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For example, fibroblastgrowth factor receptor1( BMPs, othergrowth factors arecrucialforsomiticdevelopment. BMP signalingplaysaroleinpatterningtheLPM.Inaddition to (Hornbruch etal.,1979;Tonegawa andTakahashi, 1998).Thus, antagonist Noggincangeneratesomitesfromlateralplatecells regulated byBMPsignaling.Hensen’s nodeortheBMP Mishina, 2003).Thedevelopment ofthe lateralplateisalso and hematopoietictissues(KishigamiMishina,2005; development ofmany embryoniccelltypes,includingtheheart unknown (Tam andBehringer, 1997). mechanisms involved in recruitment oftheepiblastcellsarelargely 1995; Tam andQuinlan,1996).However, themolecular trunk (Kinderetal.,1999;Parameswaran andTam, anterior ,respectively, andbecomecommittedto (PXM)cellsarerecruitedtothemiddleand streak stage,prospective lateralplatemesoderm(LPM)and Lawson etal.,1991;Parameswaran andTam, 1995).Bythelate they arecommittedtoyolksacmesoderm(Kinderet al.,1999; mesodermal cellsarerecruitedtotheproximalprimitive streakand early tomidstreakstage,primarilyfutureextra-embryonic the primitive streakclosesttotheextra-embryonic tissues.Atthe and Behringer, 1997).Inthiscase,‘proximal’referstotheendof primitive streakextends tothedistal tipoftheegg cylinder (Tam and thedistalendsofprimitive streak,andultimatelythe progresses, cellsarerecruitedtointercalatebetweentheproximal at itsjunctionwiththeextra-embryonic .As primitive streak,atransientstructure,formsintheposteriorepiblast Gastrulation begins aroundembryonicday6.5(E6.5), whenthe INTRODUCTION KEY WORDS:,Primitivestreak, Paraxialmesoderm,,BMP, FGF, Mouse development. rescues thedevelopmentofsomites.ThissuggeststhatBMPandFGFsignalingfunctionantagonisticallyduringparaxialmesoderm columns. InhibitionofFGFsignalingrestoresthenormaltimingrecruitmentprospectiveparaxialmesodermandpartially paraxial mesodermcharacterwithintheprimitivestreak,resultinginalateralexpansionofsomitictoformmultiple of prospectiveparaxialmesodermcellstotheprimitivestreakisdelayed.Thisdelaycausesamoreproximaldistributionce development. Unlike inactivation of Bmpr1a Shigeto Miura development ofthesomites recruitment oftheprospectiveparaxialmesodermand BMP signalingintheepiblastisrequiredforproper Development 133,3767-3775(2006)doi:10.1242/dev.02552 Accepted 26July2006 1 10, C458,Research Triangle Park,NC27709,USA. Environmental HealthSciences,111T. W. AlexanderDrive,POBox12233, MDC4- *Author forcorrespondence (e-mail:[email protected]) Duke UniversityMedicalCenter, DurhamNC27710,USA. Laboratory ofReproductive andDevelopmentalToxicology, NationalInstituteof It isnow wellestablishedthatBMPsignalingregulates the encodes theBMPtypeIAreceptorforbonemorphogeneticproteins(BMPs),including2and4.Here,weusemosaic Bmpr1a 1 , ShannonDavis Bmpr1a in theepiblastofmouseembryo( -null ,whichfailtogastrulate, 2 , JohnKlingensmith 2 Department ofCellBiology, Fgfr1 ) deficient Bmpr-MORE 2 and Yuji Mishina and useofanimals inresearch. accordance withNIHandinstitutional guidelinescovering thehumanecare previously (Davis etal.,2004).Allmouseexperiments were performedin The straininformationandgenotyping proceduresweredescribed Mouse studied in (Davis etal.,2004).Here,thedevelopment ofmesodermaltissuesis ,andthatendodermalmorphogenesiswas defective that theanteriorneuralectodermwas enlarged attheexpense of embryos. Ourprevious studiesusing mosaic MATERIALS ANDMETHODS gastrulation. interact forproperdevelopment of the mesodermduring development. Ourresultsalsoindicatethat BMPandFGFsignaling PXM cellscorrectlyandconsequentlytodirectnormalsomite in theepiblastisrequiredtoregulate therecruitmentofprospective Bmpr1a Soriano, 2000).For simplicity, werefertoembryosinwhich expression intheepiblast(Hayashietal.,2002;Tallquist and useful toolforreducingbut notcompletelyabolishinggene Bmpr1a and aftergastrulation,wehave generateda conditionalalleleof To elucidatefunctionsofBMPsignalingthroughBMPR1Aduring function of embryos donotinitiategastrulation(Mishinaetal.,1995),the mouse development. However, because molecules constituteamajorBMPsignalingcomponentinearly known tobeareceptorforBMP2andBMP4(Mishina,2003).These mesodermal tissues. about how they mightinteracttoregulate paraxialversus other during mousemesodermdevelopment. However, littleisknown high potentialforinteractionbetweenBMPandFGFsignaling Yamaguchi etal.,1994).Thus,onecanspeculatethatthereisa the development ofthePXMlineage(CirunaandRossant,2001; embryos donotformsomites,andFGFsignalingisinvolved in Bmpr-MORE Bmpr1a embryos) toassessfunctionsofthisgeneinmesoderm Cre (Mishina etal.,2002).The is ablatedasaresultofMOREactivity as Bmpr-MORE Bmpr1a encodes thetype1BMPreceptorBMPR1A,whichis -mediated recombinationintheepiblast,providing a embryos initiategastrulation,buttherecruitment 1, * during gastrulationinmiceislargely unknown. embryos. Theresultsindicatethat Mox2-Cre RESEARCH ARTICLE Bmpr-MORE Bmpr1a (MORE) straindrives embryos showed Bmpr-MORE -null mutant Bmpr1a lls with 3767

