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The Vg1-Related Protein Gdf3 Acts in a Nodal Signaling Pathway in the Pre

The Vg1-Related Protein Gdf3 Acts in a Nodal Signaling Pathway in the Pre

08854, USA. and DentistryofNewJersey-RobertWood JohnsonMedicalSchool,Piscataway, NJ atnM Matzuk M. Martin Baylor CollegeofMedicine,Houston,TX 77030,USA. Medicine, Houston,TX77030,USA. members tobeimplicatedinvertebrate development hasremained patterning,thefunctionofonefirstTgf importance forAPaxispatterninginthemammalianembryo. AVE inductionandNodalsignalingactivities isthereforeofcentral axis following movement oftheAVE. Themolecularregulation of the proximalepiblast,andtogetherpatternanteroposterior(AP) the distalvisceralendodermbecomeopposedtoNodalsignalsfrom Yamamoto etal.,2004).Asaconsequence,anti-Nodalsignalsfrom (Brennan etal.,2001;Ding1998;Perea-Gomez2002; antagonists thatareessentialforpatterningoftheanteriorepiblast Nodal signalingpathway, whereastheAVE itselfproducesNodal formation aswellmovement oftheAVE requireactivity ofthe 2004; Thomasetal.,1998),priortotheonsetofgastrulation.Both anterior sideby6.0dpc(Rivera-Perez etal.,2003;Srinivas etal., (dpc),but thentranslocatestotheprospective days postcoitum the mostdistalregion ofthepost-implantationegg cylinder at5.5 by Luetal.,2001;RossantandTam, 2004).TheAVE isformedin thatoverlies andpatternstheadjacentepiblast(reviewed specialized populationofcellswithintheextra-embryonic visceral embryo istheinductionofanteriorvisceralendoderm(AVE), a A key event informationoftheanteroposterioraxismouse INTRODUCTION KEY WORDS:Tgf (growth-differentiation factor3),acloserelativeof patterning oftheanterior-posterior axis.Here,weshowthatthetransforminggrowthfactor The formationoftheanteriorvisceralendoderm(AVE) inthepre-gastrulationmouseembryorepresentsacrucialevent Canhe Chen pathway inthepre-gastrulationmouseembryo The Vg1 Development 133,319-329doi:10.1242/dev.02210 Accepted 11November 2005 † 1 *These authorscontributedequallytothis work Baylor CollegeofMedicine,Houston,TX 77030,USA. Cincinnati, OH45229,USA. Hospital MedicalCenterandUniversityofCincinnatiCollegeMedicine, [email protected]) School, Piscataway, NJ08854,USA. University ofMedicineandDentistryNewJersey-RobertWood JohnsonMedical Medicine, Houston,TX77030,USA. activity inmice. a Nodal-likesignalingpathwayinpre-gastrulationdevelopment,andprovideevidenceforthefunctionalconservationofVg1 anddefinitiveendodermformation,aswellabnormal and nullmutantsfrequentlyexhibitabnormalformationorpositioningoftheAVE. Thisphenotypecorrelateswithdefectsin elongation andmesendodermformationinanimalcaps.Inmouseembryos, by Leftyantagonists.In its roleinAVE formationinvivo.Thus, cellculture,Gdf3signalingrequirestheEGF-CFCco-receptorCriptoandcanbeinh Authors forcorrespondence (e-mail:[email protected]; Center forAdvancedBiotechnologyandMedicineDepartmentofPediatrics, However, despitemany recentadvances inunderstanding early 4 Department ofMolecularandHumanGenetics,BaylorCollege 1, *, StephanieM.Ware ␤ - signaling, EGF-CFCproteins, Anteriorvisceralendoderm,Mesendodermformation related proteinGdf3actsinaNodalsignaling 4,5,6 3 Department ofBiochemistry, UniversityofMedicine , MichaelM.Shen 2 6 5 Department ofPediatrics,CincinnatiChildren’s Department ofMolecularandCellularBiology, Department ofPathology, BaylorCollegeof , Gdf3misexpressionresultsinsecondaryaxisformation,andinducesmorphogenetic 7 2, Department ofPediatrics, *, AkiraSato 1,† and ChesterW. Brown ␤ Xenopus superfamily 3 , DianneE.Houston-Hawkins Vg1, resemblestheTgf through aNodal-like pathway. Yeo andWhitman,2001),indicatesthatVg1 andGdf1signal (Gritsman etal.,1999;Reissmann etal.,2001;Yan etal.,2002; , whichactasputative co-receptorsforNodalligand (Tdgf1) (Chengetal.,2003). ThisrequirementforEGF-CFC interact withOepaswellthe mouseEGF-CFCproteinCripto CFC proteinOep,andthatmature Vg1andGdf1proteinscan of Vg1andGdf1overexpression inzebrafishrequiretheEGF- Whitman, 1999).Notably, recentwork hasshown thattheeffects that mediatesmany key aspectsofNodalsignaling (Watanabe and negative formofFoxh1/FAST, awinged-helixtranscription factor overexpression in 1996; Hoodlessetal.,1999).Inaddition,theeffects ofVg1 be antagonizedbydominant-negative Smad2mutants(Graff etal., that ofactivin andNodal,resultsinactivation ofSmad2,andcan Whitman, 2001).Inparticular, signaling by processedVg1,like right axis(reviewed bySchier, 2003;SchierandShen,2000; endoderm andaxialmesendoderm,specificationofthe left- roles inAPpatterning,formationofmesoderm,definitive through apathway similartothatforNodal,whichplayscrucial Newfeld etal.,1999). with membersoftheBmpsubfamily (e.g.Changetal.,2002; (Burt andLaw, 1994),but morerecentanalyseshave groupedGdf3 was mostcloselyrelatedto 1992). EarlyphylogeneticcomparisonshadsuggestedthatGdf3 which shares79%aminoacidsimilaritywithVg1(Jonesetal., ee nmc a sltdgot-ifrnito atr3( inmicehadisolatedgrowth-differentiation factor 3 identified inmammaliangenomes.Aninitialscreenfor (Seleiro etal.,1996;Shah1997),noclearorthologhasbeen (Dohrmann etal.,1996;HeldeandGrunwald, 1993)andchick subsequent analyseshave identified Thomsen andMelton,1993;Weeks andMelton,1987).Although mesoderm inducer(Kessler andMelton,1995;1987; blastula stageembryo,andwas proposedtoactasanaxial in ascreenforvegetally localizedmaternaltranscriptsinthe poorly understood.The Nodal Several linesofevidence have suggestedthatVg1cansignal xrsinlvl.Orfidnsidct htGf actsin expression levels.OurfindingsindicatethatGdf3 Gdf3 4,7,† is expressedintheinnercellmassandepiblast, ␤ ligand Nodalinbothitssignalingactivityand Xenopus eou Vg1 Xenopus ␤ 4 , RaymondHabas (Tgf Xenopus can beblocked byadominant- ␤ ) familymemberGdf3 RESEARCH ARTICLE was originallyidentified Vg1 Vg1 andmammalianGdf1 orthologs inzebrafish 3 , Vg1 ibited -related Gdf3 319 ) ,

