not express (McDiarmid andAltig,1999),suggestingthatearlytadpolesmight the entirefamily ofpipidfrogsdoesnotdevelop atongue 2000; SchaferandBraun,1999). undergo normaldevelopment (Brohmannetal.,2000;Gross attributed tomigrationdefects,whilehypaxialbodywall muscles mutation inthemousecausesaspecificlackoflimbmusculature long-range migrationtotheirtarget sites(Dietrich,1999).Anull mesenchymal transitioninthedermomyotomebeforeundergoing limb, tongueanddiaphragmmyoblastsundergo anepithelialto of control ofcellproliferationislinkedtoastrongdownregulation overexpression of are seenjustventral todifferentiated muscle oftheventral bodywall. the somiteandmigrateventrally. Lateron, is amarker ofproliferative myoblasts, isexpressed incellsthatexit of amniotelimbmyoblasts(MartinandHarland,2001). myoblasts thatformtheventral bodywall musclesissimilartothat 1999). Thetemporalexpression ofmuscle-specificmarkers in the inter-limb bodywall musclesofamniotes(McDiarmid andAltig, Martin andHarland,2001;Ryke, 1953).Thesearehomologous to muscles, whichformtherectusabdominusmuscles(Lynch, 1990; indicate thataprimaryfunctionof epithelial extensions ofthedermomyotome,whereas different modesofdevelopment. Body-wall musclesformas expression, thesetwo typesofhypaxialmusclesarecharacterizedby 1999; Dietrichetal.,1998).Inadditiontothedifference in excluded frominter-limb bodywall hypaxialmyoblasts(Dietrich, the limbs,tongue,anddiaphragm.Bycontrast, in asubsetofhypaxialmyoblasts,whichthemousemigrateinto domain(Jaglaetal.,2001).Inamniotes, characterized byahomeoboxDNA-binding domain andanengrailed transcriptionfactors. The ladybirdhomeobox( INTRODUCTION KEY WORDS:Hypaxial,Rectusabdominus,cervicus,Geniohyoideus, tadpoles targeting and hypoglossalmusclesoriginatingfromthesomites.AlthoughmyoblastmigratorydefectsareobservedinantisenseMOinjected the functionof contribute tothebodywallmusculature,aswellinagroupofcellsthatmigrateintohead.Despitethisdifferentexpr We haveexamined Benjamin L.MartinandRichard M.Harland* A novelrolefor Development 133,195-208doi:10.1242/dev.02183 Accepted 25October 2005 *Author forcorrespondence (e-mail:[email protected]) Berkeley, CA94720-3204,USA. Development, andtheCenterforIntegrative Genomics,UniversityofCalifornia, Department ofMolecularandCellBiology, DivisionofGenetics,Genomics, and before theeventualonsetofterminaldifferentiation. The pre-metamorphic myoD mRNA with lbx1 lbx1 . However, thesetadpolesdocontainhypaxial lbx1 lbx1 lbx1 appears tobeconserved.Morpholino(MO)knockdownof lbx1 mRNA resultsinenlargedsomites,anincreasecellproliferation,butalackofdifferentiatedmuscle.The X. laevis , thisresultsatleastinpartfromalackofmyoblastproliferationthehypaxialmuscledomain.Conversely, lbx1 expression inearly mRNA eliminatestheoverproliferationphenotypeobservedwhen ) geneisamemberoftheNK-class lbx1 tadpole doesnotcontainlimbsand lbx1 lbx1 and otherNK-classgenesare in hypaxialmuscledevelopmentistorepress X. laevis -positive myoblasts in lbx1 lbx1 Xenopus is completely lbx1 tadpoles. Incontrasttoamniotes, pax3 is expressed -positive , which lbx1 lbx1 myoD but hasnoeffect on myoblasts byactivated Notchresultsinadownregulation of example, theinhibitionofmuscledifferentiation inchicklimb et al.,1991;MartinandHarland,2001;Montarras1991).For proliferative myoblaststhathave notexited thecellcycle (Hopwood in mouseskeletal muscleformation, Myf5 differentiation ofskeletal muscle(Halevy etal.,1995).Although an integral factor incellcycle arrestduringtheterminal Weintraub etal.,1991).Indeed, Hopwood etal.,1992;Montarras1989;Tapscott etal.,1990; terminal differentiation ofskeletal muscle(Hopwood etal.,1989; skeletal muscle(MartinandHarland,2001). and they overlap with12/101staining,amarker ofdifferentiated Transcripts of is absentfromhypaxialmyoblastsasthey exit thesomite. to thatof The expression of transient upregulation of myoblasts. Whenectodermisremoved from chicksomites,a overlying ectodermofsomitesprovides aproliferative signalfor differentiation (Amthoretal.,1999;Amthor1998).The muscles requiresafinebalancebetweenproliferation and Forced expression of migration, but 1999). migrating, andlimbtonguemusclesdonotform(Amthoret al., premature differentiation prevents muscleprecursorsfrom ectoderm isremoved fromsomitesatlimband tonguelevels, the of muscledifferentiation, but furthermusclegrowth ishalted.When myoblast cells. lbx1 (Mennerich andBraun,2001). These resultssuggestthataroleof This excess canberescued byinhibitingcellproliferation increases explants increasescell proliferation.Inchicklimbbuds, The expression of The precisetimingofthedevelopment oflong-rangehypaxial Work on hypaxial myogenesis lbx1 in hypaxialmyogenesismaybe toexpand thepopulationof and expression ingain-of-functionexperiments.Co-injection , pax3 myoD myoD Myod1 lbx1 lbx1 myoD in thehypaxialmyoblasts.However, theMRF lbx1 , expression andtheamount ofdifferentiated muscle. causes aspecificreductionofbodywallmuscles in hypaxialmyogenesishasfocusedmainlycell have beenshown tohave partiallyredundantroles myoD myf5 lbx1 myf5 has alsobeenimplicatedincellproliferation. myoD do notappearuntilcellsbegin todifferentiate, , , amuscleregulatory factor (MRF),issimilar Xenopus laevis lbx1 , allowingmyoblaststoproliferate is expressedinallofthemyoblaststhat myoD or lbx1 has beenshown tobeassociatedwiththe pax3 in chickmesodermandneural tube is injectedalone.Theresults is observed correspondingtoaburst myoD expression (Delfinietal.,2000). RESEARCH ARTICLE , Cellproliferation, has beendemonstratedtobe Myf5 is expressed in ession, myoD myoD lbx1 195 ,

DEVELOPMENT amplify the5 TAGG-3 CTAG-3 TCGCTGTG-3 of thecodingregion. TheprimersXlbx5up(5 cDNA library. Asinglecloneof subsequently. two-cell stage.Anoptimaldoseof33ngwas determinedandused 17,33and57ngofantisenseMO wereinjectedintoonecellatthe Initially, ATCTCAGTC-3 control zebrafish GGAC-3 by immersionin0.05%benzocainesolution. (General Valve). Tadpoles wereimmobilizedwhileinjectingandforviewing backfilled withDiIsolutionandpressureinjectedusingaPicospritzer M sucrose.Micropipetteswerepulledandbroken toatipof~20 made upin100%ethanol.Aworking solution of0.1%DiIwas dilutedin3 A 0.25%stocksolutionofCellTracker CM-DiI(MolecularProbes)was Microinjection ofDiI TLBX-3 (5 The primersTLBX-2(5 Isolation of (Nieuwkoop andFaber, 1967). modified Ringer(MMR)solutionandstagedaccordingtothenormaltable (Sive etal.,2000).Embryoswereallowed todevelop in0.3 laevis Xenopus General methods 196 CAGATTGGTGCTGGATATGC-3 two out oftwo cells.Thefollowing primerswereused:ef1 ng ofsplice-MOintooneouttwo cells,orinjectedwith33ngofMOinto (Wilson andMelton,1994).Tadpoles usedwereuntreated,injectedwith33 RNA was extracted fromwholetadpolesatstage28andused forRT-PCR RT-PCR TGCTGC-3 sequences wereused:spliceblocking,5 Antisense andcontrolMOswereorderedfromGeneTools. Thefollowing Microinjection ofMOs of 1:1000. rabbit IgGsecondaryconjugatedtoHRP(BioRad)was usedatadilution dilution in2mg/mlBSAPBSplus0.1%Triton X100.Agoatanti- histone H3antibody(UpstateBiotechnology)was usedata1:1000 followed immediatelybyimmunohistochemistry. Theanti-phospho- were