DEVELOPMENT normal embryos,but no analogousstructureformedin LPM (Fig.1F and medialtotheyolksac,suggesting abnormaldevelopment of masseswereobserved anteriorandlateraltotheparaxialsomites embryos (Fig. 1G,G In formed lateraltosomitesandmedial tovisceralyolksac(Fig.1E,G). mesenchymal structures(Fig.1F visible orultravioletlight,andimaged. (PBS) andobserved withastereo-dissectionmicroscope(Leica)under N al., 1996), according tomanufacturers’ protocols. (Cell SignalingTechnology) usinganABCkit(Vector Laboratories) (7 paraformaldehyde overnight, dehydratedandembeddedinparaffin. Sections Cultured embryoswerewashed in coldPBSandfixed with4% Immunohistochemistry lengths oftimeinrotatingglassbottles. were culturedwithDR50containingeitherSU5402orDMSOforindicated (Sigma) asa10mMsolutionandkept at –20°Cuntiluse.E6.5-6.75embryos SU5402 (CALBIOCHEM)was suspended indimethylsulfoxide(DMSO) Inhibition ofFGFsignaling 3,3,3 Beddington, 1996).Briefly, a0.5%stocksolutionof1,1 Cell lineageanalysiswas carriedoutasdescribedpreviously (Wilson and Embryos wereharvested indissectingmedium(MiuraandMishina,2003). Cell lineageanalysis (McMahonetal.,1998). Foxf1 used werebrachyury(Wilkinson etal.,1990), Whole-mount insituwas performedasdescribed(Beloetal.,1997).Probes Whole-mount insituanalysis 3768 (Fig. 1F developed andextended laterallytoformmultipleirregular columns (Fig. 1F,G). In somites developed asasinglecolumnoneach sideoftheneuraltube these andothermesodermalphenotypes.Incontrolembryos, stage in 1A poorly formedamnioticfoldandacuteposteriorcurvature (Fig. (Fig.1A),whilemutantembryoswerecharacterizedbya bud stage,controlembryoshadadistinctlyformedamnionand apparent in 6.75 (Davis etal.,2004).However, morphologicaldefectswere morphologically indistinguishablefromcontrolembryosatE6.5- Bmpr-MORE mesodermal development Bmpr-MORE RESULTS glass bottle(BTCengineering)suppliedwith5%O to labelthemesoderm.TheseembryoswereculturedwithDR50inarotating the ,torethroughvisceralendodermandthenDiIwas released (HUMAGEN). Thetipoftheneedlesoftlytouchedposteriorregion of solution (0.05%)was sucked intoaglassneedleof25 in 100%ethanolwas dilutedtenfoldwith0.3Msucrosebeforeuse.DiI cardiac crescentdidnotdevelop in (Wallin etal.,1996), al., 2000), including astrikinglateralexpansion ofsomites(Fig.1D 1C,C 2 ␮ Histological sectioningatE8.5revealed important aspectsof Bmpr-MORE at 37°C.Afterculture,embryoswereplacedinphosphate-buffered saline Ј m) weredeparaffinized andstainedwithanti-phosphoErk1/2antibody ). Amnionandchorionwerenotformedyetatthehead-fold Ј ,3 Ј (Mahlapuu etal.,2001), Ј RESEARCH ARTICLE arrowheads). AtE8.5,multipledefectswereobserved, -tetramethy-indocarbocyanine perchlorate(DiI)(MolecularProbes) Ј ,G Bmpr-MORE Pcdh8 Mox1 Ј Bmpr-MORE ). Themutantsomitesformedtypicalball-like epithelial- Ј ,G (Candia etal.,1992), (Yamamoto etal.,2000), embryos showmultipledefectsin embryos initiatedgastrulationandwere embryos, lateralplatewas apparentlylacking,but Bmpr-MORE Ј ). Theheartformedatthemost anteriorendof Foxa2 Ј ). Notochordwas formedandexpressed embryos (Fig.1B (Sasaki andHogan,1994), embryos startingatE7.5.Attheallantoic Bmp4 embryos, ectopicmutantsomites (Winnier etal.,1995), Ј Uncx4.1 ,G Ј Bmpr-MORE ). Incontrolembryos,LPM Epha4 Ј Lefty2 ). Inaddition,atypical (Leitges etal.,2000), (Durbin etal.,1998)and 2 and CO (Meno etal.,1999), Tbx6 embryos (Fig. 2 Lim1 Bmpr Ј balanced with ␮ -dioctadecyl- (Chapman et m diameter Ј ,F (Tsang et - Ј MORE ). Pax1 in themutantembryos(Fig.1H was formedatthisstage,but theallantoiswas notablysmall and oftenweak(Fig.2G al., 2001).In normally expressed inLPM startingatE8.5(Fig.2G)(Mahlapuuet allantois, theposterior primitive streakandLPMatE8.5 (Fig. 2H) of themosaicnaturemutant embryos. suggesting somevariability ofphenotype;thisis possibly aresult all, cells (Soriano,1999).Thisisareporterlinefor We usedamouseline were abiasintheirdistribution, itmightaffect themutantphenotype. unevenly duringdevelopment amongallthreegermlayers.Ifthere therefore importanttoknow ifbothcelltypesdistribute evenly or (mutant) cellsandheterozygous(Davis etal.,2004).Itis markers suchassonichedgehog(Fig.1G Bmpr1a the similarphenotypebothin embryos, namelylateralexpansion ofsomites(Fig.1N,P). Having embryos showed aremarkablysimilarphenotypeto in theepiblast-derived tissues(Fig.1N,P).Many ofsuchmutant allowing ustoobtainembryoswithtotallossofBMPR1Asignaling loxP signaling inmost,but notall,epiblast-derived cells. ingressed intomesodermaltissues,resultinginalossofBMPR1A heterozygous cellssimultaneouslytraversed theprimitive streakand layer ormajortissueintheearlyembryo.Thus,bothmutantand signaling intheepiblastdidnotrestrictcellstoorfromany germ (Fig. 1I-L)(Davis etal.,2004).Theseresultsindicatethat distributed evenly amongtissuesincontrolandmutantembryos expression of which marksthosecellsthathave undergone recombinationby LPM domain(Fig.2D was notonlyexpressed inexpanded somites but alsoincellsofthe et al.,1992),whichisnormallyexpressed inthesomites(Fig.2D,E), patterned correctlyin lateral edgesoftheembryobut individual somitewerelargely 2F (Wallin etal.,1996)was alsoexpanded inmutantembryos(Fig. fate. Theexpression domainof 2C broadened, indicatingtheexpansion ofpresomiticmesoderm(Fig. et al.,1997)(markers ofrostralpresomiticmesoderm)was laterally expression domainof inbothcontrolandmutantembryos(Fig.2B,B shown). short mediolateralrows ofexpression domains(Fig.2A multiple irregular columnsoneachsideoftheembryo, forming shown). Inmutantembryos, expressed asacolumnoneachsideoftheembryo(Fig. 2A;datanot (a marker forcaudalsomiteandpresomiticmesoderm)was each somite)(Leitgesetal.,2000)or examined. Normally, expression forseveral markers forPXMdevelopment was To furtherexplore how mutantsomiteswerepatterned,the improper oflateralplatemesoderm patterning Correct