DEVELOPMENT detection ofmature Lefty1protein (~30 kDa)inconditionedmedia.In allpanels,assayswere performed intriplicate;error ba (Inse and Foxh1,inthe presence orabsenceofLefty1; controls correspond tothesecellswithoutNodal orbf-Gdf3transfection. ~19 kDa;FLAG-Oep,~21kDa).( ~20 kDa;FLAG-,~21and antibody (FLAG-Cryptic,~22and proteins incelllysatesusing Western blotdetectionofEGF-CFC without EGF-CFCproteins. (Inset) Nodal orbf-Gdf3andFoxh1 correspond tocellstransfectedwith Cripto orOep;control samples FLAG-epitope-tagged Cryptic, for Nodalorbf-Gdf3,Foxh1and transfected withexpression vectors but notCryptic.293Tcellswere co- Gdf3 activityisfacilitatedbyCripto, antibody or antibody (Sigma). mouse Lefty1(ChenandShen,2004),oramonoclonalanti-FLAG M2 against mouseNodal(Yan etal.,2002),polyclonalantiserumagainst mouse Gdf3polyclonalantiserum(R&DSystems), expression was detected bywesternblottingusingagoatanti- vector was usedtomaintain constantamountsoftransfectedDNA. normalize fortransfectionefficiency, andadditionofempty conditioned mediausing mature Gdf3(~17kDa)proteins in mature Nodal(~12kDa)and Westernblotdetection of (Insets) with the transfected into293Tcellstogether indicated plasmidswere co- is CriptoandFoxh1dependent.The indicated (arrows). ( the sitesofprodomain cleavageis Gdf3 andmodifiedproteins; transfection ofanexpression constructfor and Nodalactivities wereperformed asdescribed(Yan etal.,2002).Co- proprotein convertase cleavage site(Fig.1A).LuciferaseassaysforGdf3 prodomain andaFLAG orV5epitope-taginsertedimmediatelyafterthe (McPherron andLee,1993),containaheterologous constructs weregeneratedbyPCRfromamouseGdf3cDNA template The mouse Cell culture assayforGdf3activity MATERIALS ANDMETHODS early mousedevelopment. activity andislikely torepresentafunctionalhomologforVg1in adulthood. OurresultssuggestthatGdf3hasacrucialNodal-like mispositioning oftheAVE, withtheremaindersurviving to anddefectsconsistentwiththeabsenceor mouse embryos,andthat~35%ofnullmutantsdisplaypre- Gdf3 to thatofNodalin receptors andtheEGF-CFCproteinCripto,hasactivities similar culture assayforNodalsignaling,canformacomplex withactivin analyses ofmouse investigated thepotentialconservation ofVg1functionthrough Vg1 hasessentialfunctions.To addresstheseissues,wehave during mammaliandevelopment, orindeedwhetherendogenous unclear whetherthereisaconservation ofVg1-related activities 320 ( Nodal signalingactivities. Fig. 1.ComparisonofGdf3and A Schematicdepictionofmouse ) Owing tothesimilaractivities ofVg1andNodal,it hasremained is expressed inpre-implantationandearlypost-implantation RESEARCH ARTICLE A3-luc ␣ -Gdf3 antibody. ( pcDNA3-bf-Gdf3 reporter. Xenopus B ) Gdf3activity Gdf3. ␣ -Nodal ␣ We findthatGdf3hasactivity inacell embryos andanimalcaps.We show that -FLAG C ) D ) Lefty1inhibitsGdf3activity. Cellswere co-transfectedwithexpression constructsforNodal orbf-Gdf3,Cripto and pcDNA3-bv-Gdf3 ␤ -galactosidase was usedto Xenopus expression pcDNA3 Bmp2 raised tostage30in0.1 extraction andRT-PCR analysis.For phenotypicassays,embryoswere blastula stages8.5-9andculturedtoearlygastrulastage11fortotalRNA of of thefour-cell embryoforphenotypicassaysorintotheanimalhemisphere (Ambion). CappedmRNA was injectedintothetwo ventral marginal zones pCS2+ vector forinvitrotranscriptionusingamMachineSP6system corresponding tomouseGdf3,bf-Gdf3andNodalwereclonedintothe carried outasdescribed(Habasetal.,2001;Kato2002).Sequences Ovulation, invitrofertilization,andembryoexplant cultureswere Xenopus microinjection andanimalcapassays (Covance) andanti-Mycantibody(SantaCruz). the antibodiesdescribedabove, aswellmonoclonalanti-HAantibody previously (Yeo andWhitman,2001).Western blottingwas performedusing ActRIIB(K217R)-Myc andActRIB(K234R)-HAhave beendescribed RIPA114 buffer (Yan etal.,2002).Thekinase-inactive receptormutants out priortoco-immunoprecipitation,usingwhole-celllysatespreparedwith (Sigma). For allotherexperiments, crosslinkingwithDTSSPwas carried directly subjectedtoimmunoprecipitationwithanti-FLAG M2antibody Lefty1 media, 293Tcellsweretransfectedwith Shen, 2004).To detecttheassociation ofGdf3andLefty1inconditioned immunoprecipitation werecarriedoutasdescribedpreviously (Chenand dithiobis(sulfosuccinimidyl proprionate)](Pierce)andco- Reversible chemicalcrosslinkingwith0.5mMDTSSP[3,3 Protein cross-linking andco-immunoprecipitation analysis germline malechimerasthatsiredmutantmicewithidenticalphenotypes. Four independentcloneswereinjectedintomouseblastocysts, giving riseto targeted, asdeterminedbySouthernblotanalysisusingboth5 two percentofclonesresistanttobothHAT andFIAU ( 1992), usinga129S6SvEv-derived EScelllinederived fromAB2.1.Sixty- Gene targeting was performedasdescribed(Bradley, 1987;Matzuketal., Gene targetingandphenotypicanalysis Xenopus ; culturesupernatantswerecollected48hourspost-transfectionand embryos atthetwo-cell stage.Animal caps wereexplanted at ϫ MMR. pcDNA3-bf-Gdf3 Development 133(2) rs represent s.d. )Westernblot t) n =52) werecorrectly Ј and and 3 pcDNA3- Ј probes. Ј -

DEVELOPMENT with HematoxylinandEosin. was performedonformalin-fixed paraffin sectionsofwholedeciduastained a C57BL/6J-129S6/SvEvmixed geneticbackground.Histologicalanalysis using aC57BL/6Jcongenicstrain,althoughsimilardatawereobtained Kit (BoehringerMannheim).Mostofthemarker analysiswas performed al., 1998;Wilkinson, 1992),usingprobeslabeledbytheDIGRNA Labeling et al.,2002),we foundthatGdf3activity was absolutelydependent if parametersofthisassaywerealtered. AsisthecaseforNodal(Yan assay, weexamined whethertheseligandswould behave identically supplementary material). significant signalingactivity (Fig.1B;seeFig.S1inthe conditioned media,whereas the modifiedbf-Gdf3resultsin consistent withthelow levels ofmatureliganddetectedin and Foxh1, native Gdf3gives alow but detectablelevel ofactivity, luciferase reporter activity canbedetectedusingtheactivin/Nodal-responsive co-transfected withCriptoandFoxh1 into293Tcells,signaling not displayactivity inthisassay(Yan etal.,2002).WhenNodal is al., 2002)(C.C.andM.M.S.,unpublished)];moreover, Bmp4does Nodal, Gdf1,Gdf3,Lefty1orLefty2(ChenandShen,2004;Yan et Cripto andFoxh1, whicharenotexpressed in 293T cells[norare relies uponreconstitutionoftheessentialsignalingcomponents Nodal activity intransfected293Tcells(Yan etal.,2002).This assay products usingaluciferasereporterassaythatwedeveloped for to thatofNodal,weexamined theactivity oftheseGdf3protein suggest thatthesespeciesdiffer intheirlevels ofN-glycosylation. mature Gdf3sequenceandintheBmp2prodomains western blotting;consensusglycosylationsequenceslocatedinthe were several Gdf3proproteinandmaturespeciesdetectedby (see Fig.S1Ainthesupplementarymaterial).We notedthatthere processed efficiently inconditionedmediaoftransfected293Tcells prodomain-FLAG-mature Gdf3)fusionproteinisexpressed and protein detection(Fig.1A).Theresultingbf-Gdf3(Bmp2 or V5epitopetagattheNterminusofmatureligandforease efficiently in293Tcells(ChenandShen,2004),addedaFLAG prodomain withthatof inefficient processingofnative Gdf3,wereplacedtheGdf3 see Fig.S1Ainthesupplementarymaterial).To circumvent the low levels ofmatureliganddetectedinconditionedmedia(Fig.1A; observed thatthisnative construct was processedpoorly, withvery expression constructinto293Thumanembryonickidney cells,we mammalian cellculture.Whenwetransfectedanative Gdf3 protein canbeexpressed andsignalinaNodal-like pathway in To investigate thepropertiesofGdf3,weexamined whetherGdf3 Gdf3 activityinaNodal-likesignalingpathway RESULTS CCA TGCCTA CAT CCAGAG CCTGC3 QPCR MasterMix(Stratagene).Theprimerpairsusedwere:for performed usingaMx4000quantitative