carriedoutonembryos,insitustainingwas performedfirst, respectively. Incaseswherebothinsituhybridizationand12/101 staining (Jackson) asasecondaryantibodyat1:500or1:200dilution, supernatant andagoatanti-mouseIgGconjugatedtoHRPorfluorescein Immunohistochemistry usedundilutedmonoclonalhybridomacell 12/101 monoclonalantibody(KintnerandBrockes, 1984). et al.,2000).Differentiated skeletal musclewas visualizedwiththe probes labeledwithdigoxigenin-UTPusingamultibasket technique(Sive for 2hoursinMEMFA. Insituhybridizationwas carriedoutwithRNA Embryos wereallowed todevelop untilthedesiredstageandthenfixed Whole-mount insituhybridizationandantibodystaining Xenopus tropicalis MATERIALS ANDMETHODS proliferate. probably resultfromthereducedcapacityofmyoblaststo p27 myoblast proliferationthroughthedownregulation of hypoglossal muscle.We furtherdemonstratethat will populatetherectusabdominusandgeniohyoideus,a tadpoles andfoundthatitisexpressed inallofthemyoblaststhat We have examined theexpression patternof . Thus,muscledefectsobserved after RESEARCH ARTICLE Ј Ј Ј , 268nucleotides; ) weredesignedfrom ; control,5 Ј -CCGAGATGGTGAGTACGGCTTC-3 Ј ; translationblocking,5 Ј eou avslbx1 Xenopus laevis end ofthesequencefrom embryos weregeneratedandculturedbystandardmethods Ј ) andXlbx5down (5 Ј lbx1 . Stocksolutionswereresuspendedat50mg/ml. genomic sequenceandusedtoscreenanarrayed translation blocking,5 Ј -CCTCTTACCTCAGTTACAATTTATA-3 Ј -AGACGGCATGACGATTTTTGGC-3 lbx1 X. tropicalis -intron, U5 lbx1 Ј clone and D5 Ј was foundthatcontainsthemajority -TCATCTTTGGAAGTCATAGTG- Ј Ј X. laevis -TCCTGACTGAGGGCTTGTT- -GAGTGAGGAACTTACCTTC- Ј Ј -TCCTAAACAAGCCCTCA- genomic sequenceandusedto -CCAAGTCAATGTATCAG- Ј Ј -TTTAGAGCTGGAGGTC- -ACTGCCTTGATGACTC- genomic DNA. lbx1 Ј ) weredesignedfrom lbx1 loss offunction lbx1 in myoD ϫ X. laevis ␣ controls X. laevis , U5 Marc’s Ј Ј ; and ) and ␮ and m, Ј - ef1 nucleotides (properlyspliced).Twenty-one cycles wereusedtoamplifyfor less than0.01wereconsideredtobestatisticallysignificant. a full-lengthPCRinsertincs108afterbeingdigestedwith from thep3plasmid(Ruppetal.,1994).Mouse digested with ordered fromRZPD(Germany), subclonedfrompSporttocs107,and inserted intothe 3 product was digestedwith amplification usingtheLBXCLAIandLBXECORIprimers.Theresulting MLBXF (5 (5 CTTGTTGG-3 AGTTCGGGTG-3 with theprimersLBXCLAI(5 The 5 Mutant (Ambion). Azebrafish Synthetic mRNA was madeusingthemMessagemMachineSP6kit mRNA synthesisandmicroinjection GTCCG-3 PCR primersdesignedfrom Identification andexpression of 1970). manner nearthemouthoftadpole(Hammond,1965;Hazelton, are presentasapairofmusclesextending inananterior-to-posterior geniohyoideus musclesareconsideredtobeofsomiteorigin, and By stage45(Fig.1B),thecranialmusclescanclearlybeseen. The of theepaxialmyotome(McDiarmidandAltig,1999;Ryke, 1953). and rectuscervicusmusclescanbeclearlydistinguishedfromthose laevis (Kintner andBrockes, 1984),was usedtoidentifymuscles ofthe The 12/101antibody, whichmarksdifferentiated skeletal muscle Identification ofhypaxialmusclesin RESULTS counting. Sections atapproximatelythelevel ofthe3rdtrunksomitewereusedfor counted usingthemicroscope,andnotfromsubsequentphotographs. immediate vicinityofthesomitethroughentire200 and visualizedusingaZeissAxioplanmicroscope.Stainednucleiinthe sections wereclearedandmountedinwellscutintoSylgardcoatedslides used, whichwerecutat200 ␮ vibratome (Oxford).Themajorityofsectionswerecutatathickness100 Tadpoles wereembeddedin4%low meltagaroseandsectioned usinga Sectioning andcountingnuclei treated H test mRNAs asalineagetracer. galactosidase mRNA (pCS2- region oftheembryo. one cellatthetwo-cell stage.Injectionsweretargeted tothemediolateral treated H myf5 clone of were usedtoscreenanarrayed region minusthe5 two conserved domainstypicalof theNKclassoftranscription predictions fromthe determined byPCRof Ј m, except forsectionsinwhichtheanti-phosphohistoneH3antibodywas Ј The synthesizedmRNA was resuspendedasastocksolutioninDEPC- amplified fragmentsweregelpurifiedandmixed together, followed by -CGGAATTCCGCCTTGCATTTCAAGTTCTTCCGTG-3 ␣ , and 50cycles for Ј myoD tadpole. Atstage37(Fig.1A),thehypaxialrectusabdominus end ofthezebrafish lbx1 2 2 O ataconcentrationof1mg/ml.Working solutionsof O. Approximately4nlofeachmRNA solutionwas injectedinto P lbx1 Ј Ј -CCAACAAGCCTCTCTCCGTTAAGAAGTC-3 values wereobtainedusing the pairedStudent’s + and D5 construction Asc lbx1 Ј was foundthatcontainsthe majorityofthecoding ). The3 Cla I formRNA synthesis. Ј and ) andMLBXR(5 I, Eco lbx1 Ј X. tropicalis lbx1 -CCAACTCATAAATCTGGTGGTTCG-3 Ј Ј myf5 end was amplifiedwiththeprimersLBXECORI RI sitesofpCS107. end. The5 Cla lbx1 X. laevis intron. I.M.A.G.E. EST(GenBankAL831789)was Nls-NlacZ + I and ␮ Ј eou tropicalis Xenopus -CCATCGATGGCGTATGAGGACTAA- lbx1 EST (GenBankAL831789)was amplified m. Theanti-phosphohistoneH3stained Eco X. laevis were dilutedto0.1mg/mlinDEPC- Ј genome. -GACTTCTTAACGGAGAGAGG- genomic DNA usingsequence 0-0 pg)was co-injectedwith , 100-200 RI andgelpurified.Thiswas then Ј eou myoD Xenopus end ofthesequencewas Myf5 cDNA library. Asingle .lei lbx1 X. laevis X. laevis lbx1 Development 133(2) was synthesizedfrom genomic sequence X. laevis ␮ was synthesized Asc m sectionswere Ј t I. Nuclear -test. Values ). The5 lbx1 Ј ) and contains , Ј myoD , 300 Ј and X. ␤ - ,

DEVELOPMENT (arrowheads). positive myoblastsare seen intheventralbodywallregion migrating around thehyoid (arrow). ( development procedes. ( geniohyoideus. (C-H)Expression of hypaxial musclesconsistoftherectus abdominus,rectus cervicusand lateral view, whileBisaventralviewwithanteriortotheleft.The ( regions of homeodomain. Thesedomainsareidenticaltothecorresponding factors, includingtheengrailedhomologydomain(eh1)and Xenopus ( Fig. 1. ( expression precedes myoblastexpression (stage17,dorsalview). lbx1 amino acididentitiesare94%,79%and63%between Harland, 2001).Atstage37/38,athinstreamofcellsappearsto thatmarkthedeveloping hypaxialbodywall (Martinand posteriorly andventrally, consistentwiththeexpression ofother arrowhead). Asdevelopment proceeds,expression expands 26 intheventrolateral region ofanteriortrunksomites(Fig.1D, shown). Initiationofmesodermalexpression canbeseenatstage neurula stagesisnotdetectablebyinsituhybridization(data (Gross etal.,2002).Expressionof consistent withinterneuronspecificexpression inthemouse stages definesadorso-intermediateregion oftheneuraltube, is firstestablishedatstage17intheneuraltissue,andlater respectively. ThemRNA expression patternof while thenucleicacididentitiesare88%,66%and61%, E A D , ) and45( Earlymyoblastexpression canbeseenatstage26 (arrowhead). ) F Myoblastexpression expands posteriorlyandventrallyas ) and Xenopus hypaxial myogenesis X. tropicalis B X. tropicalis ) tadpoleswere stainedwiththe12/101antibody. Aisa hypaxial musclesand G , mouseandzebrafish ) Atstage37, , mouseandzebrafish lbx1 H lbx1 ) Bystage42,veryfew from stage17to42.