ofectopicsomitesand patterning regulate earlymouseepiblastdevelopment. signalingactinanon-cell-autonomous mannerto BMPR1A Bmpr-MORE By contrast, By contrast,mutantLPMwas very poorlypatterned. Ј Ј ). Thesedatashow thatsomitesextended abnormallytothe ; datanotshown). sequence intheepiblast(Fig.1M,O)(Hayashietal.,2002), flox/null Uncx4.1 Bmpr-MORE lacZ. Sox2-Cre embryos impliesthatdownstream targets of embryos weremosaicforrecombined was correctlyexpressed incaudalregion ofeach As observed previously, bothcelltypeswere Bmpr-MORE Mox1 Ј Epha4 Uncx4.1 R26R ,E Ј Ј mice catalyzecompleterecombinationof ). Somemutantsdidnotexpress ), suggestingtheLPMhadacquiredPXM embryos, expression of (a paraxialmesodermmarker) (Candia to visualizemutantandheterozygous (Durbin etal.,2000)or Uncx4.1 Ј (a marker forthecaudalregion of ). Pax1 embryos. Dll1 Bmpr-MORE (a marker forsclerotome) or Ј (Bettenhausen etal.,1995) ) (Davis etal.,2004).The Dll1 Development 133(19) Bmp4 Cre was expressed in Foxf1 was expressed in and recombinase, Bmpr-MORE Mesp2 was patchy Bmpr1a Ј Foxf1 Sox2Cre ; datanot Bmpr1a Foxf1 Ј ). The (Saga was –/– at ;

DEVELOPMENT ( and heterozygous cells(pink)were distributedevenlyamongtissuessuchassomites(yellowarrowheads), LPM(bluearrowhead) o carrying and allantois(al)were formedatthisstageinmutantembryos(H the mutantembryoconsistsofneuraltissues.Somitesdeveloponlyinposteriorhalfembryo.(H,H developed intheanteriorregion ofcontrol embryos(G,red arrowhead) butnotin the somitesandvisceralyolksac(G (black arrowhead), LPMwasobservedincontrol embryos(bluearrowhead) (G).In the lateraledgeofembryo(F accumulated betweensomites(yellowarrowheads) andvisceralyolksac(blackarrowhead) inmutants(E between somites(so)(yellowarrowhead) andvisceralyolksac(vy)(blackarrowhead). Inmutantembryos,mesenchymalcells(blu ( arrowheads). Whitelinesshowapproximate levelofsectionforF,F somites oneachsideoftheneuraltube(arrowheads). (D arrowheads), whereas Amnion andchorionwere notformedin embryos (A Lateral view. Amnionandchorionwere formedincontrol embryosatthisstage(A,arrowheads). Thesetissues were notformedin BMP signalingformesodermpatterning (Winnier etal.,1995).In 2H expressed inallantoisandtheprimitive streakbut notinLPM(Fig. Fig. 1.Multipledefectsofmesodermdevelopmentin somites (N,P, arrowheads). Scalebars:170 formation ofthe allantoicbud), brachyury was expressed inthe stages ofgastrulation.Attheno allantoicbud stage(justpriorto was consistentlyshorterthan thatofcontrolembryosduringlate described above. However, theprimitive streakofmutantembryos In Delay inrecruitment ofprospective PXM patterning ofLPMinnormaldevelopment. shown). Thesedataindicatethat was eithernotexpressed orlower than normal(Fig.2I 2I) (Tsangetal.,2000).In recombination of M-P F-H Ј Bmpr-MORE ). Ј ) Transverse sectionofcontrol and ) Histologicalanalyses(HematoxylinandEosinstaining).(F,F Lim1 R26R Ј , arrowhead). Anacutecurvature wasobserved(A expression was normallyobserved inLPM atE7.75(Fig. loci afterstainingfor R26R embryos, gastrulationinitiated normallyas Bmpr-MORE was observedinthegermlayers,namelysomites(O,P). Bmpr-MORE Bmpr-MORE Ј , yellowarrowheads). (G,G embryos didnot(C ␤ -galactosidase activity. KandLare magnifiedimagesofIandJ,respectively. Recombined(mutant)cells(blue) Bmpr1a Ј Sox2Cre , blueasterisks).Somitesdevelopedasmultiplerows (G Bmpr-MORE ␮ embryos, m forA;200 is requiredforproper ; embryos, Bmpr1a Lim1 embryos (B Ј , arrowheads). (D-E Ј flox/null ) Ventral view. Mutantembryosdevelopedmultiplecolumnsofsomitesoneachside(D ␮ Bmpr-MORE m forA Bmp4 Ј expression Ј ) Transverse section.Betweenthesomites(yellowarrowhead) andthevisceralyolksac ; datanot Ј , arrow). (B-C embryos carrying Ј ) Frontal section.Incontrol embryos,lateralplatemesoderm(LPM)developed Ј Ј , orangearrowheads). ( Ј , arrowheads). Control embryosdevelopedatypicalheartcrescent (C, . (E,E Ј was ; 250 Ј ) Lateralview. Whitelinesshowapproximate levelofsectionforGor embryos. ␮ Ј m forB-C ) E8.5.(D)Dorsalview. Control embryosdevelopedonecolumnof was inthedistalregion oftheembryo(Fig.3A MORE reached theanteriorendofembryos(Fig.3A).However, in (Fig. 3A)(Wilkinson etal.,1990).Inthenotochord, theexpression entire primitive streak,thenodeandnotochord incontrolembryos primitive streak. In Hogan, 1994).Thenodeisformed atthemostdistalregion ofthe andanteriordefinitive (Fig.3B)(Sasakiand embryo attheallantoicbud stage, distoanterior extension oftheprimitive streakwas defective in al., 1998;Tsanget2000). Theseresultsindicatethatthe Lim1 (Fig.3C observations were madeusingprobesfornoggin more proximallythanincontrol embryos(Fig.3B Ј ) Thehead-foldstage.(B,B R26R Sox2Cre Bmpr-MORE (see Fig.S1A Bmpr-MORE ( embryos, themostanteriordomainofbrachyuryexpression A-E Ј ,I,I loci afterstainingfor Ј Ј ,M,N; 500 I-L ) Whole-mountviews.(A,A ; Bmpr1a ) Transverse sectionofcontrol or Ј embryos, mesenchymalcellmassesexistedbetween embryos (G , yellowarrowheads andinset).Heart(he) Ј Bmpr in thesupplementarymaterial) (McMahonet flox/null ␮ m forD-E Ј - Ј ) Lateralviews.(C,C , seeinset).Multiplesomitesdevelopedto MORE embryos developedmultiplecolumnsof Ј , red arrowhead). Theanteriorhalfof ␤ -galactosidase activity. Complete Ј ,F-H Foxa2 embryos, thenodewas formed RESEARCH ARTICLE Ј Ј Ј ,K,L,O,P. ) Sagittalsection.Amnion(am) ) Thenoallantoicbudstage. was expressed inthenode, Ј Bmpr-MORE ) Frontal views. e asterisks) r neuraltube(nt).