PCRinstrumentwithSYBRGreen generate first-strandcDNA (Invitrogen). Real-timePCRamplificationwas isolated usingtheRNeasyMicroKit(Qiagen)andreverse-transcribed to Total RNA fromindividual embryosintheC57BL/6strainbackgroundwas Real-time RT-PCR analysis Gdf3 actsinaNodal-likepathway AGG ACC CTGCC3 and 5 normalization against on eachtotalRNA, followed byreal-timePCRamplificationintriplicateand 3 GAC TTCAAC AGC AAC TC CC 3 hl-on insituhybridizationwas carriedoutasdescribed(Dinget Whole-mount As Gdf3andNodaldisplayedsimilar activities inthiscellculture Given thebodyofevidence thatVg1signalsinapathway similar Ј Ј Threeindependentreverse-transcription reactionswereperformed . GGA GCAGCTGCGTGCATG AG 3 A3-luc Gapdh Ј ; for Gdf1 (Fig. 1B).Whenco-expressed withCripto levels. Xenopus ,5 Ј and 5 Ј TTC TGCCAG GGCACG TGC G3 Ј GCC TCTCTTGCTCAG TGT Bmp2, whichisprocessed Ј and 5 Ј ; andfor Ј TGG TGTTCCAGG Gapdh Nodal , 5 Ј TGC , 5 Ј Ј marginal zonecandorsoanteriorize studies have shown thatmisexpression ofmouse compare theeffects of performed gain-of-functionassays in in 2000). Following microinjection ofmRNAs intothetwo ventral caps (Jonesetal.,1995;Joseph andMelton,1997;Takahashi etal., dose-dependent expression ofdorsalmesodermmarkers inanimal To examine whetherGdf3signalsinaNodal-like pathway invivo Similar effects of (Chen andShen,2004). proprotein (Fig.2C),similartoourprevious findingswithNodal immunoprecipitate thematureLefty1protein,but not theLefty1 crosslinking. We found thatGdf3couldspecifically interact withGdf3inconditionedmediatheabsence of Shen, 2004).Consequently, weexamined whetherLefty1could as wellwithmatureNodalligandinconditionedmedia(Chen and with EGF-CFCproteins(ChenandShen,2004;Chengetal.,2004), proteinscaninhibitNodalsignalingthroughaninteraction Gdf3 signalingactivity (Fig.2C).We andothershave shown that Gdf3 ligandwithactivin receptors. enhances and/orstabilizesreceptorcomplex formation bymature combination, thesefindingsindicatethatCriptospecifically prodomain-V5-mature Gdf3)aswiththematureligand(Fig.2B).In Cripto interactedequallywellwiththebv-Gdf3 proprotein (Bmp2 in theabsenceofco-transfectedactivin receptors,wefoundthat Furthermore, incrosslinking/co-immunoprecipitationexperiments are notaconsequenceoftheuseheterologousBmp2prodomain. complexes werelow (datanotshown), indicatingthatourfindings assay, althoughthelevels ofmatureligandpresentinactivin receptor 2001). We alsoobtainedsimilarresultswithnative Gdf3inthis compare lanes4and6)(Chengetal.,2003;Yeo andWhitman, complex formationwithmatureNodalinfroganimalcaps (Fig.2A, similar toprevious observations ofEGF-CFCdependentreceptor activin receptorswas greatlyenhancedinthe presenceofCripto, Shen, 2004).However, theinteractionofmatureGdf3ligandwith similar toourprevious observations withNodal(Fig.2A)(Chenand well withactivin receptorsintheabsenceorpresenceofCripto, 2004). with themembrane-impermeableagentDTSSP(ChenandShen, immunoprecipitated togetherwithActRIIBfollowing crosslinking (f-Cripto) in293Tcells,andexamined theproteins (ActRIIB) receptorstogetherwithbf-Gdf3andFLAG-tagged Cripto expressed epitope-taggedtypeI(ActRIB/ALK4)andII reversible proteincrosslinking/co-immunoprecipitation.We co- and examine theformationofaGdf3receptorcomplex, weused To addresswhetherGdf3signalingismediatedbyactivin receptors, complex Interaction ofGdf3andCriptoinareceptor Gdf3 canuseasignalingpathway highlysimilartothatforNodal. (Chen andShen,2004).Taken together, theseresultsindicatethat signaling activity (Fig.1D;datanotshown), analogoustoNodal we foundthatco-expression ofLefty1orLefty2couldinhibitGdf3 Cryptic (Cfc1)orfishOepthanwithmouseCripto(Fig.1C).Finally, observed thatGdf3displayedlessactivity incombinationwithmouse whether otherEGF-CFCproteinscouldmediateGdf3signaling,and further explored theEGF-CFCdependenceofGdf3byexamining upon expression ofEGF-CFCproteinsandFoxh1 (Fig.1B).We Finally, weinvestigated thebasisbywhichLefty1caninhibit Interestingly, wefoundthatbf-Gdf3proproteininteractedequally Xenopus embryos Gdf3 Gdf3 and and Nodal RESEARCH ARTICLE Xenopus Nodal Xenopus laevis misexpression. Previous misexpression embryos, andinduce Nodal in theventral embryos to 321 , we

DEVELOPMENT proprotein, ~49kDa;mature bf-Gdf3,~17kDa); Westernblotsofimmunoprecipitatedinternalization. andinputproteins are shown,detectedusingthefollowingantibodies: tagged ActRIIBfrom celllysatesandreversal ofcrosslinking; kinase-inactiveactivinreceptor mutantswere usedinorder tod with theindicatedexpression constructs,followedbycrosslinking withthemembrane-impermeablereagent DTSSP, immunoprecipita Fig. 2.InteractionsofGdf3withactivinreceptors, CriptoandLefty1. 322 elongation (79%, pg) orbf-Gdf3(50inanimalcapsinducedmorphogenetic (Fig. 3A-D;Table 1).Similarly, expression of low dosesofNodal(50 and higherlevels ofGdf3orbf-Gdf3(100pgand50pg,respectively) of partialsecondaryaxes inresponsetolow levels ofNodal(10pg) marginal zonesoffour-cell stageembryos,weobserved theformation dorsal mesoderm[ expression ofmarkers ofearlypan-mesoderm[ ( antibodies: prodomaingenerated byuseofanalternative cleavagesite(asterisk)are indicated.Proteins wereblotting detectedbywestern mature formofLefty1ispreferentially immunoprecipitated; theexpectedpositionsofLefty1proprotein (arrow) andamatur Gdf3 proprotein, ~50kDa)and of FLAG-taggedCripto(f-Cripto)from celllysatesandreversal ofcrosslinking. Westernblotsare shownusing thefollowingan extra-embryonic ectodermorvisceral endoderm(Fig.4C-F). throughout theepiblastat5.5and 6.0dpc,but isnotdetectableinthe post-implantation development, expression inmouseand humanEScells(Satoetal.,2003).During mass attheblastocyst stage (Fig.4B),consistentwithitsconserved and earlypost-implantationstages.We foundthat expression patterninwild-typemouseembryosatpre-implantation To gaininsightintopotential rolesfor and Restricted expression of embryos, but possessessimilaractivity tobf-Gdf3(Fig.3I). suggesting thatunmodifiedGdf3ispoorlyprocessedin (76%, morphogenetic elongationwithhighdosesofnative Gdf3(500pg) ( not shown). Subsequently, with noexpression detected atearlierpre-implantationstages(data initiates inapopulationofinnercellsatthemorulastage(Fig. 4A), B Mixer CriptointeractswithGdf3.Expression constructswere co-transfectedinto293Tcells,followedbycrosslinking withDTSSP, im ) ), but notthedorsalmarker n RESEARCH ARTICLE =33), aswellinductionof ␣ -FLAG (bf-Gdf3proprotein, ~49kDa;mature bf-Gdf3, ~17kDa)and Xwnt8 n =33 forNodal;39%, , , Gdf3 ␣ -FLAG (f-Cripto,~21kDa).