( lbx1 lbx1 during gastrulaandearly -positive cellscanbeseen expression. lbx1 lbx1 lbx1 , respectively, . Overall, the (Fig. 1C-H) Stage 37 X. laevis lbx1 C ) Neural - 12/101 imagesin G. shown inD,DiIlabelingE,12/101 inFandthemergeofDiI with thegeniohyoideusmuscleat stage45.Alight-fieldimageis population ofcellscomingfrom theanteriortrunksomitesco-localize ros CG Ventral viewsofthehead.( arrows. (C-G) while labeledcellsthathavemigrated intotheheadare markedwith and 45( (Niewkoop andFaber, 1967)].Alive tadpoleisvisualizedatstage37( injections [modified,withpermission,from NiewkoopandFaber seen asathinband(Fig.2C,arrows). Co-localization ofDiI-and At stage45,thefinallocationoflabeledcellsinheadcanbe around thehyoidarchandintohead(Mackenzie etal.,1998). myoblasts inotherorganisms, wherecellsmigrateinathinstream arrowhead) atstage37.Thepathofmigrationissimilar totongue ventrally andanteriorlyaway fromthesiteofinjection(Fig.2B, (Fig. 2B)and452C).DiI-positive cellscanbeseenmoving somites 1-3)(Fig.2A).Labeledcellswerevisualizedatstage37 domain oftheanteriortrunksomites(approximately migrate intothehead,DiIwas injectedintotheventrolateral In ordertodeterminethefate of anterior trunksomites The geniohyoideusmusclesoriginatefrom eventually lostinthisregion. of thebodywall muscles(Fig.1H,arrowheads) beforeitis By stage42,expression becomesvery weakintheventral domain be migratingintothehead(Fig.1G,arrow, Fig.3Carrowheads). from theanteriortrunksomites.( immunofluorescence (green) wasusedtotrackthemigration ofcells from theanteriortrunksomites. Fig. 2.Thegeniohyoideusisahypaxialmuscleandoriginates C ). Theoriginalsiteoftheinjection ismarkedbyanarrowhead, A ) Aschematicofthestage28 lbx1 DiI labeling(red) and12/101 RESEARCH ARTICLE -positive cellsthatappearto D - G ) Themigratory 197 B )

DEVELOPMENT wall region asthey extend asepithelia, whereas of hypaxialmarkers spantheentirewidthofsomiteinbody specific markers suchas lbx1 somite cleftsofastage37tadpole,intheposterior-most region of myoblasts ofamniotes. myoblasts migrateinamannersimilarto rectus abdominusin mesenchymal cells(Neyt etal.,2000). zebrafish finmyoblastsemerge atsomitecleftsandmigrateas hypaxial myoblasts. lbx1 extensions ofthedermomyotomeinamniotesandfail toexpress Rectus abdominusandotherbodywall musclesformasepithelial tadpole hypaxialmuscledevelopment Conserved cell-typeof shown). tadpole inwhichthesomiteswerelabeled(controlsidenot DiI-labeled cellsonlygive risetothesemusclesonthesideof found togive risetotherectuscervicusmuscle(datanotshown). geniohyoideus (Fig.2D-G).Thesamepopulationofcellswas also cells comingfromtheanteriortrunksomitesgives risetothe 12/101-labeled cellsindicatesthatthemigratingpopulationof 198 with 12/101(brown). ( clefts inastage31tadpole(arrows). MyotomesinAandBare labeled marker, emerging myoblastsshown).( tadpole emergeatthesomitecleft(arrows, mostposteriorsomitewith mesenchymal cells. Fig. 3.Ventral bodywallmyoblastsappeartomigrateas hindlimb ofastage53tadpole(E, arrows), similarto with 12/101(D).( a ventralviewofstage42tadpole (C,arrowheads), whichco-localizes end towards theleft). the samestage(F, arrows) (limbs are shownfrom alateralview, distal expression (Fig.3A,arrows). Thisisalsotrueforotherhypaxial (Dietrich, 1999).Because RESEARCH ARTICLE , canbeseenexpressed incellsmigratingoutofthesomite E , F lbx1 ) X. laevis ( lbx1 A C ) , D expression inthegeniohyoideus canbeseenin lbx1 lbx1 tbx3 ) is alsofoundinthemigratingmuscles ofthe lbx1 -expressing myoblastsofastage37/38 B , wewanted todeterminewhetherthese -positive myoblastsarepresentatthe ) Anotherhypaxialmyoblast-specific (Fig. 3B).Inamniotes,theexpression expression isfoundinlong-range type lbx1 lbx1 is expressed inprecursorsofthe -positive myoblastsin lbx1 -positive long-range pax3 lbx1 expression at -positive geniohyoideus muscles(Fig.3D).Inaddition, arrowheads) inaregion thatco-localizeswith12/101stainingofthe expression of type of al., 2000).Taken together, theseresultssuggestconservation inthe development oflimbmusclemassesinothertetrapods(Uchiyamaet expressed inthedorsalandventral regions, consistentwiththe region similarto myoblasts arepresentinstage53limbbuds (Fig.3E,arrows) ina head. Indeed, migratory myoblastssuchasthosethatmigrateintothelimbsand migration, wewould expect ittobeexpressed inthelong-rangetype directed againstzebrafish control MO,whichisfamously non-toxic,otherMOs,suchasone 40 (Fig.4B-E,N,arrow) (16%withdefects, tadpoles arelargely unaffected ontheinjectedsideatstage37and side (Fig.4O,arrow). Bycontrast,thecontrolMOinjected tadpoles show alossofthegeniohyoideusmuscleoninjected body wall muscledefects,anteriorventral views ofstage40 4F,G) and40(Fig.4H,I)(87%withdefects, muscle whencomparedwiththeuninjectedsideatstages37(Fig. with thespliceblockingMOexhibit asevere decreaseinbodywall ng ofacontrolMOandthenstainedwith12/101.Thoseinjected two-cell stagewitheither33ngofthespliceblockingMOor led toa91%decrease.Embryoswereinjectedintoonecellatthe transcript comparedwiththecontrol,whiletwo-cell injection 85% decreaseinradioactive signalfromtheproperlyspliced the radiolabeledbandsshowed thattheone-cellinjectioncauseda decrease inproperlysplicedtranscript(Fig.4A).Quantificationof cell wereanalyzedbyRT-PCR. Theresultsindicateaclear or intobothcellsatthetwo-cell stagewith33ngofMOintoeach lbx1 development, asplice-blockingantisenseMOwas usedtoinhibit To determinewhether development Conserved functionof musculature. designed toblockthetranslationof defects (datanotshown). Asafurthercontrol,secondMOwas muscles in due toaberrantmigrationofmyoblasts.Thelackbody wall 1999). (Brohmann etal.,2000;GrossSchaferandBraun, derived from required fortheproperdevelopment ofhypaxial musclesthatare (compare Fig.4I,Mwith4H,L).Theseresultsindicatethat of muscleisalways significantlylessthanontheuninjected side some development ofhypaxialmuscles(Fig. 4I,M),but theamount injected witheitherthespliceortranslationblockingMOexhibit 4J-M) (92%withdefects, hypaxial musclephenotypewas seenatbothstage37and 40(Fig. was injectedintoonecellatthetwo-cell stage,thesamelossof knockdown phenotype.When33ngoftranslationblockingMO indicate theposition of Merged imagesshowing both 12/101 antibody, whichmarksdifferentiated skeletal muscle. stained with myoblasts. Fig.4P-Sshows stage37tadpolesthathave been splice MOcanalsobepartially linked toabnormalmigrationof If The lackofhypaxialmusclesobserved in function. Embryosinjectedintoonecellatthetwo-cell stage lbx1 lbx1 has atrulyhomologousrolein Xenopus -positive myoblastmigrationin lbx1 pax3 lbx1 lbx1 pax3 -positive myoblastsmigrateintothehead(Fig.3C, , amarker ofproliferative myoblasts,andthe -positive myoblasts,similartomouse -positive cells(Fig.3F, arrows). Bothgenesare in myoblaststhatpopulatethebodywall tadpoles thathave beeninjectedwith pax3 lbx1 lbx1 n =40). Laterindevelopment, tadpoles -positive hypaxialmyoblasts relative pax3 lbx1 have never caused hypaxialmuscle is requiredforhypaxialmuscle and 12/101staining(Fig.4Q,S) in hypaxialmuscle lbx1 , inordertoconfirmthe n n Development 133(2) =85). Apartfromthe =197). Inadditionto lbx1 X. laevis X. laevis mutant miceis lbx1 myoblast , despite -positive lbx1 lbx1 lbx1 is -