Bmpr-MORE Ј ). Inacontrol Ј ). Similar embryos Bmpr Ј ) and 3769 Ј . Ј , -

DEVELOPMENT was notexpressed (I cells; thisallowed ustolabelmesodermal cellswithDiI,thento performed acelllineageexperiment usingDiI,atracerofliving extension oftheprimitive streakin primitive streak was delayed.This,inturn,causedadelaythe suggest thattherecruitmentofprospective LPMand/orPXMtothe in material). Thus,areducedamountofLPMand/orPXMwas induced Bmpr-MORE decreased andobserved inonlytheproximalembryonicregion in material) (Chapmanetal.,1996),but itsexpression was also PXM atthelatestreakstage(seeFig.S1Cinsupplementary ␮ arrowheads showtheposteriorprimitivestreak andallantois,respectively (H proximal primitivestreak (blackarrowhead) and allantois(red arrowhead) (H). Foxf1 embryonic region (Fig.3E of LPMmarkers.(G,G domain for 2002; Menoetal.,1999).In expressed inLPMandPXMatthisstage(Fig.3E)(Iratnietal., in sclerotome incontrol embryos(F, arrowheads). expression of expanded in embryos (blackarrow). Thebracketshowsonesomite. expressed intheentire region ofeachsomite (red arrow) and Fig. 2.CorrectofectopicsomitesandimproperLPMin patterning 3770 altered in and/or PXM,whichisnormallyinducedbythelatestreakstage, was latestreakstage,weexamined iftheproductionofLPM the around during gastrulation streak extension was temporallydelayedin 3D primitive streakof Bmpr-MORE Dorsal orventralviewisshownforcontrol or of mutantembryos(A column oneachsideofcontrol embryos(A,arrowheads). Three ormore irregular columnsofsomitesoccurred andexpressed m forI,I To furtherprobethetemporalnatureofmesodermformation, we Because thedelayofprimitive streakextension becameapparent Bmpr-MORE Ј ; seeFig.S1B was patchyandweakin RESEARCH ARTICLE Ј ,E,E Bmpr-MORE Lefty2 Bmpr-MORE Mox1 Ј . embryos. However, bythehead-foldstage, embryos (seeFig.S1C embryos bythelate-streakstage. Theseresults Ј was decreasedandobserved onlyintheproximal Bmpr-MORE Ј was observedinsomites(D . in thesupplementarymaterial).Thus,primitive ) orwasdecreased inmutantembryos.Scalebars:300 Ј Ј ) Ventral viewatE8.5.Uppersideisanterior. , arrowheads). Doubleinsituanalysesfor embryos (C embryos. Incontrolembryos, Ј ). Bmpr Bmpr-MORE Tbx6 appeared tobefullyelongated(Fig. - Ј MORE was alsoexpressed inLPMand Bmpr-MORE , arrowheads). embryos (G Ј Bmpr-MORE embryos, theexpression in thesupplementary Ј Bmpr-MORE ,E Pax1 Ј , arrows) andinLPM(D embryos. Mox1 is expressed inexpandedsomitesof Epha4 Ј , arrows). (H-I embryos, respectively. was expressed insomites(D,E,arrows) butnotinLPMcontrol embryos.Inmutants, Lefty2 Uncx4.1 is arostral presomitic mesodermmarker(C,arrowheads). Theexpression domainwas embryos Uncx4.1 Foxf1 was Ј ) Posteriorview. Incontrol embryos, was expressed inthecaudalpartofeachsomitebothcontrol andmutant was expressed inLPMofcontrol embryos(G,arrow). Theexpression of (black) and Ј ,E Ј distal region oftheprimitive streakofcontrolor sac (3/5)(Fig.4B mesoderm cellsofcontrolor injected intotheproximalregion oftheprimitive streaktolabel analyze theirfate afterculturingthemforadefinedperiod.DiIwas probably axialmesoderm(3/3)(Fig.4C labeled cellscontributed tosomitesandanaxialtissue, whichwas embryos atthelatestreakstage(Fig.4C,D).Incontrolembryos, embryos, wefound two domainsofexpression for of prospective LPMandPXMoccurscontinuously. In expression werebasically contiguous,suggestingthatrecruitment migrating LPMandPXMcells (Fig.3F).Thetwo domainsof streak stagein recruitment ofprospective PXMhadnotoccurredyetatthelate embryo (4/4)(Fig.4D region but rathertotheanteriorregion andanaxialtissueofthe mutant embryos,labeledcellsdidnotcontribute to theparaxial somites (Fig.4B embryo anteriorandlateraltotheparaxialregion, but nottothe embryos (5/5),labeledcellsweredistributed inregions ofthe approximately 30hoursuntiltheorganogenesis stage(Fig.4A stage(Fig.4A,B).The embryoswerethenculturedfor streak plate (3/3)andyolksac(Fig.4A As expected, incontrolembryos,labeledcellscontributed tolateral ␮ ). Theexpression of Ј , red arrowheads, seeinset).(E,E Bmp4 m forA-A At theallantoicbud stage, Uncx4.1 was notexpressed inmutantLPM(H Mox1 Ј Bmpr-MORE ,C,C acaudal markerofeachsomite,wasexpressed ina , Bmpr-MORE (yellow) (B,B Bmpr Ј Ј ,D,D ,B Ј ,B Lim1 Љ ). Labeledcellswerealsodistributed intheyolk - Ј Љ MORE ,F,F ). DiIwas alsoinjectedintotheanteriorand Ј (I,I embryos. ; 600 Ј Ј embryos (F ,D Ј ). , arrowhead) inmigratingLPM.The Mox1 Bmp4 embryos. Љ ). Theseresultsindicatethatthe ␮ Lefty2 Ј Bmpr m forB,B ) SectionsofD,D’. , amarkerofsomites,was ( was expressed inLPM(arrow), A-F Ј , arrowheads). ( - Ј MORE Ј ,A was normallyexpressed in ) E8.5.Uppersideisanterior. Ј Љ ; 250 Ј ). Similarly, inallmutant ro) Blackandred , arrow). Ј Development 133(19) ,C Uncx4.1 Љ embryos atthelate ␮ ). However, inthe m forG-H Pax1 Lefty2 G-I Bmpr Bmpr-MORE Ј is expressed on eachside ) Expression that were Ј ; 125 - MORE Lim1 Ј ,B Ј ).

DEVELOPMENT regions oftheprimitivestreak (G the migratingLPMexpressed arrowhead, respectively). regions oftheprimitivestreak (F expressing black arrowhead). More recently recruited mesoderm Bmpr-MORE PXM (F, blackandred arrowheads, respectively). In bud stage. between arrowheads). ( In (E) expressed inLPMandPXM(betweenarrowheads). stage. (D) (distance betweenarrowheads). ( of theprimitivestreak wasshorterinmutantembryos expressed inthenode(lowerarrowheads). Thelength arrowheads). ( control and of theprimitivestreak appeared tobesimilarbetween (betweenarrowheads). (D mutant embryo(B arrowheads). Definitiveendodermdevelopsinthe compared withthatofcontrol embryos(B primitive streak of the lengthbetweentwoarrowheads. Thelengthofthe streak, thelengthofprimitivestreak corresponds to node isformedatthedistalregion oftheprimitive indicate embryonic/extra-embryonicborder. Asthe notochord ofcontrol embryos.Upperarrowheads expressed inthenode(B,lowerarrowhead) and ( in expression of was attheanteriorofembryo(A,arrowhead). The of control embryos.Therostral endofthenotochord in theprimitivestreak (A,arrow), nodeandnotochord 4E-E primitive streak contributed toLPMincontrolembryos(3/3)(Fig. (3/3) (datanotshown). Mesodermcellsinthemiddleregion ofthe contributed tosomitesinbothcontrol(2/2)andmutant embryos head-fold stage.Asexpected, cellsintheanteriorprimitive streak