( expression islimitedto the innercell Gdf3 Gdf3 Xnr3 Gsc Gdf3 expression occursuniformly (Fig. 3E-I).We alsoobserved Xbra (goosecoid)] andendoderm in innercellmass n in vivo, weassessedits =31 forbf-Gdf3),and and ␣ -FLAG (f-Cripto,~21kDa); Xbra Mixer Gdf3 (brachyury)], expression (1000 pg), C ) Gdf3interactswithLefty1inconditionedmediatheabsenceofcrosslinking. The Xenopus ( To investigate thebiologicalfunctionsof homozygous nullmutants Early embryoniclethalityinasubsetof the pluripotentcellsofinnercellmassandepiblast. primordial germcells.Thus, hybridization duringgastrulationorthrough9.5dpc,includingin onset ofgastrulation,withnofurtherexpression detected byinsitu Notably, and healthy supplementary material).Althoughwecouldreadilyrecover fertile with anullmutationatthe in embryonic lethality, aswellembryonicphenotypeswereobserved mesoderm derivatives (21%)(Fig.5B-G).Similar frequenciesof junction, poorlydifferentiated anteriorstructuresandabnormal including prominentconstriction attheembryonic/extra-embryonic embryos exhibited characteristicmorphologicalphenotypes, visceral yolksacsat8.5dpc(11%)(Fig.5A).Lessseverely affected embryos wereeitherresorbing(5%),orcorrespondedtoempty phenotypes at6.0to8.5dpc(Table 2).Themostseverely affected ~35% ( phenotypes of intercrosses (datanotshown). Consequently, weexamined the significantly under-represented amongprogeny fromheterozygote suggesting that thephenotypicheterogeneity isnotstrongly been backcrossedforseven generations(Table 2;datanotshown), A ␣ ) FormationofaGdf3receptor complex.293Tcellswere transfected Gdf3 -HA (ActRIB-HA,~54kDa); ␣ -Lefty1 (Lefty1proprotein, ~40kDa;mature Lefty1, ~30kDa). –/– n Gdf3 =127) of 129SvEv inbredandC57BL/6J congenicmicethathad Gdf3 expression appearstoceaseby6.5dpc,priorthe Gdf3 –/– mutants atembryonicstages,andfoundthat homozygous mice,weobserved thesewere Gdf3 ␣ Gdf3 homozygotes displayedabnormal -Myc (ActRIIB-6xMyc,~75kDa). Gdf3 expression ishighlyrestrictedto locus (seeFig.S2inthe Gdf3 ecrease receptor Development 133(2) ␣ tibodies: usingthefollowing , weproducedmice -Gdf3 (bf-Gdf3 e Lefty1variant munoprecipitation tion ofMyc- Gdf3 ␣ -V5 (bv-

DEVELOPMENT transcriptase added. decarboxylase ( and endoderm( ( undergo morphogeneticelongation, asscored atstage20. the indicateddosesofNodal(F),Gdf3(G)orbf-Gdf3(H)mRNAs explants. Unlikecontrol uninjectedcaps(E),animalinjectedwith formation. ( mRNAs;arrows indicatesecondaryaxis Gdf3 (C)orbf-Gdf3(D) embryo (A)andembryosinjectedwiththeindicateddosesofNodal (B), ventral marginalzoneinjection.Morphologyofcontrol uninjected (500-1000 pg)result inweak inductionof of a from homozygousbyheterozygouscrossesexcluded thepossibility influenced bystrainbackground.Phenotypicanalysisofembryos Gdf3 actsinaNodal-likepathway induction ofmarkersformesoderm ( doses ofNodal(50pg)andbf-Gdf3 (50-100pg)result instrong in Gdf3inducessecondaryaxisandmesendodermformation Fig. 3. specific markers atgastrulationandpre-gastrulationstages.Initially, hybridizationusingtissue- we performedwhole-mountinsitu To analyzethephenotypicabnormalitiesobserved in Gdf3 Defects inAVE andprimitivestreak derivativesin I Mesendodermformationinanimalcapassays.Highdoses ofGdf3 ) Xenopus Gdf3 mutants maternal effect (C.C.andM.M.S.,unpublished). E-H embryos. Odc Gdf3inducesmorphogeneticelongationinanimalcap ) Mixer ) is used as an internal control.) isusedasaninternal –RT, noreverse ), butnotthedorsalmarker ( A - D ) Secondaryaxisformationfollowing Xbra , Xbra Gsc , chordin and and Xnr3 Mixer . Ornithine Gdf3 , whilelow Xwnt8 mutants, ) of probably duetoabsenceofthelateralplatemesoderm. embryos lacked left-sided shown). Notably, theseanalysesidentified for in theAVE (31%, embryos, lateral platemesodermat8.0dpc(67%four- toeight-somitestage homozygotes displayedcorrectleft-sided not shown). Inparticular, morphologicallynormal development, includingleft-rightaxisspecification(Fig.6N,O;data reveal additionaldefectsinbodypatterningatlaterstagesof prospective anteriorside. n mislocalization oftheAVE inthedistalvisceralendoderm(8%, reduction orabsenceoftheAVE (19%, expression of Additional analysesshowed that in 50%( mesoderm emerging fromtheprimitive streakwas reducedorabsent (Fig. 6A,B).We alsofoundthatexpression of absent in60%of( observed thatdefinitive endodermformationwas greatlyreducedor 64% ( diminished orabsentin50%( and subsequentlyinthenodeaxialmesendoderm,but isgreatly Gdf3 and movement aswelllossofdefinitive endoderminasubsetof of by theseverely decreasedorabsentexpression of However, thisearlyexpression was oftennotmaintained,asshown unaffected in expression inprospective forebrainandmidbrainregions was neural specificationandpatterningappearedtobenormal,as the markers Similar defectsinnascentmesodermformationwereobserved using expression of Although Foxa2 we examined theexpression ofearlymesodermmarkers, suchas (Fig. 6U),whereas 3/15(20%) at 6.0dpc,withlimitedstaining intheproximoposteriorepiblast homozygotes showed apparentdownregulation of (Fig. 6T,Z). Interestingly, wefoundthat2/15(13%) epiblast, withhighestlevels intheposteriorandproximalregion prior togastrulation, itself in potential effects onthispathway, weexamined expression of genes intheNodalsignalingpathway (seeDiscussion).To assess mutants stronglyresemblethoseobserved inmutantsforseveral Overall, thephenotypicabnormalitiesobserved inaffected embryos Altered expression of consequence of indicating thatsecondaryaxisformationisaninfrequent corresponding toduplicatednotochordsinsections(Fig.6R,S) and embryos (5%, and axialmesodermat8.0dpc(Fig.6P,Q), rarehomozygous =26) (Fig.6M),suggestingafailure inAVE movement tothe Surprisingly, ourmarker analysesalsorevealed anunusualsubset Examination oflessseverely affected Further analysesdemonstratedsevere defectsinAVE formation Gdf3 Gdf3 Lhx1, homozygotes. Usingthemarker n (Hnf3 =11) of mutants at8.5dpc(datanotshown). Gdf3 n =4) of Gdf3 homozygotes thatdisplayapartialaxisduplication. which isalsoexpressed intheAVE (Fig.6C,D;datanot n =9) (Fig.6N);afew morphologicallyabnormal ␤ Wnt3 Gdf3 Hesx1 homozygotes at6.0-6.75dpc.In wild-typeembryos T ) , homozygotes frequentlydisplayedreducedorabsent n Gdf3 (brachyury) Gdf3 which isexpressed intheanteriorprimitive streak Gdf3 =57) showed twinnedaxialmesodermstaining, n n and homozygotes ( =13) (Fig.6I-M);similarresultswereobserved =5) at 6.75dpc(38%, inactivation. homozygotes at6.75dpc(Fig.6C,D),andin homozygotes at7.5dpc(datanotshown). Nodal Fgf8 Gdf3 Nodal , n Nodal (data notshown). Bycontrast,early homozygotes at7.0dpc(Fig.6G,H). which isnormallyfoundinthenode =6) of displays gradedexpression inthe Gdf3 expression (Fig.6O),but thiswas n =15) at7.0-7.5dpc(Fig.6E,F). Gdf3 RESEARCH ARTICLE Gdf3 in homozygotes haddefectsin n =8) and Gdf3 Cerl mutants showed strong homozygotes at6.75dpc n Gdf3 Nodal Gdf3 =26) (Fig.6D,J,L),or Lhx1 (-like) mutant homozygotes with Otx2 Hex mutants failed to expression inthe Nodal ( Lim1 at 6.5-7.0dpc in 63%( expression inlateral ) Nodal Gdf3 Gdf3 Gdf3 Gdf3 Otx2 n , 323 =8) we