DEVELOPMENT In additiontothelossofhypaxialmuscle, manipulations The expression of of thesomites(80%withmigrationdefect, but moreposteriorcellsremainadjacenttotheventrolateral edge anterior myoblastshave begun tomigrate away fromthesomites, from thesomites(Fig.4Q).InMOinjectedtadpoles4R,S), Xenopus control tadpoles, to thesomitesfromwhichthey werederived (12/101,green).In marked by et al.,1997;Denetclaw andOrdahl,2000).Thisexpansion is this region expands inthedorsalandventral domains(Denetclaw myogenesis intheepaxialdomain ofsomites,themusculature stage illustratesthisloss(Fig. 5A,B).Aftertheinitialonsetof that was injectedwith anterior trunksomites.Atransverse sectionofastage40tadpole also causesaspecificlossofventral epaxialmusculatureinthe ventrolateral anddorsomediallips of thedermomyotome.We hypaxial myogenesis myf5 pax3 expression inproliferative myoblastsatthe -positive cellshave moved ventrally away lbx1 myf5 -splice MOinonecellatthetwo-cell is affected by n =25). lbx1 lbx1 -splice MO 5M,N, comparewith5D),in addition toupregulating rescue, other regions ofthe somite targeted bytheinjection (100% of injection of muscle, asdeterminedby12/101 staining(Fig.5L).Theco- increased sizeofthesomite, thereisalackofdifferentiated (100% withincreased expression isobserved, aswellanenlarged somite(Fig.5K) is overexpressed insomites,adramaticincrease expression inthisregion (Fig.5G-J,arrows). When ventrolateral domain(Fig.1),whichnormallyoverlaps with expression). Thefirst eighttrunksomitesexpress myf5 expression, ventral domains(Fig.5D,arrow) (96% withreduced somites, but isexpressed atnormallevels indorsalandposterior is specifically reducedintheventrolateral region ofanterior examined myf5 expression (Fig.5F)( n expression intheventrolateral domainofthesomite(Fig. =7). myf5 lbx1 n =26). InjectionofacontrolMOhasnoeffect on expression ininjectedtadpolesandfoundthatit mRNA withthe adjacent tothedifferentiated myotome. which showsthemovementof Q,S). Acontrol tadpoleispictured inPandQ, then mergedwiththe12/101antibody(green, and Rare stainedfor large numberof myotome. In myoblasts awayfrom thedifferentiated epaxial geniohyoideus muscleislostonthe into onecellatthetwo-cellstage).The injected sidedownwards (embryoswere injected are shownfrom aventralview, withtheMO injected (N)and in arrow). Myoblastmigrationdefectsare observed both are present inthecontrol injectedMO(N, injected sideofthetadpole(O,arrow), whereas appear butare greatly reduced inthe (compare FwithG).Atstage40,thesemuscles cervicus musclesontheinjectedsideatstage37 exhibit alossofrectus abdominusandrectus injected tadpoles(J-M).( results were observedforthelbx1-transMO- MO-injected tadpoles(compare HwithI).Similar E). Tadpoles injectedwiththe (compare BwithC)orstage40(Dcompared to compared withtheuninjectedsideatstage37 of rectus abdominusmusclesontheinjectedside tadpoles donotshowadifference inthepresence towards theright.Thecontrol MO-injected uninjected sidesare pictured withanterior sides ofeachtadpoleare shown,where the uninjected (B,D,F,H,J,L) andinjected(C,E,G,I,K,M) stained withthe12/101antibody(brown). The mRNA asalineagetracer(red). Tadpoles were splice blockingMO( blocking MO( two-cell stagewithacontrol MO( is seen.Embryoswere injectedintoonecellatthe specific lossoftheproperly spliced Fig. 4. cells orbothatthetwo-cellstage.( injected with formation. lbx1 myf5 -splice injectedtadpoles.( lbx1 n =6, onewithslightlyreduced RT-PCR wasperformedontadpoles is required forhypaxialmuscle lbx1 expression, lbx1 J - lbx1 M lbx1 pax3 RESEARCH ARTICLE -splice-injected tadpoles(R,S),a -splice MOintooneoutoftwo ) alongwith -splice MOrescuestheloss F -splice injected(O)embryos pax3 - -positive myoblastsremain I ) orthe N , O mRNA (purple)and n ) Stage40control =13). Despitethe lbx1 ␤ lbx1 pax3 -galactosidase P - -splice MO B S -translation lbx1 - ) Tadpoles inP lbx1 -positive lbx1 E lbx1 ), the lbx1 transcript A -splice ) A myf5 -splice in their mRNA lbx1 myf5 myf5 myf5 myf5 199 - in

DEVELOPMENT the ventrolateral region ofdeveloping somites,where unmanipulated tadpoles,anabundance ofmitoticcellsarefoundat used tovisualizemitoticcells(SakaandSmith,2001).Innormal, expression. AnantibodytophosphorylatedhistoneH3(pH3)was myoblast proliferationratherthandirectlyupregulating (Fig. 6I,J;resultssummarizedinFig.10).Atlaterstages number ofmitoticcellsortheamountdifferentiated muscle and lack ofdifferentiated muscle(Fig.6H).Injectionof acontrolMO region ofinjectedtadpoles(Fig.6G);thisalsocorrespondstoa dramatic increaseinmitoticcellsisobserved inthesomitic is foundin side, aswellaslightlossofmuscleinthisregion. Theopposite is seenintheventrolateral region ofthesomiteoninjected and 12/101(Fig.6F)antibodies.Aspecificlossofmitoticcells stage and31tadpoleswerestainedwiththepH3(Fig.6E) Fig. 5G). myoblasts arenormallypresent(Fig.6A-D,compareFig.6Bwith in Because oftheenlarged somiteswithalackofdifferentiated muscle lbx1 200 lbx1 The ␤ controls myoblastproliferation -galactosidase mRNA hasnosignificant effect onthe -injected tadpoles,weinvestigated whether lbx1 RESEARCH ARTICLE -splice MOwas injectedintoonecellatthetwo-cell lbx1 mRNA-injected tadpoles(Fig.6G,H),wherea lbx1 lbx1 is controlling -positive myf5 lbx1 muscle onthe tadpoles (Fig.6L).Bystage41,theamountofdifferentiated the excess differentiated musclecrossesthemidlineofinjected terminal differentiation ofthesecells(Fig.6K,L). Insomecases, mRNA injectedtadpoles,mitosisdecreases,correspondingtothe lack ofdifferentiation throughtheinhibitionof investigated whether Pownall etal.,2002;Weintraub etal.,1991).We therefore Duprez, 2004;Halevy etal.,1995;Hopwood andGurdon,1990; Lbx1 downregulates increased cellproliferation. thus confirms thatincreaseinmusclemassisdueto of theembryo(duetodefective convergence andextension), and the increaseinareaofmusclesectionisduetodistortion muscle isseenatbothlevels. Thisrulesoutthepossibilitythat (Fig. 6M)andtaillevel (Fig.6N).Anincreaseindifferentiated shows transverse sectionsthroughastage41tadpoleattrunk muscle isnever observed outsidethesomiticregion. Fig.6M,N exceeds theamountonuninjectedside,thoughectopic of myoblastscanbecausedbytheexpression of In many cases,exit fromthecellcycle andterminaldifferentiation injected cells. is rescued when in MandinjectedsideN.Thelossof splice MOand the right.( into onecellatthetwo-cellstage.Theinjectedsideisto with 12/101staining(L,green). Embryoswere injected stage 28tadpole,showing stained for approximately theleveloftrunksomite3.Thissectionis transverse sectionfrom astage28tadpoleat differentiated muscle.( myf5 Overexpression ofzebrafish myoblast region ofthesomite(H,J,arrows). green). Bothtranscriptsare foundintheventralhypaxial with 12/101staining(H,green). ( somite 3.Thesectionisstainedfor of astage28tadpoleatapproximately theleveloftrunk myoblasts canbeseeninG-J.( expression domainsfor tadpole ontheinjectedside(D,arrow). Thesimilar normally expressed isseeninthe loss of All ofthesepanelsare stainedfor where EistheuninjectedsideandFinjectedside. the injectedside.( injected tadpolewhere CistheuninjectedsideandD corresponding lossof domain ofepaxialmuscle(B,arrowhead). A muscles (B,arrow), butalsoalossofmore dorsal injected) reveals notonlyalossofventralbodywall splice-injected tadpole(leftsideuninjected,right 28 Fig. 5. loss of lbx1 lbx1 expression andlargersomites,butadecrease in myf5 myf5 mRNA-injected sideofthetadpolesgreatly -splice injectedtadpoles.( lbx1 lbx1 M myf5 . , expression intheregion that may promotemyoblastproliferationand N expression isaffected bythegainor ( A myoD lbx1 ) Stage29tadpoleco-injectedwith , lbx1 B (I) andmergedwith12/101staining(J, E Atransverse sectionthrough an ) , mRNA. Theuninjectedsideisshown F ) Control MO-injectedtadpole mRNA isaddedbackto myf5 K lbx1 and , L ) Atransversesectionofa myf5 expression isseeninstage lbx1 and p27 G , causes anincrease in myf5 staining (K)merged H I C lbx1 Development 133(2) , myf5 J , ) Atransversesection lbx1 D ) Similarly, a myoD ) An myoD -splice-injected in thehypaxial myf5 expression. A (G) andmerged lbx1 lbx1 (Delfini and expression. expression lbx1 -splice is -splice lbx1 lbx1 - -