used DiItotracethemesodermofcontrolandmutantembryosat the the middleregion oftheprimitive streakdevelops intoPXM,we embryos relative tocontrollittermatesofthe samestage. production ofpresumptive PXMwas delayedin MORE Lefty2 bars: 100 In BMP signalingformesodermpatterning mesoderm marker atthisstage(Fig.3G for theexpression ofparaxialprocadherin(PCDH8),atrunk of theprimitive streak(Fig.3F mesoderm morerecentlyrecruitedattheanteriorandmiddleregion represented migratingLPM(Fig.3F separated (Fig.3F ( during gastrulationin Fig. 3.Recruitmentofprospective PXMisdelayed contributed tothemorelateralcolumns ofsomites(Fig.4F (1/5).Importantly, thesemesodermcellslargely (Fig. 4F-F region oftheprimitive streakmainlycontributed tosomites (5/5) B-C A , Bmpr-MORE Bmpr-MORE To determineifthemesoderm thatwas formedrelatively latein A Ј Ј ) Theearlytolateallantoicbudstage. Noallantoicbudstage.Brachyurywasexpressed ) Љ Bmpr-MORE ). In was actively transcribedeven atthehead-foldstagein embryos (Fig.3H ␮ Tbx6 Lefty2 Lefty2 Bmpr- m forA,A Bmpr-MORE Љ , themigratingLPMexpressed ) andonlyoccasionallyto LPM (1/5)andhead Brachyury E embryos (A embryos, , was expressed inthemesodermexcept E’ was observedatthemiddleanddistal , was expressed inmigratingLPMand MORE embryos,themesoderm cellsinthemiddle Ј ) Thelatestreak stage. Bmpr-MORE , arrow). (C,C Lefty2 Ј Ј ). Themoreanteriordomainmostprobably ,E,E F-G’ did notextendtotheanterior Bmpr-MORE embryos (distancebetween Lefty2 Pcdh8 expression wasdecreased (E Ј ; 250 Ј , arrow andarrowhead). ) Theearlytolateallantoic Ј Pcdh8 ). Theseresultsfurthersuggestthat embryos wasshorter was stillstrongly expressed atdistalandmiddleembryonicregion ( Ј (PAPC onfigure) wasalsoexpressed inLPMandPXM(G,blackred arrowheads, respectively). In Ј Ј ␮ Ј , red arrow and D ) Nogginwas ). Thesameobservations weremade , red arrow andarrowhead, respectively). m forB-D , D’ (G ) Thehead-fold Ј embryos. , blackarrowhead). More recently recruited mesodermexpressing Ј ). Theotherdomainrevealed Lefty2 Ј Ј Ј , ) Thelength Lefty2 Foxa2 ) (Yamamoto etal.,2000). Ј ; 200 was was (F ␮ Ј m forF-H , Ј , Bmpr-MORE Ј . Bmpr- Ј ,F Љ ). Lefty2 (Mohammadi et al.,1997).We reasoned thatiftheessentialfunction embryos inthepresenceof the FGFR1antagonistSU5402 character. Thishypothesiswas testedbyculturingE6.5-6.75 signaling, whichotherwisemight causetheLPMtoacquirePXM us tohypothesizethatBMPsignaling protectsLPMfromFGF LPM acquiredPXMcharacter(Fig. 2D Rossant, 2001;Yamaguchi etal.,1994).In involved inthedevelopment ofthePXMlineage(Cirunaand (FGFR1) donotformsomites,indicatingthatFGFsignaling is Embryos deficientforfibroblastgrowth factor receptortype1 Bmpr-MORE rescues theabnormal expansion ofsomitesin recruitment ofprospective PXMandpartially Inhibition ofFGFsignalingrestores proper somites in primitive streak,whichprobablyleadstothe lateralextension of distribution ofmesodermcellswithPXMcharacter withinthe recruitment ofprospective PXMresults inamoreproximal to PXM.Collectively, ourresultssuggestthatthedelayin in themiddleregion oftheprimitive streakweremostlycommitted mesodermalcellsformedrelatively late These resultsindicatethat was notexpressed atthehead-foldstageincontrol embryos( Bmpr- embryos MORE embryos. H’ , arrowhead andarrow, respectively). Scale Pcdh8 was observedatmiddleanddistal RESEARCH ARTICLE Ј ,E Ј ). Theseobservation led Bmpr- MORE embryos, Bmpr-MORE 3771 H , ).

DEVELOPMENT 5A; datanotshown). Whencultured with40 poorly developed amnioticfoldsandashorterprimitive streak(Fig. patterning ofLPMin signaling, SU5402shouldbeabletomitigatetheabnormal Mutant embryosculturedwith DMSO or10 to theallantoicbud to head-foldstage(Fig.5A;datanotshown). 20-24 hours,mostcontrolembryos reachedastagecorresponding SU5402. WhenculturedwithDMSO or10 sulfoxide (DMSO)(vehicle forSU5402)orvarious concentrationof DMEM with50%ratserum(DR50)suppliedeitherdimethyl 3772 of Mutant andcontrolembryosatE6.5-6.75werecultured in Bmpr1a RESEARCH ARTICLE in termsofLPMdevelopment istoantagonize FGF Bmpr-MORE mutants. ␮ M SU5402foraround ␮ M SU5402showed ␮ M SU5402,all somites (arrow). Scalebars:100 (arrowhead) andfewermesodermcellscontributedtomedialrow of middle primitivestreak mainlycontributedtolateralrow ofsomites (asterisks). (F contributed tolateralplatemesoderm(arrowheads), butnottosomites embryos, mesodermcellsderivedfrom middleprimitivestreak 24 hours.(E’,E the primitivestreak. EmbryosinjectedwithDiIwere cultured foraround mutant embryosofthehead-foldstage.Arrows indicatethelengthof the middleregion oftheprimitivestreak (E,F, arrowhead) ofcontrol and (arrowhead) andanaxialstructure (arrow). (E-F distal primitivestreak contributedtoaregion anteriortosomites Bmpr-MORE respectively), butnottolateralplate(bluearrows) (seeinset).(D (asterisks) andtoanaxialstructure (red arrows andarrowheads, to-distal primitivestreak ofcontrol embryoscontributedtosomites and D with DiIwere cultured foraround 30hours(C (C) ormutant(D)embryosatthelatestreak stage.Embryosinjected anterior-to-distal region oftheprimitivestreak (arrowheads) ofcontrol the paraxialregion (B region (B primitive streak contributedtoregions anteriorandlateraltoparaxial In arrowhead andyolksac,A primitive streak ofcontrol embryoscontributedtolateralplate(A (A border. EmbryosinjectedwithDiIwere cultured foraround 30hours the latestreak stage.Arrows indicatetheembryonic/extra-embryonic primitive streak (arrowheads) ofcontrol (A)ormutantembryos(B)at light ( culture, embryoswere observedundervisiblelight( cells. Fig. 4.Celllineageanalysesofcontrol andmutantmesoderm at 10-20 FGFR1 activity. Asa50%inhibitionofFGFR1functionisachieved supplementary material),asexpected, withastrongreduction in embryos showed abnormaldevelopment (seeFig.S2inthe ␮ However, whencultured with 20 expression was shifted proximallyanddecreased(3/3)(Fig.6B). Lefty2 Lefty2 MORE signaling fromFGFR1was inhibitedinbothcontroland expression inthechorion was significantlyreduced,indicatingthat still observed stronglyintheectoplacentalcone.However, the (Fig. 5C,D).Whenculturedwith20 was observed inthechorionoramnioticfoldandectoplacentalcone et al.,2003).InembryosculturedwithDMSO,strongexpression embryonic ectodermderived tissuesandectoplacental cone(Corson al., 2003).Theexpression was observed most stronglyinextra- to evaluate thelevel ofsignalingtransducedbyFGFR1(Corsonet embryos weretestedfortheirexpression ofphosphorylatedErk1/2 normally in suggesting thattherecruitmentofprospective PXMhadoccurred streak appearedtoextend normallyinsuchembryos(Fig.5B), chorion andallantois(12/12)(Fig.5B,F).Moreover, theprimitive concentration. Bycontrast,mutantembryosdeveloped anamnion, 20 m forA-B Ј Bmpr-MORE ,A ␮ Ј M ofSU5402.Controlembryosdeveloped normallyatthis A DiI wasappliedtotheprimitivestreak mesoderm( Љ ,B,B , ventralview).