DEVELOPMENT Scale bars:50 ; expression disappears priortoprimitivestreak formation. (E,F), andisnotfoundinthevisceral endoderm(ve)orextra-embryonic and6.0dpc restricted totheembryonic epiblast(epi)at5.5dpc(C,D) oa 1 g 21/2(7)6/2(3)0/72(0%) 90/90(100%) 1/65(2%) 3/76(4%) 60/72(83%) 0/90(0%) 28/65(43%) 33/76(43%) 12/72(17%) of 0/90(0%) 36/65(55%) individual embryosat6.0dpcrevealed significantdownregulation 40/76(53%) 6V,A 72 upregulation of 65 90 76 pigmented protrusion intheposteriorregion thatcould notbeclassifiedasapartialaxis. *Embryos withposteriordefectshadabnormalblastopore closure, resulting inanopenneuraltubetheposterior;addition Nodal (10pg) bf-GDF3 (50pg) GDF3 (100pg) Uninjected Table 1.Phenotypesresulting from ventralmarginalzoneinjectionsin 324 Fig. 4. 35%, respectively; Table 2).Thiscorrelationsuggeststhatthe percentage withmorphologicaldefectsat6.75and7.5dpc(29% AVE defectsbymarker analyses(27%, additional 47arenotshown), issimilartothepercentage displaying is shown inFig.S3Athesupplementarymaterial; dataforan expression byreal-timePCR(34%, the percentageofembryosshowing alteredlevels of upregulation (seeFig.S3Ainthesupplementarymaterial).Notably, controls, while3/26 primitive endoderm(pe).( the blastocyststage(B),withnoexpression inthetrophoblast or andislaterexpressed athigher levelsintheinnercellmass(icm)at (A), low levelsintheinnercellsofcompacted morulaatthe16-cellstage implantation mouseembryos. Nodal Ј ). Similarly, quantitative real-timeRT-PCR analysisof Gdf3 RESEARCH ARTICLE expression in5/26 expression inpre-implantation andearlypost- ␮ m. Nodal Gdf3 C expression throughouttheepiblast(Fig. - homozygotes displayedsignificant F ubrPrilscnayai otro eet Normal Posteriordefect* Partial secondaryaxis Number ) Afterimplantation,expression is Gdf3 ( A homozygotes relative towild-type , B ) Expression of n =73) (analysisof26embryos n =26), aswelltothe Gdf3 is foundat Nodal Nodal Gdf3 upregulation of cells ofmesodermalappearanceanddisplayedcircumferential cross-section (Fig.6Y,D morphology thatwas associatedwitharadiallysymmetricshapein two outofnine(22%)homozygousmutantsdisplayeda‘conical’ axis duplicationsobserved atlaterstages(Fig.6R,S).Furthermore, streak insections(Fig.6X,C Gdf3 abnormal morphologyand data notshown). Theexpression patternsof showed strongupregulation throughouttheepiblast(Fig.6E epiblast, withtheexception oftheunusual‘conical’mutants, which most Nodal formation andmovement, withconsequenteffects onexpression of interpretation that and becomemesoderm. suggesting thatmostoftheepiblasthadundergone streakformation observed moreintenseposterior a significantnumberof and becomesupregulated at~5.75dpc(Wall etal.,2000)(this which isexpressed atlow levels throughouttheepiblastat5.5dpc through aNodal-like pathway, weexamined theexpression of remains atleastpartiallyintact.Finally, asGdf1canalsosignal (Fig. 6G on the adjacentproximalepiblast(Brennanetal.,2001),but noeffects the extra-embryonic ectodermisdependentuponNodalsignalingin respectively (datanotshown). Furthermore,expression of described above forotherAVE andmesodermalmarkers, dpc and visceral endodermoverlying thestreak(Fig.6W,B the ,but isslightlyupregulated intheposterior expression persistsintheposteriorepiblastandisdownregulated in In wild-typeembryosatearlyandmid-streakstages, ai 7 9 5 37% 127 35% 27 144 29% 40 20 119 17% 117 11 31 20 3 analysis. analyses, andinaC57BL/6J-129S6/SvEv mixedgeneticbackground forthe8.5dpc Mice were inaC57BL/6Jcongenicbackground forthe6.0,6.75and7.5dpc 0 Ratio Total Empty yolksac/resorbed Abnormal Table 2.Frequency ofembryonic phenotypesin Nodal remaining expression at6.0dpcandundergo abnormaldevelopment, whilethe homozygotes withAVE defectsalsodisplayaltered Additional marker analyseswereconsistentwiththe At laterstages,wealsoobserved considerablevariation inthe Bmp4 Xenopus Gdf3 homozygotes, whichshowed agreatlyexpanded primitive . Examinationof expression patternsandmorphologiesof Lefty2 Ј ,H expression wereobserved in Gdf3 homozygotes showed normalexpression intheposterior Ј ), suggestingthatNodalactivity intheproximalepiblast embryos in nascentmesodermat6.75dpcweresimilartothat homozygotes arephenotypicallywildtype. Nodal Gdf3 Ј Cripto ). Strikingly, theseembryoswerefilledwith Gdf3 expression inthevisceralendoderm, mutants have primarydefectsinAVE . p .5dc75dc8.5dpc 7.5 dpc 6.75dpc 6.0 dpc Ј , manyembryoscontainedasmalldarkly Nodal ); suchembryosmaygive risetothe expression at6.75dpcshowed that homozygotes (44%, Nodal expression at6.75dpc.We Gdf3 expression inoneoutofnine mutants at6.5dpc( Lefty1 Development 133(2) Gdf3 Gdf3 in theAVE at6.0 Ј ). We foundthat homozygotes. n –/– =9) showed embryos Bmp4 Nodal Nodal Gdf1, n Ј ,F =7) in Ј ;

DEVELOPMENT controls ( of work). We observed asimilarexpression patternandtemporalonset Gdf3 actsinaNodal-likepathway Nodal in Second, wehave demonstratedthatmisexpression ofbf-Gdf3and Cripto andLeftyproteinsinamannerhighlyreminiscentofNodal. form complexes withactivin receptors,andinteractswithboth inhibited byLeftyproteins.Atthebiochemicallevel, Gdf3can pathway initsrequirementforEGF-CFCproteinsandability tobe signaling incellculturedisplaysthedefiningfeaturesofNodal in aNodal-relatedpathway. First,wehave shown thatGdf3 Our studiesprovide threeprincipallinesofevidence thatGdf3acts functionsinaNodal-related pathway Gdf3 in mouseembryogenesisthanhithertoappreciated. the possibilitythatNodalpathway activity may playanearlierrole functions of at pre-gastrulationstages Gdf3 representsanimportantcomponentofNodalpathway activity The characteristicfeaturesofthe function have provided strongevidence foritsNodal-like activity. Our combinedbiochemicalandmoleculargeneticanalysesofGdf3 DISCUSSION levels of addition, quantitative real-timePCRanalysisconfirmedsimilar (arrowhead). ( structures (arrow) andanectopicprojection embryo (right)withreduced anterior wild-type embryo(left)andanabnormal embryos at8.0dpc,withaphenotypically mutant embryos. Fig. 5.Morphologyandhistologyof ␮ less-affected embryoinGlacksanallantois(arrowhead) andhasvisceralyolksac-liketissueinplaceofanamnion(arrows). an embryonic/extra-embryonicboundaryconstriction(arrowheads) andanabnormalembryonicregion containingmesoderm(arrows). (D,F), theseverely affected mutantinEshows with thephenotypicallywild-typeembryos dpc (D,E)and8.0(F,G). Incomparison ( consists ofanemptyvisceralyolksac(right). type embryo(left)andasevere mutantthat at 8.5dpc,showingaphenotypicallywild- structure (al?)ispresent. ( boundary (arrowheads); anallantois-like constriction attheembryonic/extra-embryonic abnormal embryonicregion andprominent decidua containing of paraffin wax-embeddedsectionsthrough