DEVELOPMENT Xenopus downregulation of lbx1 One cellofeachembryowas injectedatthetwo-cell stagewith myoD 7C-E), while galactosidase staining), are seen.Inregions where on theinjectedside(Fig.7G).Aslateasstage33,sameresults somite (Fig.7B),againthereisanexpansion of loss of Transverse sectionsthroughstage24 tadpolesshow acomplete side (Fig.7A),while not shown). hypaxial muscles,suchas n endogenous mis-expression of opposed totheuninjectedside (Fig. 7K).Atstage33,thetargeted endogenous repression ofendogenous injected cells,they donothave ahypaxial identityasshown by =10). Despite therapidproliferationand mRNA andanalyzedatvarious stages.Atstage22,astrong expression, myoD hypaxial myogenesis lbx1 lbx1 expression ontheinjectedside,despiteenlarged pax3 expression (Fig.7M).Other markers expressed in expression ontheinjected side(Fig.7L)as n myoD is expanded (Fig.7H-J)(100%withreduced lbx1 =9; 100%withincreased pax3 myoD to posteriorsomitesdoesnot induce lbx1 expression isobserved ontheinjected lbx1 expression isexpanded (Fig.7F). . Astage24tadpoleshows alossof and expression isdownregulated (Fig. is expressed (marked byred tbx3 pax3 show similarresults (data expression in pax3 pax3 expression, expression lbx1 ␤ - - lateral edgeofthe somitewhereactive differentiation isoccurring. (Vernon andPhilpott,2003).Normalexpression isfoundalongthe shown toberequired fortheproperdifferentiation ofthemyotome injected side(Fig.7P).Bystage41,ectopic tadpole, thepresenceofinjected coincide withthepresenceofectopic cycle inhibitor from thecellcycle andterminal differentiation. In expression isobserved atthisstage(Fig.7S). on theinjectedside(Fig.6M,N).Acorrespondingincreasein stage atwhichamuchlarger differentiated myotomecanbedetected longer bedetectedbyinsituhybridization(Fig.7R).Thisisalso the situ hybridization(Fig.7O).Atthisstage, repression of increased ventrolateral region ofthesomite(Fig.7N, arrow) (64%with slight increaseof MO intoonecellatthetwo-cell stage.Atransverse sectionshows a observed instage26tadpolesthatwereinjectedwiththe function shouldresultinatransientincrease As previously mentioned, theexpression of If lbx1 myoD normally represses splice MO( injected inonecellatthetwo-cellstagewitheither the somite(arrows). Theremaining panelsshowtadpoles proliferating cellscanbeseenintheventrolateral region of (B), 33(C)and37(D),anincreasing numberof ventrolateral region ofthesomite(A,arrow). Atstages28 is oninmyoblasts,showsverylittlecellproliferation inthe larger. ( differentiated musclemassontheinjectedsideismuch mRNA havethesameamountofcellproliferation, butthe section through astage24tadpole,before in boththetrunk(M)andtail(N). has alargerdifferentiated musclemassontheinjectedside tadpoles injectedwith the samesectioninF(12/101,green). ( slightly smallerdifferentiated musclemasscanbeseenin (E). Consistentwiththelossofproliferating cellsinE,a proliferating cellsintheventrolateral region ofthesomite injected with side ontheright.Stage33tadpolesthathavebeen All ofthesepanelsare transversesectionswiththeinjected in thehypaxialmuscleregion ( combination with12/101(green) showscellsproliferating lbx1. Fig. 6.Myoblastcellproliferation iscontrolled by muscle. ( difference incellproliferation orsizeofdifferentiated of thelineagetracer(red). ( increase inthenumberofproliferating cellsintheregion ( in Ginhibitsmuscledifferentiation (12/101,green). I , J myoD Control injectedtadpolesatstage33showno ) p27 expression oninjectedside, An anti-phosphohistoneH3antibody(brown) in M is expressed indeveloping somitesandhasbeen myoD K , caused bytheectopic N , L E ) Astage41tadpoleinjectedwith ) Bystage37,tadpolesinjectedwith , F lbx1 ), expression ontheinjectedsidein lbx1 -splice MOshowadecreased numberof myoD mRNA (G,H,K-N)oracontrol MO(I,J). lbx1 lbx1 RESEARCH ARTICLE H mRNA exhibitadramatic mRNA canbedetectedbyin expression, thelossof ) Activecellproliferation seen lbx1 A - myoD D lbx1 , arrows). Atransverse n mRNA. Inastage31 myoD =11). Inaddition,the lbx1 myoD G expression should is repressedonthe ) Stage28 Xenopus mRNA canno lbx1 can leadtoexit . Thisresultis lbx1 Lbx1 expression lbx1 , thecell mRNA lbx1 -splice myoD lbx1 201 -

DEVELOPMENT the injectedside(rightside)(G).Atstage33,expression of complete lossof expression ishigher(F).Theseembryosare shownfrom adorsalview, withtheinjectedsideontop.Transverse sectionsofsta injected side(S,T). right). Atstage24,endogenous stage 33inregions ofthelineagetracer(H,I).TheuninjectedsideisshowninH,injectedI,andatransversesectio uninjected sideisshowninC,injectedD,andatransversesectionE(injectedtotheright). expression, theoppositeresultswere obtained.Atransverse expression ontheinjected sideobserved afterwild-type Instead ofthedecreasein mutant constructresultedinaslight dominant-negative phenotype. that spanstheentireeh1domain. Theover-expression of this PCR-based mutagenesiswas usedtodeletea12aminoacidstretch lbx1 The engrailedrepressor domainisrequired for (Fig. 7T, rightside). cells thatwereoverproliferating arenow terminallydifferentiating side, mRNA isnolongerpresentand is absentontheinjectedside).Atstage41,when indicates aregion ofstrongexpression ontheuninjected side,which is alsorepressedcomparedtotheuninjectedside(Fig.7Q,arrow myoD myoD sections ofstage31(O-Q)and41(R-T)tadpolesinjectedwithlbx1intoonecellatthetwo-cellwere stainedforinject 33, ectopic J injected with Fig. 7.Lbx1downregulates 202 stage 31,wheninjected We examined theexpression of ) or lbx1 p27 and (P,S) and function RESEARCH ARTICLE ( is alsoupregulated ontheinjectedside,indicatingthat K p27 lbx1 - M lbx1 ). Redstainingisthe are repressed ontheinjectedside(P,Q). WhentheinjectedmRNAisnolongerdetectable(R), p27 expression intheposteriorsomites(red staining)failstoupregulate endogenous myoD -splice MO(rightside)showsamildupregulation of (Q,T, arrow inQindicatesregion ofstrong expression ontheuninjectedside).Wheninjectedlbx1mRNAisstillpresent (O), expression ontheinjectedside(rightside),despitethere beinganenlargedsomite(B),while lbx1 myoD myoD is presentand lbx1 myoD p27 ␤ expression andincrease in . -galactosidase lineagetracer. Atstage22, expression