(C Љ in mutantembryosculturedwith DMSOwas abnormal;the theexpression of in culturedembryos.Asobserved invivo, -F embryos (Fig.5E,F).We thenexamined theexpression of Ј ␮ ,B Ј Љ , ventralview).Mesodermcellsderivedfrom proximal ). (A-B M (Mohammadietal.,1997),wealsotestedtheeffect of Љ Љ ,D-F , arrowheads andarrow, respectively) butapparently notto Ј embryos, mesodermcellsderivedfrom theanterior-to- ,F Љ Bmpr-MORE Љ ) Dorsalviews.(F embryos, mesodermcellsderivedfrom proximal ) In Љ ; 600 Љ ) DiIwasinjectedintotheproximal region ofthe Bmpr-MORE Ј ,B ␮ Љ Ј m forC , asterisks).(C-D ,C Љ Љ , arrow), butnottosomites(A ) Mesodermcellsderivedfrom theanterior- embryos inculture.Next, cultured Ј Ј ␮ embryos, mesodermcellsderivedfrom ,F ,C m forA-D;250 Љ ␮ Љ ) Ventral view. (E . M SU5402, ␮ Љ ) DiIwasinjectedintothe M SU5402,expression was Ј Љ Development 133(19) and C ) DiIwasinjectedinto Lefty2 ␮ A m forandE,F;300 Ј ,E Ј Љ - , dorsalview;D Љ F ) Incontrol Ј A ) orultraviolet was expressed Ј - ,A F ). After Љ , asterisk). Ј ,D Bmpr- Ј , Љ ) In Ј

DEVELOPMENT embryos. the developmentofsomitesin Fig. 6.InhibitionofFGFsignalingpartiallyrescues for A,B;100 SU5402 byextensive washing, andcontinuedtoculturetheembryos with 20 cultured withDMSOforaround 48hoursintotal.Inthiscondition,somitesdeveloped normally(arrowheads). (H)Mutantembryos edge oftheembryos(arrows) ( showed asignificantexpansionof somitestothelateral (arrowheads). (F)Mutantembryos cultured withDMSO embryos cultured withDMSO normallyexpressed examined fortheexpression of E6.5-6.75 were cultured foraround 48hoursand arrowheads). ( expression of arrowheads). Exposure toSU5402normalizedthe SU5402 showednormalexpression of arrowheads). Control embryoscultured with20 shift anddecreased expression of Mutant embryoscultured withDMSOshowedaproximal with DMSOnormallyexpressed examined for E6.5-6.75 grown inculture for20-24hourswere Expression intheectoplacentalconewasnotaffected (arrow) butthatofthechorionwassignificantlydecreased (arrowheads). ectoplacental conewasnotaffected (arrow), butthatofthechorionwassignificantlydecreased. (F)Mutantembryoscultured wi ectoderm intheectoplacentalcavitywasweaklystained(blackarrowheads). (E)Control embryoscultured with20 cultured withDMSO.Ectoplacentalcone(arrow) andtheamnioticfold(orangearrowhead) showedstrong expression. Theextra-emb Control embryoscultured withDMSO.Ectoplacentalcone(EPC)(arrow) andchorion(arrowheads) were strongly stained.(D)Mutant heterozygous for developed amnion,chorionandallantois(red arrowheads), andawell-extendedprimitivestreak (yellowarrow). Embryospositive BMP signalingformesodermpatterning cultured embryoswith20 embryos (seeFig.S2inthesupplementarymaterial).Therefore,we 48 hours.However, somitedevelopment was inhibitedinsuchmany somite development, weculturedembryoswith20 Bmpr-MORE activity rescuedthedelayedrecruitmentofprospective PXMin indicate thatinhibitionofFGFsignalingviareductionFGFR1 at anormallevel inmutantembryos(2/2)(Fig.6D).Theseresults tested, partialrescue ofsomitedevelopment wasobserved(arrowheads). Scalebars:85 Bmpr1a- (yellow arrows) inculture, aswasobservedinvivo.Insetshowsgenotypingresults fortheseembryos.Embryospositive streak toallantoicbudstage. Fig. 5.InhibitionofFGFsignalingbyaspecificantagonistforFGFR1in To furtherexplore theimpactofFGFsignalinginhibitionon ␮ null alleleare M SU5402for12-15hours,extensively washedandthenwithDMSOforaround 48hoursin total. Insixmutantembryosoutof10 ( A-D ␮ Lefty2 Lefty2 E-H m forC-F. embryos. ) Control and Bmpr1a-null ) Control and in expression. Control embryoscultured Bmpr-MORE Bmpr-MORE ␮ M SU5402for12-15hours,removed the Bmpr-MORE Bmpr-MORE allele are n Uncx4.1 Lefty2 Bmpr-MORE =9). (G)Control embryoswere cultured with20 Lefty2 Bmpr-MORE embryos (inset,pinkstars).( embryos (2/2)(D, Lefty2 (A, arrowheads). . (E)Control Bmpr-MORE 33 (B, (3/3) embryos showedapoorlydevelopedamnioticfold(red arrowheads) andlessextendedprimitivestreak embryos at embryos at (C, ␮ M Uncx4.1 embryos (inset,pinkstar).( ␮ M SU5402for B ) Control embryosdevelopedtotheallantoicbudstage. development was observed ononesideofeachembryo(seeFig.S3 (Fig. 6H).Inthreeothermutantembryos,therescueinsomite embryos developed onlyoneortwo rows ofsomitesonbothsides 6H, seeFig.S3inthesupplementarymaterial).Threemutant somite development insixoutoftenmutantembryostested(Fig. treatment withSU5402resultedinavariable level ofrescuefor DMSO (9/9)(Fig.6F, seeFig.S3inthesupplementarymaterial), significantly expanded laterallyinallmutantembryoscultured with developed normally(12/12)(Fig.6G).Althoughsomiteswere with DMSOfor48hoursintotal.Undertheseconditions,somites Bmpr-MORE C-F ␮ M SU5402for12-15hours,extensively washedandthen ) Cultured embryosstainedwithphospho-Erk1/2antibody. (C) ␮ m forA,B;100 embryos. ( A ) Control embryosdevelopedtothelate ␮ m forC,D;600 ␮ RESEARCH ARTICLE M SU5402.Expression inthe Bmpr-MORE Cre ␮ m forE-H. and heterozygous for Scale bars:500 th 20 for were cultured embryos Cre ryonic embryos ␮ M SU5402. and 3773 ␮ m