yolk sacphenotype (Brennanetal.,2001;Waldrip etal.,1998), absence ofanteriorstructuresand isoftencorrelatedwithanempty Yamamoto etal.,2004).Failure to formtheAVE leadstothe the prospective anteriorside(Dingetal.,1998;Lowe etal., 2001; 2002), aswellitsmovement fromitsoriginaldistalpositionto is essentialforAVE formation (Brennanetal.,2001;Norris signaling pathway. Inparticular, Nodal signalingfromtheepiblast with loss-orreduction-of-functionforcomponentsoftheNodal majority ofaffected activity isspecificforaNodal-like pathway invivo similarity ofGdf3andNodalactivities, andsuggeststhatGdf3 to inducedorsalmesodermmarkers inanimalcapsunderscores the indistinguishable phenotypes.Inparticular, theabilityofbf-Gdf3 embryos at6.0dpc(seeFig.S3Binthesupplementarymaterial). B m. Abbreviations: al,allantois;am,amnion;ch,chorion;fb,forebrain; mes,mesoderm;ne,; vys,visceralyolks )A Gdf1 Gdf3 upregulation in Gdf1 Xenopus n –/– =17) at5.5-6.0dpc(Fig.6I Vg1 mutant at8.5dpcdisplaysan D-G expression inindividual wild-typeand -related genesinvertebrate development, andraise ) Hematoxylin-Eosinstaining ( embryos andanimalcapsresultsinessentially Gdf3 A Gdf3 ) Two –/– . Gdf3 C Moreover, ourfindingsuncover conserved )Two embryos at7.5 mutants displayphenotypesassociated Gdf3 homozygotes ( –/– Gdf3 Gdf3 embryos –/– Gdf3 null phenotypeimplythat Ј ,J Ј ; datanotshown). In n =22) andwild-type . Gdf3 Finally, the mutant throughout the epiblast(Fig.7B),similarto thephenotypeof fails toform, definitive endoderm formation(Fig.7B).However, iftheAVE epiblast, resultinginreduction orabsenceofmesodermand to move, leading toretentionof the absenceof by therequirementof These apparentlyoppositeoutcomescanpotentiallybeexplained either bedownregulated orupregulated intheabsenceof 2005). important roleatearlierstagesofdevelopment (Saijohetal., raisesthepossibilitythatautoregulation mayplayan however, therecentelucidationofasecondNodal-responsive in arelatively mildleft-rightpatterningdefect (Norrisetal.,2002); plausible asdeletionofthe the Nodalpositive autoregulatory loop.Thismodelisperhapsless upregulate second, non-mutuallyexclusive possibilityisthatGdf3mayactto primary orsecondaryconsequencesof whether thegastrulationphenotypesof perturbed intheabsenceof definitive endodermformation;however, as Gdf3 sufficient pathway activity tocompensatefortheabsenceof 7A). Thevariability ofthe formation andmovement inpre-gastrulationdevelopment (Fig. a Nodal-like pathway toprovide acrucial signalforAVE Yamamoto etal.,2001). 2001; Lowe etal.,2001;Song1999;Vincent etal.,2003; Nodal pathway components(Dunnetal.,2004;Hoodless axial mesendoderm,whichiscommonlyobserved in mutants for display reducedanteriorstructures,mostprobablyowing tolossof to stochasticdifferences intheabilityof Norris etal.,2002;Zhou1993).Inaddition, and hypomorphicmutants(Conlonetal.,1994;Lowe etal.,2001; mesoderm anddefinitive endodermformation,similarto Less severely affected similar tothatobserved inthemostseverely affected Unexpectedly, wehave observed that Based onourfindings,weproposethatGdf3functionsthrough may alsoberequiredsubsequentlyformesodermand Nodal Gdf3, Nodal expression itself,throughitspotentialtoactivate the AVE maybeabletoform, but beunable Gdf3 expression would remainathighlevels Gdf3 Gdf3 Nodal for AVE inductionandmovement. In Gdf3, mutant embryosexhibit defectsin -null phenotypemightthusbedue Nodal autoregulatory enhancerresults RESEARCH ARTICLE we areunabletodistinguish expression intheproximal Gdf3 Nodal Gdf3 Nodal Nodal mutants represent Scale bars:200 inactivation. A expression can Gdf3 Gdf3 expression is ac. to provide Nodal mutants. mutants The Gdf3 Gdf3. 325 null .

DEVELOPMENT A-Y,E unaffected in Abnormal frequently absent(D endoderm (ave)(C),butis mesoderm andanteriorvisceral primitive streak, nascent (H often absentin but thedefinitiveendodermis AVE anddefinitiveendoderm(de), most (M absent (L)orismisplaceddistally the AVE issometimescompletely observed in lateral platemesoderm(lpm)is expression of Nodal pathway upregulation, suchasinmutants forthe 2002). Similarphenotypeshave beenobserved inothercasesof secondary axis(BertocchiniandStern,2002;Perea-Gomezetal., inhibitors intheAVE isnecessarytosuppressformationofa well aspartialaxisduplications,expression ofNodalpathway splitting oftheprimitive streakatearlygastrulationstages,as ( ( in neuroectoderm (ane)isunaffected Otx2 greatly reduced (D absent in the AVE issometimesreduced or abnormal is absentorgreatly reduced in notochord (not)at8.0dpc(P),but marks thetailbud(tb)and (Q (R reveals apartialaxisduplication homozygotes, expression of (S formation oftwinnednotochords expressed intheposterior epiblast andinthevisceralendoderm(ve)overlyingprimitivestreak (W,B posterior epiblast(ep)at6.0dpc(T,Z) Nodal expansion oftheprimitivestreak (X,C (J Gdf3 often absentorgreatly reduced in primitive streak (ps)(A),butis normally expressed intheanterior Gdf3 abnormal Fig. 6.Phenotypicdefectsand In addition,overexpression of Smad2 326 Gdf3 surrounding athickmesodermal layer(mes)(D ( the node(nd)(O plate, butretain expression around Nodal a fewabnormalmutantslack normal morphologyat8.0dpc(N); (arrowheads). ( C G P , , , , , , , , Gdf3 Q arrow). ( D , H arrows) andmultiplesomites arrows), corresponding to arrow). ( arrow). ( arrow). ( ) Expression of ) Expression of ) Ј in theanterior Gdf3 Lhx1 homozygotes (B mutants. ( mutants. -J is foundintheproximal- expression inthelateral Ј -null mutants(Brennanetal.,2001;Waldrip etal.,1998). RESEARCH ARTICLE homozygotes. ; 50 Gdf3 Gdf3 Gdf3 is expressed inthe K Nodal homozygotes, butisupregulated in‘conical’mutants(F I R Gdf3 N , - ␮ J M , Nodal Gdf3 S ) , m forZ-D O –/– T –/– ) Inrare Hesx1 ) –/– Gdf3 E - ( , ) Asymmetric Hex D A , –/– embryos embryos arrow). F , embryos withthe‘conical’morphology (Y)showhigh-level expression in Ј , Expression of ) T B ) Expression of arrowhead) in mutants. ( Cerl embryos with , in theleft ) (brachyury) expression in homozygotes arrow) or , expression in Foxa2 arrow). Gdf3 Ј marks the . T is I Ј , J Ј ) Expression of Nodal , Ј double-headedarrow) thatis accompanied byincreased expression inthevisceral endoderm(C , but isdownregulated insome might leadtoexpansion and Ј arrow). ( , Gdf1 in thepre-gastrulation epiblast isunaffected in E Ј , F Ј ) Expression of Ј ). ( Gdf3 G Ј , H mutants (U)whileupregulated inothers(V,A Ј ) possibility ofsuchheterodimerizationwithNodalhasbeen diminished activity throughheterodimerizationwith Nodal.The the possibilitythatGdf3mightcreateanovel enhancedor by signalinginparalleltoNodalitself.However, wecannotexclude Gomez etal.,2002). inactivation oftheNodalantagonists transcriptional repressor Cripto Bmp4 Nodal In thesimplestcase,Gdf3mayupregulate Nodalpathway activity in theproximal-posterior epiblast at6.75dpcisunaffected in expression intheextra-embryonic ectoderm(exe)at6.5dpcis expression circumferentially inthevisceralendoderm Gdf3 Ј ), butsome Drap1 homozygotes. Scalebars:100 (Iratni etal.,2002),orcombined Gdf3 Ј ). At6.75dpc, Lefty1 –/– embryos showan Development 133(2) and Cerl Ј Nodal arrow). , ␮ m for (Perea- is