islostontheinjectedsideofembryo(L),whencompared withtheuninjectedside(K).Atstage Embryos were injectedwith in is upregulated ontheinjected lbx1 myoD -injected tadpoles.At is repressed, myoD is stilldownregulated inregions ofthe lbx1 pax3 lbx1 lbx1 p27 myoD mRNA inonecellatthetwo-cellstageandstainedfor lineages, rather thananincreaseintheproliferation ofmyoblasts expansion ofmyotomethrough therecruitmentofnon-myogeniccell overexpression of should eliminatetheoverproliferation ofmyoblasts.In downregulation of If myoblast proliferation Co-injection of 29 tadpoles,having ashorterbut widershape (Fig.8D,F, arrows). In addition,themyotomeisabnormaloninjectedsidesofstage of stage29tadpoles,asjudgedwiththepH3antibody(Fig.8C,E). of cellproliferationdoesnotappeartoincreaseontheinjected side arrow) (78%withreduced downregulated ontheinjectedsideofastage 31tadpole(Fig.8B, increased expression ontheinjectedside(Fig.8A,arrow) (90%with section throughastage24embryoshows anincreaseof in theventrolateral domainofthesomite(arrow). ( myoD lbx1 is downregulated ontheinjectedside(A),while is controllingmyoblastproliferation throughthe myoD myoD myoD on injectedside, lbx1 myoD expression (M).( , thentheco-injectionof or ␤ pax3 pax3 -galactosidase lineagetracer(C-E).The with myf5 myoD expression isalsostillupregulated at on injectedside, in thesomiticregion causes an lbx1 and pax3 N n p27 ) Astage26,tadpole =20), whereaspax3is n inJ(injectedsidetothe represses expression ishigheron ed lbx1mRNA(O,R), are upregulated onthe Development 133(2) ge 24embryosshowa myoD O n - =9). Theamount T myoD ) Transverse ( A Xenopus pax3 - E ), with pax3 myoD lbx1 , the ( F -

DEVELOPMENT the recruitmentofnon-myogeniclineages. tadpoles aretheresultofincreasedmyoblastproliferationandnotfrom Figs 6and9,indicatethattheenlarged myotomesof (arrows). Theseresults,combinedwiththecellproliferationresultsof on boththeuninjected(Fig.11E)andinjected11F)sides with normal, well aspax8inthepronephrictubules (Fig.11D,arrow) (100% NCAM intheneuraltubeoninjectedside(Fig.11C,arrow), as through tadpolesinjectedwith uninjected sides(Fig.11G,I,arrows). However, transverse sections tadpoles (Fig.11H,J).Thepronephrictubules arepresentonthe n shows thatpronephrictubules arelostonthe cell proliferation.Insituhybridizationforpax8,apronephricmarker, antibody exhibit larger somitesontheinjectedsidebut noincreasein myoD Transverse sectionsofstage29tadpolesthathave beeninjectedwith is notduetoincreasedcellproliferation(Ludolphetal.,1994). tadpoles istheresultofrecruitmentnon-myogeniclineages,and As previously described,theenlarged myotomesof lineages the result oftherecruitment ofnon-myogenic Enlarged somitesin mitotic nucleiontheinjectedside. tadpoles, donotshow asignificantdifference intheamountof including thecontrolMO+ Fig. 10,indicatedwithanasterisk).Allotherinjectionsshown, splice MOinjectedsidesoftadpoles(pairedStudent’s observed ontheinjectedsidesof tadpoles isshown inFig.10.Significantlymoremitotic nucleiare immediate somiticregion ofinjectedversus uninjectedhalves of and (Fig. 9N)but thereisalsoanincreaseincellproliferation(Fig.9M) injected tadpoles,whilesignificantlyfewer areobserved onthe to theinjectionof 9B) andanexpansion of of mitoticcells(Fig.9A),adecreasedifferentiated muscle(Fig. (Ludolph etal.,1994).In Xenopus The expression of DISCUSSION similar to a slightdecreasein cells (Fig.9D),alarge increaseindifferentiated muscle(Fig.9E),and Expression of tetrapods However, co-expression of cannot promotemyoblastproliferationinthepresenceof decreased ontheinjectedsideoftadpole(Fig.9L).Thus, no increaseincellproliferation(Fig.9J). observed. Thereisanincreaseindifferentiated muscle(Fig.9K)with injected with proliferation (Fig.9G)or a slightexpansion ofthemyotome(Fig.9H),but hasnoeffect oncell and head,those thatextend asepithelia toformtheinter-limb between myoblaststhatundergo long-rangemigrationintothe limbs the mouseandchicken. In amniotes, avery cleardistinctionismade tetrapods; itisonlyfoundinprospective limbandtonguemusclesin muscles in =11) or The summaryofthenumbersphospho-histoneH3nucleiin pax3 lbx1 (Fig. 11A)or n myoD expression (Fig.9O). =8). Similarly, lateralviews ofastage31tadpoleinjected hypaxial myogenesis show normalexpression ofpax8inthepronephric tubules lbx1 Xenopus laevis myoD lbx1 alone, whereaslightlysmallermyotomeisobserved + lbx1 myoD in myoblaststhatwillformtherectus abdominus pax3 myoD , aphenotypethatisthesameas (80% absent, pax3 lbx1 expression (Fig.9F). pax3 lbx1 alone, whichcausesnochangeinmitotic + lbx1 is different fromits expression inother lbx1 myf5 expression (Fig.9C).Thisisincontrast -injected tadpoles,thereisanincrease lbx1 expression (Fig.9I).When in (Fig. 11B),andstainedwiththepH3 -injected tadpolesare not ␤ X. laevis with -galactosidase mRNA-injected alone show normalexpression of lbx1 n =10) injectedsidesofstage31 lbx1 and pax3 produces aphenotype lbx1 is unusualfor myf5 myoD expression isalso + injection causes myoD myoD myf5 t (91% absent, -test, lbx1 lbx1 -injected -injected mRNA- P alone is myoD <0.01. is co- lbx1 lbx1 - . the issueofwhetherornot The uniqueexpression of Conservation of animal. body wall muscleshasbeenlostorsignificantlymodified inthis laevis expression of the long-rangetypeofmigratorymyoblasts(Jaglaetal.,1995). The In amniotes (mesenchymal versus epithelial)andbythegenesthatthey express. on themechanismbywhichthesecellspopulatetarget sites abdominal muscles(Dietrich,1999).Thisdistinctionisbasedboth rather thanasepithelial extensions (Fig. 3).Thus, show thatbodywall muscleprecursorsmigrateasmesenchymalcells X. laevis zebrafish limbs/finsisalsousedfor bodywall muscledevelopment in appears thatthelong-rangemigration mechanismusedinamnioteand body wall andhypoglossal muscles(Fig.4).Fromthesedata,it knockdown of 2000; Grossetal.,SchaferandBraun,1999).Likewise, severe loss,but notdepletionof musclesinlimbs(Brohmannetal., development ofthesemuscles.The a slightlyshorter, yetwider, differentiated myotome(arrows). observed ontheinjectedsides(C,E),and12/101staining(D,F)indicates with 12/101(D,F).Noincrease inthenumberofmitoticnucleiis type stage. Theinjectedsidesare ontheright.Phenotypesoppositeofwild- homologydomainwasinjectedintoonecellatthetwo-cell function. Fig. 8.Theengrailedrepressor domainof 29 tadpoles( section reveals anincrease in decrease inPax3staining( arrow), whileatransversesectionthrough astage31tadpoleexhibits lbx1 suggests thatthemechanismofepithelialextension toform tadpoles. Insupportofthisidea,in situhybridizationpatterns overexpression were observed.Atstage24,a transverse (A-F) mRNAfrom amutant , lbx1 C - F lbx1 lbx1 ) were stainedwiththepH3antibodyalone(C,E)or was thefirstmolecularmarker thatwas specificto function in in bothlimb,headandbodywall musclesin lbx1 B lbx1 , arrow). Transverse sectionsthrough stage myoD function X. laevis in bodywall muscleprecursorsraises lbx1 staining ontheinjectedside( lbx1 lbx1 plays aprominentroleinthe RESEARCH ARTICLE using antisenseMOsdepletes mutation inmousecausesa construct thatlacksthe lbx1 is necessaryforits lbx1 -positive A , 203 X.