DEVELOPMENT form ectopicsomites(yellowsquare). LPMisreduced owingtolossofcontributingcells(green hexagon)resulting from reduced arrow andhexagon).(C)Alternatively, someoftheprospective PXMcellsare abnormallyrecruited tothemiddleprimitive strea arrow), whichshouldformLPM,changetheirfatetoectopicsomites(yellowarrow andyellowsquare); LPMisconsequently form somitesasinwild-typeembryos(red arrows andred square). However, manyepiblastcellspassingthrough themiddleprimi of themesodermalregion oftheembryo,suchassomites. streak. This,inturn,hasimportantconsequencesfordevelopment essential forproperrecruitmentofepiblastcellsintotheprimitive In thispaper, wehave shown thatBMPRIAintheepiblastis DISCUSSION not rescuetheabnormaldevelopment oftheLPM(datanotshown). these mutantembryos,indicatingthatinhibitionofFGFsignalingdid embryos culturedasdescribedabove. embryos. Therefore,wealsoexamined theexpression of was duetonormaldevelopment oftheLPMin development ofsomitesin of FGFsignalingduringgastrulationpartiallyrescuedabnormal in thesupplementarymaterial).Thesedataindicatethatinhibition respectively, atthelatestreak stage.( primitive streak (red arrow) ortothemiddleprimitive streak (green arrow) are fatedtoformsomites(red square) orLPM(gre Fig. 7.Modelsfortherole forBMPRIAinmesodermdevelopment. 3774 altered morphogenesisofsomites in part oftheprimitive streakmigratemoremedially, thuscausingan primitive streak migratemorelaterallyandPXMcellsatthedistal (Smith etal.,1994).Presumably, PXMcellsinthemiddlepart ofthe ofcellsfromtheprimitive streak intothemesoderm mesoderm cellswithintheprimitive streak ismaintainedduring the primitive streak.Thedistoproximalorder oftheprospective PXM characterariseinthemiddleadditiontoanteriorpart of anterior primitive streak.Ourresultsindicate mesodermcellswith Normally, theprospective PXMpopulationarisesmainlyatthe the allantoicbud toheadfoldstagein during themidtolatestreakstagewas delayedandtookplace during indicate thatrecruitmentofprospective PXM thatnormallyoccurs which was duetoadelayofprimitive streakextension. Ourresults characteristic morphology:anacutecurvature intheposteriorside, first modelproposes thatlossofBMPsignaling causes afate change how ectopicsomites develop in Bmpr It was possiblethattheobserved rescueofsomitedevelopment We proposetwo possiblemodelstoexplain themechanismof RESEARCH ARTICLE - MORE embryos attheallantoicbud stageshowed a Bmpr-MORE Bmpr-MORE B , C Bmpr-MORE ) Mutantembryos.(B)Inmutantembryos,epiblastcellsrecruited totheanteriorprimitivestreak probably Foxf1 embryos. Bmpr-MORE was notexpressed in mutant embryos.The embryos. Bmpr-MORE embryos. Foxf1 in ( A ) Inwild-typeembryos,epiblastcellsthatare recruited totheanterior involved in antagonisticeffects. downstream targets ofBMPandFGFsignaling areprobably expressed normallyinmutant embryos(datanotshown). Rather, level. Inaddition, major signaltransducersofFGF signalingappearsatthenormal In sufficiently inhibitFGFsignalingtoelicitthismigratorydefect. Probably, theconcentrationofSU5402usedheredoesnot cells outoftheprimitive streakduringgastrulation (Sunetal.,1999). deficient miceexhibit amarked decreaseinmigrationofmesoderm apparently normallyuptotheallantoicbud stage.However, and thusforsuccessfulestablishmentofmousebodyplan. be presentthatareresponsibleforcontrolledallocationofmesoderm (this study).Theseobservations suggestthatamechanism(s)might of prospective LPMcells,but they areessentialforLPMpatterning Interestingly, wefoundthatBMPsdonotregulate therecruitment prospective PXMcellsbut is requiredfortheirpatterning. indicate thatFGFsignalingnegatively regulates recruitmentof normal timingofprospective PXMrecruitment.Together, thesedata inhibition ofFGFsignalingin brachyury and streak toformectopicsomites(Fig.7C,yellow arrow). embryos, thesecellsareabnormallyrecruitedtothemiddleprimitive anterior primitive streak(Fig.7A,redarrow). Inthemutant are recruitedtotheanteriorprimitive streakandexit fromthe recruitment totheprimitive streak(Fig.7C).Normally, thesecells abnormal morphogeneticmovement ofprospective PXMcellsupon posits thatthedelayofrecruitmentin form ectopicsomites(Fig.7B,yellow arrow). Thesecondmodel would normallyformtheLPM,andthatthesecellsconsequently of epiblastcellspassingthroughthemiddleprimitive streak,which It isunlikely thatBMPsignaling directlyinhibitsFGFsignaling. Control embryostreatedwith20 FGF signalinginducesPXMpatterningviaexpression of Bmpr-MORE embryos, thephosphorylationof ERK1andERK2, Tbx6 Snai1 (Ciruna andRossant,2001).However, oneoftarget genesofFGFsignaling,is , Bmpr-MORE Bmpr-MORE ␮ M SU5402developed Development 133(19) en hexagon), k (yellowarrow) and BMPsignaling. embryos restored tive streak (green reduced (green embryos causes Fgf8 -

DEVELOPMENT understanding ofmousegastrulation. the assessmentoftheirfunctionsarecrucialforamorethorough effects onthisprocess.Theidentificationofsuch mechanismsand although weshowed thatBMPandFGFsignaling have antagonistic recruitment ofepiblastcells(prospective PXM)isunknown, issues remaintobeaddressed.Themechanismthatexplains delayed novel functionsofBMPsignalingingastrulation.However, many Durbin, L.,Brennan, C.,Shiomi,K.,Cooke,J.,Barrios,A.,Shanmugalingam, Davis, S.,Miura,Hill,C.,Mishina,Y. andKlingensmith, J. Belo, J.A.,Bouwmeester, T., Leyns,L.,Kertesz, N.,Gallo,M.,Follettie,M. References http://dev.biologists.org/cgi/content/full/133/19/3767/DC1 Supplementary materialforthisarticleisavailableat Supplementary material (R01DE013674 andP01HD39948). Environmental HealthSciencestoY.M. andbyNIHawards toJ.K. supported bytheIntramuralResearch Program oftheNIH,NationalInstitute Shimono forinformationaboutthecelltraceexperiments.Thisresearch was Lacy forcriticalreading ofthismanuscript;andNorikoOsumiAkihiko Manas Ray, MitchEddy, AkihikoShimono,Yu-Ping Yang, BrigidHoganandLiz M. Robertisforinsituprobes. We alsothankXinSun,MichelleTallquist, R. P. Harvey, C.V. Wright, A.Kispert,R.Balling,D.L.Chapman,Y. SagaandE. R26R transgenicmice;andB.G.Herrmann,H.Hamada,P. Carlsson,B.Hogan, We thankPhillipeSorianoandMichelleTallquist forproviding us BMP signalingformesodermpatterning Hayashi, S.,Lewis,P., Pevny, L.andMcMahon,A.P. Chapman, D.L.,Agulnik,I.,Hancock,S.,Silver, L.M.andPapaioannou,V. E. Candia, A.F., Hu,J.,Crosby, J.,Lalley, P. A.,Noden,D.,Nadeau,J.H.and Durbin, L.,Sordino, P., Barrios,A.,Gering,M.,Thisse,C.,B.,Brennan, Corson, L.B.,Yamanaka, Y., Lai,K.M.and Rossant,J. Bettenhausen, B.,HrabedeAngelis,M.,Simon,D.,Guenet,J.L.and Hornbruch, A.,Summerbell,D.andWolpert,Hornbruch, L. Ciruna, B.andRossant,J. Kishigami, S.andMishina,Y. Kinder, S.J.,Tsang, T. E.,Quinlan,G.A.,Hadjantonakis,A.K.,Nagy, A.and Iratni, R.,Yan, Y. T., Chen,C.,Ding,J.,Zhang,Y., Price,S.M.,Reinberg, D. Lawson, K.A.,Meneses,J.andPedersen, R.A. morphogenesis and ectodermal patterning. morphogenesis andectodermalpatterning. receptor IAisrequired inthemammalianembryoforendodermal for segmentationanddifferentiation