DEVELOPMENT embryos, Our functionalanalysisof ligands inearlyembryogenesis Potential overlappingactivitiesofNodalpathway remains tobeelucidated. 2002). Whethersuchheterodimerizationisphysiologicallyrelevant well asforthe demonstrated inprincipleforBmp7(Yeo andWhitman,2001),as Gdf3 actsinaNodal-likepathway shown inred. Intheabsenceof is depictedinblue,whilethatofNodalantagonists Nodal pathwayactivity(includingGdf3)intheepiblast oftheanteriorepiblast;distribution patterning antagonists bytheAVE provides essentialsignalsfor ( streak formationandmaintenanceof and movement,mayadditionallybenecessaryfor feedback loop. maintains itsexpression through anautoregulatory movement aswellprimitivestreak formation,and Fig. 7.Modelfor the primitivestreak andaxisduplication. upregulation of Alternatively, iftheAVE failstoform(bottom),theabsenceofNodalantagonistswillresult in in embryoswithdownregulated form butbeunabletomove(middle),thereby resulting Similarly, expression (Bertocchinietal.,2004;SkromneandStern, 2001). and FGFsignalsinprimitive streakformationandinitiationof XII (Shahetal.,1997),andhasbeenproposedtocooperatewithWnt stages intheepiblastofposteriormarginal zonefromstageXto embryos. Inparticular, chick related genesatpre-gastrulationstagesareconserved in coordinating left-rightpatterningin (Rankin etal.,2000),consistentwiththeproposedrolefor Null mutantsfor be detectedthroughouttheepiblast(Wall etal.,2000)(thiswork). after 6.0dpcand strikingly complementary, with been divided between definitive endoderm. mutants displaydefectsinformationoftheAVE, mesodermand genes aregenerallyconsistentwithourfindingsthatmouse Weeks andMelton,1987).Theseputative functionsof 1998; Kessler andMelton,1995;Thomsen1993; dorsal mesodermandendodermformation(JosephMelton, the blastulastage,andhave beensuggestedto playacentralrolein phenotype of Hyatt andYost, 1998;KramerandYost, 2002).Notably, the The patternsof patterns of development. Thispossibilityissupportedbytheexpression understood rolesinpre-implantation andearlypost-implantation suggests thattheNodalsignalingpathway mayplayasyet poorly facilitated byCrypticinvivo. mutants (Yan etal.,1999),suggestingthatGdf1signalingmaybe 2003). Moreover, recentstudieshave alsosuggested a rolefor Bhattacharya etal.,2004;Ramalho-Santos etal.,2002;Sato undifferentiated mouse andhumanEScells(Besser, 2004; by theconserved expression of B ) Inwild-typeembryos(top),expression ofNodal Interestingly, theconserved functionsof The potentialoverlapping functionsofNodal, Gdf3 andGdf1 Nodal eou Vg1 Xenopus Gdf3 Gdf1 Gdf3 Nodal activity promotes AVE inductionand Xenopus and Gdf3 Gdf1 Gdf1 Gdf1 mutants isindistinguishablefromthatof is alsorequired forAVE formation expression throughout theepiblast,potentiallyleadingtoexpansionof Nodal Gdf1 function. are defective forleft-rightaxisspecification being upregulated at~5.75dpc,whenitcan and Tgf transcripts arelocalizedtovegetal cellsat Gdf3 ␤ and in pre-gastrulationembryos,as wellas Gdf3 Gdf3 Nodal factor Derriere(EimonandHarland, Vg1 ( Gdf3, Nodal, Cripto,Lefty1 Gdf3 suggests thattheactivities of A , theAVE may ) Inwild-type Nodal expression intheepiblastare is expressed atearlypre-streak expression thatisrestricted totheproximal epiblast. Xenopus being downregulated shortly its mammaliancounterparts expression. eou Vg1 Xenopus (Hyatt etal.,1996; and Vg1- may have Gdf3 Cryptic related Vg1 Nodal Gdf3 Vg1- in in . Chen, C.andShen,M. Brennan, J.,Lu,C.C.,Norris, D.P., Rodriguez,T. A.,Beddington,R.S.and Bradley, A. Bhattacharya, B.,Miura,T., Brandenberger, R.,Mejido,J.,Luo,Y., Yang, A. Cheng, S.K.,Olale,F., Bennett,J.T., Brivanlou,A.H.andSchier, A.F. Burt, D.W. andLaw, A.S. Conlon, F. L.,Lyons, K.M.,Takaesu, N.,Barth,K.S.,Kispert, A.,Herrmann, Bertocchini, F., Skromne, I.,Wolpert,C.D. L.andStern, Cheng, S.K.,Olale,F., Brivanlou,A.H.and Schier, A.F. Chang, H.,Brown, C.W. andMatzuk,M. Besser, D. http://dev.biologists.org/cgi/content/full/133/2/319/DC1 Supplementary materialforthisarticleisavailableat Supplementary material Wellcome FundCareer Award intheBiomedicalSciences. Robert Wood JohnsonFoundation.C.W.B. wasarecipient ofaBurroughs HD42837 toM.M.S.,andHD01156HD27823C.W.B.), andbythe Institutes ofHealth(HL67355andHD41648toS.M.W., HD32067toM.M.M., the AmericanHeartAssociation(R.H.),March ofDimes(R.H.)andtheNational and commentsonthemanuscript.Thisworkwassupportedbygrantsfrom real-time PCRanalysis,andCoryAbate-ShenRickPadgettfordiscussion Hua Changforexperttechnicalassistance,MariannadeJulioadviceon Jin LeeandMalcolmWhitmanforplasmidreagents, PeiWang, LeiChenand We wouldliketothankBrigidHoganforsharingunpublishedinformation,Se- mammalian embryogenesisthanpreviously conceived. lead ustospeculatethatthispathway mayplayabroaderrolein ES cells(Jamesetal.,2005;Vallier etal.,2004).Theseobservations Nodal signalinginmaintainingtheundifferentiated stateofhuman Bertocchini, F. C.D. andStern, References Nodal signaling. GDF1. EGF-CFC proteins are essentialcoreceptors for theTGF-betasignalsVg1and 823. mammalian transforminggrowth factor-beta superfamily. beta superfamily. mouse embryo. Robertson, E.J. 151. Oxford: IRLPress. StemCells:APracticalApproach and Embryonic 2956-2964. in humanembryonicstemcelllines:uniquemolecularsignature. X., Joshi,B.H.,Ginis,I.,Thies,R.S.,Amit,M.etal. 45076-45084. human embryonicstemcellsrequires activationofSmad2/3. E30. subset ofTGFbetasignalsbyantagonizing EGF-CFCcoreceptors. Development acting sequentiallyduringprimitivestreak formationinthechickembryo. of embryonicpolarityinaregulative system:evidenceforendogenousinhibitors 744. positions theprimitivestreak byantagonizingnodalsignaling. Genes Dev. (2004). Expression ofnodal,lefty-a,andlefty-Binundifferentiated (1987). Production andanalysis ofchimericmice.In 131 Nature Curr. Biol. (2001). Nodal signalling in the epiblast patterns theearly (2001). Nodalsignallingintheepiblastpatterns Prog. GrowthFactorRes. , 3381-3390. 17 , 31-36. 411 (2004). Two modesbywhichLeftyproteins inhibit (1994). Evolutionofthetransforminggrowth factor- 14 , 965-969. , 618-624. (2002). Thehypoblastofthechickembryo RESEARCH ARTICLE 5 , 99-118. (2002). Geneticanalysisofthe (ed. E.J.Robinson),pp.113- (2004). Geneexpression (2004). Leftyblocksa Endocr. Rev. (2004). Determination J. Biol.Chem. Teratocarcinomas Dev. Cell Blood PLoS Biol. 23 103 3 (2003). , 735- , 787- 279 , 2 327 , ,