DEVELOPMENT myoblasts in 3C,E). Taken together, thesedatasupporttheideathatbodywall the long-rangetype,namelylimbandhypoglossalmyoblasts(Fig. also expressed inhypaxialmyoblaststhataretraditionallyconsidered Braun, 1999;Uchiyamaetal.,2000).UsingantisenseMOsto migration (Brohmannetal.,2000;GrossSchaferand Several resultssupportarolefor secondary toreduced myoblastproliferation Migration defectsin and tonguemyoblastsofamnioteszebrafish. in thetypeofmigration(Neyt etal.,2000).Finally, migration ofzebrafishmyoblastsintothefin,suggestingconservation the bodywall muscle(Fig.3A,B).Thisissimilartowhatseeninthe myoblasts emerge fromthesomitecleftsbeforemoving ontopopulate 204 RESEARCH ARTICLE X. laevis are migratinginamannersimilartolimb/fin lbx1 lbx1 loss offunctionmaybe in mousehypaxialmyoblast .lei lbx1 X. laevis is knockdown would expect theretobemoresevere defectsintongueand extent. If suggesting thatmyoblastshave notproliferatedtotheproper positive. Althoughthey form,thesemusclesarereducedinsize, 1999). Theprecursorstoallofthesemusclesarenormally (Brohmann etal.,2000;GrossSchaferandBraun, limb musclesform,inadditiontotongueanddiaphragm (Amthor etal.,1999).Furthermore,inmouse tongue levels causesalackoflimbandtonguemusculature capacity ofmyoblastsbyremoving theectodermatlimband Amthor etal.,1998).Inthechick,reducingproliferative needed forhypaxialmyoblastmigration(Amthoretal.,1999; proliferation, andthatthismaybeitsprimaryrole. However, wealsofoundthat A properbalancebetweenproliferationanddifferentiation is (B,E,H,K,N) or anti-phospho-histone H3antibody(A,D,G,J,M),12/101 stage 29,sectionedinthetransverseplane,andanalyzedwith different tadpoles.Injectionof on thesamesections,while Anti-phospho-histone H3and12/101stainingwere performed each tadpoleisontheright,markedby and Injection of differentiated muscle(H)andnochangein significant changeincellproliferation (G),aslightincrease in increase in proliferation (A),decrease indifferentiated muscle(B)and with injectionof decrease in proliferation (D),anincrease indifferentiated muscle(E)anda with, Embryoswere injectedintoonecellatthetwo-cellstage O) eliminates overproliferation and Fig. 9.Co-injectionof (M-O) causesaphenotypelikethatof lbx1 lbx1 lbx1, lbx1 is onlyinvolved inthemigrationofmyoblasts,one + ( pax3 A myf5 we have observed asimilareffect in pax3 lbx1 - C pax3 ), (C). Injectionof + myoD ( myoD expression (F).Injectionof M myoD expression (C,F,I,L,O). Theinjectedsideof - O ). Injectedembryoswere grown until lbx1 alone, whileinjectionof ( myoD D (J-L) produces aphenotypeconsistent - F ), pax3 plays acentralroleinmyoblast myf5 lbx1 mRNA with myoD staining wascarriedouton ( causes anincrease in G pax3 - lbx1 I causes nochangein ), Development 133(2) ␤ lbx1 -galactosidase (red). lbx1 upregulation. alone. pax3 myf5 lbx1 + mutants, some lbx1 myoD expression (I). causes no mRNA + Xenopus. ( myf5 J - L (A- ) lbx1

DEVELOPMENT Xenopus # mitotic nuclei 10 15 20 25 30 35 40 45 0 5 morpholino Control = N=9 N=8 hypaxial myogenesis morpholino lbx1-splice ** mRNA lbx1 N=16 mRNA myoD N=8 mRNA myf-5 N=9 mRNA lbx1+myoD N=8 Injected side Uninjected side mRNA lbx1+myf-5 N=9 * MO. test, mitotic nuclei,indicatedbyanasteriskonthe plus represented. Injectionofthe the uninjectedversusinjectedhalvesofmanipulatedtadpolesare phospho-histone H3-positivenucleiintheimmediatesomiticarea of Fig. 10.Summaryofcellproliferation data. myf5 P <0.01). Thecontrol MOusedisthezebrafishtranslationblocking mRNA causedasignificantdifference inthenumberof (G,H) or the myotome,despitethere beingalargersomite. alone donotshowalackoftissuessurrounding sides (H,J).However, tadpolesinjectedwith sides (G,I,arrows), butare lackingontheinjected show normalpronephric tubulesontheuninjected ( the uninjected(E)andinjected(F)sides(arrows). show thepresence ofpronephric tubulesonboth ( arrow) are present ontheinjectedside. and Pax8staininginthepronephric tubules(D, both NCAMstainingintheneuraltube(C,arrow) sections through stage31tadpolesindicatethat (A) or non-myogenic lineages. tadpoles isnottheresult ofrecruitment of Fig. 11.Enlargedmyotomesof recruited tothemyotome.( non-myogenic lineagessuchasthepronephros are transverse sectionsofstage29tadpoles.Instead, (right side),asjudgedbypH3stainingon increase incellproliferation ontheinjectedside G E , - F J Lateralviewsofatadpoleinjectedwith ) ) Stage31tadpolesinjectedwith myoD lbx1 myoD -splice MO, + lbx1 + lbx1 RESEARCH ARTICLE (B) mRNAdoesnotcausean (I,J), andstainedwithpax8, lbx1 x -axis (paired Student’s ( The numberof A,B C , mRNA or D Injection of ) ) Transverse lbx1 myoD -injected lbx1 lbx1 mRNA alone myoD lbx1 205 t -

DEVELOPMENT musculature (McDiarmidandAltig,1999).In rectus abdominus,whichisthehypaxialcomponentoftrunk representing theepaxialdomain.Theventral region containsthe muscle inthetrunk.Indorsalregion, thereisthedorsalis trunci, At earlytadpolestages, musculature lbx1 shown). fact, therearefewer hypaxialmusclesinthese tadpoles(datanot overexpressed, thereisnoincreaseinnumberofmigratorycells.In also explain whyinmicemutantfor mediated means,suchas cells having hadalongertimetoproliferatethroughnon- have migratedventrally (Fig.4R,S).Thismaybetheresultofthose stuck adjacenttothesomite,thoseoriginatingfromtrunksomite1 pax3 progression asthepoolofmyoblastsexpands. Thus,atthetimethat Xenopus mass ofcells.Themigrationdefectsseenin progressively bymigratingmyoblasts(Kardonetal.,2003). compartments arespecifiedattheperipheryandfilled the lackofrestrictionmyoblastfates, suchthatmuscle musculature, except inreducedsizes.Thisisalsoconsistentwith proximal regions, aswellthediaphragmandtongue,have regions oflimbscompletelylackmusculature,but themore defects. Thisiswhatitseeninmousemutants,wherethedistal sites farthest fromthesomitewould exhibit themostsevere differentiate atvarious pointsalongthemigratorypath,thanthose myoblasts hasareducedabilitytoproliferate,andsome diaphragm muscles.Ifthestartingpopulationofhypaxial 206 a casewhere to theformationofepaxialmusculature.Thisisfirstexample of (Fig. 5B,D).Thisindicatesthat is reduced,withacorrespondinglossof injected tadpoles,theventral componentofanteriorepaxial muscle migration comesfromtheoverexpression of 1999). (Brohmann etal.,2000;GrossSchaferandBraun, which originatefromposteriorsomites,completelyfail toform originate fromanteriorsomites,form,whereashindlimbmuscles, ectopic show thatthisisalsotrueinthecontext of the wholeembryo.When myoblasts intissueexplants (MennerichandBraun,2001).Here,we Previous work hasshown that proliferation In vivoevidencethat myoblast proliferation,andnotmigration. The roleof these two muscletypesaremoresimilarthanpreviously suggested. hypaxial muscle,suggestingthatthedevelopmental programsof the injected smaller amountofdifferentiated muscle(Fig.6H).Lateron,after somites wereformed.Initially, theseenlarged somitescontaineda earlier arguments thatthe primary roleof However, somite, asmyoblastscandivide ordifferentiate, but not do both. the firstindicationthat differentiated into a large amountofmuscle(Fig. 6M,N).Thiswas The abilityofmyoblastcellstomigratemayalsorequireacritical Finally, evidence that -positive myoblastsoriginatingfromtrunksomite4maybe -positive myoblastscontributetoepaxial RESEARCH ARTICLE lbx1 tadpoles occurearly, andrecover inananteriortoposterior