ofthesomites. S., Guthrie,B.,Lindberg,R.andHolder, N. 130 ofERKsignalingduringmouseembryogenesis. temporal patterns gastrula. neutralizing activityexpressed intheanteriorprimitiveendodermofmouse and DeRobertis,E.M. Dev. modulation inmouseepiblastusingaSox2Cre transgenicmousestrain. gastrulation. (1996). Tbx6,amouseT-Box geneimplicatedinparaxialmesodermformationat mouse embryos. subfamily andare differentially expressedin duringearlymesodermalpatterning Wright, C.V. zebrafish. required withinsegmentsforsomiteboundaryformationindeveloping C., Green, A.,Wilson,S.and Holder, N. 37-49. Development embryogenesis ofDll1,amurinegenecloselyrelated toDrosophila Delta. Gossler, A. Morphol. the earlychickembryofollowinggraftsofHensen’s node. specification andmorphogeneticmovementattheprimitivestreak. mouse embryo. extraembryonic structures and theanteroposterior axisduringgastrulationofthe Tam, P. P. patterning. patterning. gastrulation bythetranscriptionalcorepressor DRAP1. and Shen,M. Through theanalysesof , 4527-4537. 119 , S97-S101. Mech. Dev. 51 (1999). Theorderly allocation ofmesodermalcellstothe Development GrowthFactorRev. (1995). Transient andrestricted expression during mouse , 51-62. Dev. Biol. 121 (1992). Mox-1andMox-2defineanovelhomeoboxgene Development Development , 2407-2418. (2002). Inhibitionofexcessnodalsignalingduringmouse 68 180 , 45-57. 127 (1997). -likeisasecreted factorwith (2001). FGFsignalingregulates mesodermcellfate , 534-542. , 1703-1713. (2005). BMPsignalingandearlyembryonic 126 116 Bmpr-MORE , 4691-4701. , 1123-1136. 16 (2000). Anteroposterioris patterning , 265-278. Dev. Biol. (1998). Ephsignalingisrequired (1979). Somiteformationin embryos, weidentified (1991). Clonalanalysisof Dev. 270 (2002). Efficient Science (2003). Spatialand , 47-63. J. Embryol. Exp. J. Embryol. 298 (2004). BMP 12 Mox2-cre Development , 1996-1999. , 3096-3109. Dev. Cell Mech. and 1 , Tallquist, M.D.andSoriano,P. Miura, S.andMishina,Y. Mishina, Y., Hanks,M.C.,Miura,S.,Tallquist, M.D.andBehringer, R. Soriano, P. Sun, X.,Meyers,E.N.,Lewandoski,M.andMartin,G.R. Saga, Y., Hata,N.,Koseki,H.andTaketo, M. Parameswaran, M.andTam, P. P. Mohammadi, M.,McMahon,G.,Sun,L.,Tang, C.,Hirth, P., Yeh, B.K., Mishina, Y., Suzuki,A.,Ueno,N.andBehringer, R. Sasaki, H.andHogan,B.L. McMahon, J.A.,Takada, S.,Zimmerman,L.B.,Fan,C.M.,Harland,R.M. Mishina, Y. Smith, J.L.,Gesteland,K.M.andSchoenwolf,G.C. Meno, C.,Gritsman,K.,Ohishi,S.,Ohfuji,Y., Heckscher, E.,Mochida,K., Mahlapuu, M.,Ormestad,Enerback,S.andCarlsson,P. Leitges, M.,Neidhardt, L.,Haenig,B.,Herrmann,B.G.andKispert,A. Tam, P. P. andQuinlan,G.A. Tonegawa, A.andTakahashi, Y. Tam, P. P. andBehringer, R. Wilson, V. andBeddington,R.S. Wilkinson, D.G.,Bhatt,S.andHerrmann,B.G. Wallin, J.,Eibel,H.,Neubuser, A.,Wilting,J.,Koseki,H.andBalling,R. Tsang, T. E.,Shawlot,W., Kinder, S.J.,Kobayashi,A.,Kwan,K.M., Yamamoto, A.,Kemp,C., Bachiller, D.,Geissert,D.andDeRobertis,E.M. Yamaguchi, T. P., Harpal,K.,Henkemeyer, M.andRossant,J. Winnier, G.,Blessing,M.,Labosky, P. A.andHogan,B.L. strain. MORE mice:atooltodistinguishembryonicvs.extra-embryonicgenefunction. embryo. disruption ofFgf8causesfailure ofcellmigrationinthegastrulatingmouse development tothegastrulationstage. during mouseembryogenesis. type Ibonemorphogeneticprotein receptor thatisessentialforgastrulation (2002). GenerationofBmpr/Alk3conditionalknockoutmice. 289. Genet. morphogenetic movementofthemesodermduringmousegastrulation. initiation. gene expressed inthepresegmented mesodermandessential forsegmentation 276 domain offibroblast growth factorreceptor incomplexwithinhibitors. Hubbard, S.R.andSchlessinger, J. mouse development. map ofthemouseprimitivestreak at7.5daysofgestation. development. 12 required forgrowthoftheneuraltubeandsomite. andpatterning and McMahon,A.P. embryonic andlateralplatemesoderm. forkhead transcriptionfactorFoxf1isrequired fordifferentiation ofextra- during vertebrategastrulation. Mouse Lefty2andzebrafishantivinare feedbackinhibitorsofnodalsignaling Shimono, A.,Kondoh,H.,Talbot, W. S.,Robertson,E.J.etal. 2267. processes andproximal ribsofthevertebralcolumn. (2000). Thepaired homeoboxgeneUncx4.1specifiespedicles,transverse 113 epiblast fateduringgermlayerformationinthemouseembryo. Genesis mammalian bodyplan. Dev. Biol. the mouseTgeneanditsrole inmesodermformation. 104-106. movement inthelatemouseprimitivestreak. required fornormalT-cell maturation. (1996). Pax1isexpressed during developmentofthethymusepitheliumandis mouse embryo. (2000). Lim1activityisrequired forintermediatemesodermdifferentiation inthe Schughart, K.,Kania,A.,Jessell,T. M.,Behringer, R.andTam, P. P. gastrulation. required forembryonicgrowthduringmouse andmesodermalpatterning essential formousedevelopment. (2000). Mouseparaxialprotocadherin isexpressed intrunkmesodermandisnot the mouse. morphogenetic -4 is required in formesodermformationandpatterning , 1438-1452. , 955-960. , 891-911. Nat. Genet. 17 26 Genes Dev. (1999). GeneralizedlacZexpression withtheROSA26Cre reporter 202 Genes Dev. , 16-28. (2003). Functionofbonemorphogeneticprotein signalingduring , 113-115. Genes Dev. Genes Dev. , 172-182. Cell Dev. Biol. 76 21 13 , 103-115. , 70-71. Front. Biosci. 11 (1998). Noggin-mediatedantagonismofBMPsignalingis , 1834-1846. Mech. Dev. 9 , 1827-1839. 8 , 2105-2116. (2003). Whole-embryoculture ofE5.5mouseembryos: 223 , 3032-3044. (1994). HNF-3betaasaregulator offloorplate (1996). Mappingvertebrateembryos. (1997). Mousegastrulation:theformationofa , 77-90. Genes Dev. Mol. Cell (2000). Epiblast-restricted Cre expression in (1998). Somitogenesiscontrolled byNoggin. (1996). Cellfateandmorphogenetic (1995). Regionalisationofcellfateand Genesis 8 68 , d855-d869. (1997). Structures ofthetyrosine kinase , 3-25. Development Genesis Development 4 , 287-298. RESEARCH ARTICLE 9 27 , 3027-3037. , 49-57. Mech. Dev. 37 (1990). Expressionof pattern (1997). Mesp2:anovelmouse , 38-43. 122 Development 128 (1994). Prospective fate Nature (1995). Bmprencodesa , 23-30. 55 , 155-166. (1995). Bone (1999). Targeted Dev. Dyn. , 79-89. Genesis (2001). The 343 (1994). fgfr-1 is Development , 657-659. Curr. Biol. 127 (1999). Genes Dev. 32 201 , 2259- Science , 69-72. Dev. , 279- 3775 6 ,

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