-Issa, R.,Smyth,G.,Smoak,I.,Yamamura, K.andMeyers,E.N. References Couly, G.F., Coltey, P. M.andLeDouarin,N. Couly, G.F., Coltey, P. M.andLeDouarin,N. and earlymorphogenesis. 2171. rhombomere identityafterlocalized Hoxb1misexpression. projections inthechickembryo. derivatives: muscle,cartilageandtendon. the epibranchialplacodes. pattern inthechickparaxialmesoderm. pattern 429. flank ofchickembryos. (1995). Fibroblast growth factorsinduceadditionallimbdevelopmentfrom the 369. (2004). 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DEVELOPMENT