lbx1 lbx1 lbx1 mRNA was targeted tothesomiticregion, enlarged lbx1 lacks theability toinducemyoblasts,asinjections in bothtypesofmuscledevelopment also supports -positive myoblastscontribute tobothepaxialand had beendepleted,theenlarged somites lbx1 lbx1 pax3 Xenopus was controllingcellproliferation inthe lbx1 lbx1 is notdirectlyinvolved in myoblast -dependent proliferation.Thismay lbx1 controls myoblast can promotetheproliferationof contain two typesofskeletal -positive myoblastscontribute lbx1 myf5 , tonguemuscles,which staining inthisregion lbx1 lbx1 lbx1 lbx1 . When is topromote -splice MO- knockdown lbx1 lbx1 is expressed and anincreaseindifferentiated muscle(Fig.9D-F).Whenweco- in thenumberofmitoticcells,aslightdecrease observation byshowing thatoverexpressed have adifferent fate (Ludolphetal.,1994).We confirmedthis result oftheinductionmyogenesisincellsthatwould otherwise has previously beenshown toproduceenlarged myotomes,asthe myf5 from enlarged somitescausedbyectopicexpression of inactivation of control ofcellproliferationby independent of that enlarged somitescausedby myoD proliferation; theproliferative effect canbereversed byrestoring to whatwas seeninthechickexperiments. greater thantheamountonuninjectedside(Fig.7S),similar Further evidencethat function experiments thatlinks inhibits In additiontothegain-of-functionexperiments showing that the intensityof injected (Jagla etal.,2001).Indeed,ourresultsdemonstratethatafterthe which hasbeenshown tomediatetranscriptionalrepression DNA-binding domain,italsohasanengrailed homologyregion, transcriptional repressor. Inaddition tohaving ahomeobox would fit withtheputative roleofLbx1proteinasa myoblast proliferationinwhich expression inthechickexperiments mayfollow aperiodof expression of overexpression of proliferation inwhich these experiments istheendresultofaperiodmyoblast 2001). We have foundthattheincreasein expanding thepopulationofmyoblasts(MennerichandBraun, process requirescellproliferation,indicatingthat increase in In thechick, downregulating lbx1 proliferation (Fig.9A-C). injected tadpolesindicatesthat pax3 myoblasts ormuscle(datanotshown). Thepresence ofexpanded targeted outsidethesomitedidnotresultinformationofectopic Instead, aweakincreasein When overexpressed, themutantconstructnolongerinhibits encompasses theentireeh1domain changesthefunctionof essential foritsfunction.Theremoval ofa12aminoacidstretchthat relieved. transcription. If side (Fig.8A).Furthermore, ratherthan Stage 26tadpolesthathave beeninjectedwiththe lbx1 7N), suggestingthattherepressionbyendogenous exhibit anincreasein We foundthatwhile We have alsoshown that theputative repressordomainof lbx1 . Theoverexpression of function shouldresultinatransientupregulation of expression aswellahighernumberofmitoticcellsin controls myoblastproliferation by expression withinjectedmRNA (Figs9,10).Thusthe myoD lbx1 does nothave theabilitytocontrolcellproliferation myoD myoD lbx1 can nolongerbedetectedbyinsituhybridization, myoD myoD expression, wealsofindevidence inMO loss-of- myoD lbx1 and myoD lbx1 overexpression inthelimbregion leadstoan expression andlimbmuscleformation.This lbx1 expression. , sowesuggestthattheincreasein has adirectroleinrepressing myoD myoD levels (Fig.9J-L).Thisindicatesthatthe in thesomiticregion stronglyinhibitsthe staining ontheinjectedsideisactually , thesameresultwas obtained,indicating myoD lbx1 lbx1 lbx1 myoD expression ontheinjectedside(Fig. myoD lbx1 expression aredifferent inorigin expression isseenonthe injected lbx1 represses lbx1 myoD represses and is amajorregulator ofcell is notexpressed. The to theinhibitionof pax3 myf5 is achieved throughthe is notexpressed. This myoD Development 133(2) myoD being upregulated on in myoD Xenopus myoD causes nochange pax3 lbx1 , andcauses expression in myoD lbx1 lbx1 -splice MO expression, myoD has been embryos , lossof may be myoD myoD lbx1 myoD myoD lbx1 lbx1 lbx1 or is - . . .

DEVELOPMENT as wellaneh1repressordomain,similarto similar phenotypeto the eh1domain,itwould notbeabletorepressthetarget .A of endogenoushomeoboxcontainingproteins.Owingtothelack maybindtohomeobox-bindingsitesandprevent thebinding the somite. pax3 may includeanotherNK-classtranscriptionfactor, orperhapseven interferes withthenormalfunctionofaproteinotherthan The oppositeactivity of lbx1 The nature ofthedominantnegativeactivity eh1 domainproducesaweakdominant-negative effect. lbx1 the injectedside,itisinhibited(Fig.8B).Theseresultsindicatethat Xenopus Amthor, H.,Christ,B.,Weil, M.and Patel,K. References (GM061952) toSharon Amacher. Association (015164).B.L.M.wasalso partiallysupportedbyaNIHgrant work wassupportedbytheNIH(GM42341) andtheMuscularDystrophy Amacher andmembersofhergroup forhelpfulcommentsand advice.This We thankmembersoftheHarlandgroup, MarvaleeWake, andSharon normal factors. Importantlythough, Krieg, 2002).BagpipeisamemberoftheNK-classtranscription bagpipe homologue expression in that central playerintheprecisetimingofmyotomedifferentiation and (Vernon andPhilpott,2003).Theseresultsindicatethat undifferentiated somitesresult,similarto somites. WhenitsfunctionisreducedusingantisenseMOs,enlarged The cellcycle inhibitor control A molecularlinkbetween a naturaldominant-negative protein. acid substitutionintheeh1domain.Thismayeffect causeittobe splice MO,the that itisactingasdominantnegative. Contrastingwiththe (Fig. 7Q).Atstage41,wheninjected mRNA isstillpresent, detectable, surrounding thesomitein myoD recruitment ofnon-myogeniclineagessimilartowhatisseen in that thisisthecauseofenlarged somites,but theremayalsobea The enhancedcellproliferationseenin injected tadpoles proliferation andnotfromtherecruitmentofnon-myogeniclineages. somites in tadpoles (Fig.11G-J).Theseresultsdemonstratethattheenlarged the pronephrosisabsentinboth the neuraltubeandpronephrosformnormally(Fig.11C-F).However, responsible forenlargedmyotomesin Recruitment ofnon-myogeniclineagesisnot tadpoles. indicate thatcell-cycle inhibitionisdisruptedin These resultsaresimilarto differentiation duringlimbmuscledevelopment. lbx1 , whichisexpressed throughouttheentiredorsoventral axisof normally functionsasarepressorandthattheremoval ofthe eh -injected tadpoles(Ludolphetal.,1994).We examined tissues – lbx1 may inhibititsexpression. We testedthisbyexamining hypaxial myogenesis p27 lbx1 Pax3 expression domain.Thisresultsuggeststhat lbx1 expression isenhancedontheinjectedside(Fig.7T). lbx1 -injected tadpolesaretheresultofenhancedcell contains bothahomeoboxDNA-binding domain -injected tadpoles.Atstage31,wheninjected koza eh– lbx1 p27 injections alsohave aneffect outsideofthe lbx1 is overexpressed in p27 lbx1 expression isinhibitedontheinjectedside eh– myoD koza eh– overexpression isobserved whenthe is expressed indeveloping -injected tadpolesandfoundthatboth relative towild-type expression inthesametadpolesand contains anon-conservative amino myoD lbx1 lbx1 (1998). Theimportance oftiming and lbx1 Curr. Biol. and cellcycle -injected tadpolessuggests Xenopus lbx1 myoD mRNA isnolonger lbx1 -injected tadpoles 8 , 642-652. lbx1 + . TheLbx1 lbx1 (Newman and lbx1 lbx1 lbx1 - indicates -injected -injected Xenopus p27 lbx1 . This lbx1 lbx1 p27 is a eh– eh– - Hazelton, R.D. Hammond, W. S. 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DEVELOPMENT