DEVELOPMENT Graff, J.M.,Bansal,A.andMelton,D. Eimon, P. M.andHarland,R. S.D.,Oxburgh,L.,Robertson,E.J.and Bikoff,Dunn, N.R.,Vincent, E.K. Gritsman, K.,Zhang,J.,Cheng,S.,Heckscher, E.,Talbot, W. S.andSchier, A. Lu, C.C.,Brennan, J.andRobertson, E.J. Hoodless, P. A.,Pye,M.,Chazaud,C.,Labbe,E.,Attisano,L.,Rossant, J.and Dohrmann, C.E.,Kessler, D.S.andMelton,A. Lowe, L.A.,Yamada, S.andKuehn,M.R. Kramer, K.L.andYost, H.J. Hoodless, P. A.,Tsukazaki, T., Nishimatsu,S.,Attisano,L.,Wrana, J.L.and Habas, R.,Kato,Y. andHe,X. McPherron, A.C.andLee, S.J. Norris, D.P., Brennan, J.,Bikoff, E.K.andRobertson,J. Newfeld, S.J.,Wisotzkey, R.G.andKumar, S. Matzuk, M.M.,Finegold,J.,Su,J.G.,Hsueh,A.andBradley, A. Hyatt, B.A.andYost, H.J. Kessler, D.S.andMelton,A. Helde, K.A.andGrunwald,D.J. Ding, J.,Yang, L.,Yan, Y. T., Chen,A.,Desai,N.,Wynshaw-Boris,A.and Jones, C.M.,Kuehn,M.R.,Hogan,B.L.,Smith,J.andWright, C.V. 328 Melton, D.A. James, D.,Levine,A.J.,Besser, D.andHemmati-Brivanlou,A. Iratni, R.,Yan, Y. T., Chen,C.,Ding,J.,Zhang,Y., Price,S.M.,Reinberg,D. Hyatt, B.A.,Lohr, J.L.andYost, H.J. Jones, C.M.,Simon-Chazottes,D.,Guenet,J.L.andHogan,B. Kato, Y., Habas,R.,Katsuyama,Y., Naar, A.M.andHe,X. Joseph, E.M.andMelton,D.A. Joseph, E.M.andMelton,D.A. induction inXenopus. proteolytic processing onDerriere andNodalactivity:implicationsformesoderm transduce distinctsubsetsofsignalsfortheTGFbetasuperfamily. formation and patterning inthemouseembryo. formation andpatterning (2004). CombinatorialactivitiesofSmad2andSmad3regulate mesoderm 108-117. mesoderm byzDVR-1,thezebrafishorthologueofXenopusVg1. signaling. F. 487. gastrulation: axisformationinthemouseembryo. streak inthemouse. Wrana, J.L. function in patterning themouseembryo. function inpatterning Dev. Cell axis formationinmigratingmesodermasacell-nonautonomousVg1cofactor. soluble, mature Vg1protein. signaling anddisruptaxisformationinXenopus. Thomsen, G.H. 418-426. suppliedanddistributedthroughoutis maternally theembryo. Cell vertebrate gastrulationandrequires anovelForminhomologyprotein Daam1. posterior axisinthemouseembryo. dependent autoregulatory enhancercontrols thelevelofNodalsignalsin 795. beta familyligands,receptors andSmadsignaltransducers. developmental pathway:phylogenetic analyses oftransforminggrowth factor- pole ofXenopusoocytes. cysteines. the transforminggrowth factor-beta of superfamilycontaininganovelpattern Nature Alpha-inhibin isatumour-suppressor genewithgonadalspecificityinmice. 384-392. organizing left-rightaxisformation. Isolation ofVgr-2, anovelmemberofthetransforminggrowth factor-beta- signalling. component oftheARC/Mediatorcomplexrequired forTGF beta/Nodal Shen, M. 120 formation andmaintenanceoftheprimitivestreak inthemouse. B. andRobertson,E.J. gastrulation. Nodal-related signalsinduceaxialmesodermanddorsalizeduring related genefamily. gastrulation bythetranscriptionalcorepressor DRAP1. and Shen,M. Vg1. axis formationbymaternal pluripotency inhumanembryonicstemcells. TGF{beta}/activin/ signalingisnecessaryforthemaintenanceof and mesodermformationinXenopusembryos. expressed intheSpemannorganizer. (1999). TheEGF-CFCprotein one-eyedpinheadisessentialfornodal , 1919-1928. 107 RESEARCH ARTICLE 360 , 843-854. 2 J. Biol.Chem. Cell , 115-124. Nature , 313-319. (1987). Translocation mRNAtothevegetal ofalocalized maternal Development (2001). FoxH1(Fast)functionstospecifytheanteriorprimitive (1998). 97 , 121-132. (1999). Dominant-negativeSmad2mutantsinhibitactivin/Vg1 418 (2002). Inhibitionofexcessnodalsignalingduringmouse Mol. Endocrinol. Genes Dev. Cripto , 641-646. Development 268 (1994). Aprimaryrequirement fornodalinthe Nature (1998). Theleft-rightcoordinator: therole ofVg1in 121 (2002). Ectodermalsyndecan-2mediatesleft-right , 3444-3449. is required forcorrect orientationoftheanterior- Development (2001). Wnt/FrizzledactivationofRhoregulates , 3651-3662. (1993). GDF-3andGDF-9:twonewmembersof (1995). Inductionofdorsalmesodermby (2002). Effects ofheterodimerization and (1998). MutantVg1ligandsdisruptendoderm (1997). Xnr4:aXenopusnodal-related gene 15 Nature (1993). TheDVR-1(Vg1)transcriptofzebrafish 328 , 1257-1271. Cell Nature 129 6 Dev. Biol. , 80-82. (1996). Initiationofvertebrateleft-right , 1961-1968. 384 93 , 3089-3103. (1996). XenopusMadproteins (2001). From fertilizationto Development , 37-46. 121 (2001). Geneticdissectionofnodal 395 , 62-65. Development 184 , 2155-2164. , 702-707. (1999). Molecularevolutionofa Development Development Dev. Biol. Curr. Opin.Genet.Dev. , 367-372. (1996). Inductionofaxial Science 128 207 132 Genetics (2002). TheFoxH1- (2002). A , 1831-1843. 125 298 , 364-379. 131 Dev. Biol. , 1273-1282. (2005). Development Dev. Biol. , 2677-2685. Cell , 1996-1999. , 1717-1728. (1992). 152 85 159 (1992). , 479- , 783- (1995). 11 175 , , , Saijoh, Y., Oki,S.,Tanaka, C.,Nakamura,T., Adachi,H.,Yan, Y. T., Shen,M. H.,Blokzijl,A.,Andersson,O.,Chang,C., Reissmann, E.,Jornvall, Sato, N.,Sanjuan,I.M.,Heke,Uchida,Naef,F. andBrivanlou,A.H. Rankin, C.T., Bunton,T., Lawler, A.M.andLee,S.J. Ramalho-Santos, M.,Yoon, S.,Matsuzaki,Y., Mulligan,R.C.andMelton,D. Perea-Gomez, A.,Vella, F. D.,Shawlot,W., Oulad-Abdelghani,M.,Chazaud, Rossant, J.andTam, P. P. Rivera-Perez, J.A.,Mager, J.andMagnuson,T. Yamamoto, M.,Meno,C., Sakai,Y., Shiratori,H.,Mochida,K.,Ikawa,Y., Shah, S.B.,Skromne, I.,Hume,C.R.,Kessler, C.D. D.S.,Lee,K.J.,Stern, Seleiro, E.A.,Connolly, D.J.andCooke, Schier, A.F. andShen,M. Schier, A.F. Thomsen, G.H.andMelton,D.A. Thomas, P. Q.,Brown, A.andBeddington,R.S.P. Weeks, D.L.andMelton, D. A. Waldrip, W. R.,Bikoff, E.K.,Hoodless,P. A.,Wrana, J.L.andRobertson,E. S.D.,Dunn,N.R.,Hayashi,S.,Norris,D.P.Vincent, andRobertson,E.J. Vallier, L.,Reynolds,D.andPedersen,R.A. Takahashi, S.,Yokota, C.,Takano, K.,Tanegashima, K.,Onuma,Y., Goto,J. Srinivas, S.,Rodriguez,T., Clements,M.,Smith,J.C. andBeddington,R.S. Song, J.,Oh,S.P., Schrewe, H.,Nomura,M.,Lei,Okano,Gridley, T. Skromne, C.D. I.andStern, Wilkinson, D.G. Whitman, M. Watanabe, M.andWhitman, Wall, N.A.,Craig,E.J.,Labosky, P. A.andKessler, D.S. asymmetric expression ofNodal. M. andHamada,H. proteins duringvertebratedevelopment. orphan receptor ALK7andtheActivinreceptor ALK4mediatesignalingbyNodal Minchiotti, G.,Persico,M.Ibanez,C.F. andBrivanlou,A.H. 265. inmicebygrowth/differentiationright patterning factor-1. Dev. Biol. with themouse. (2003). Molecularsignature ofhumanembryonicstemcellsanditscomparison cells. A. multiple primitivestreaks. Nodal antagonistsintheanteriorvisceralendodermprevent theformationof C., Meno,Pfister, V., Chen,L.,Robertson,E.,Hamada,H.etal. mouse embryo. inductive eventsinXenopusNieuwkoopcenter. and Asashima,M. in earlymousedevelopment. 470-487. morphogenetic eventscharacterizethemousevisceralendoderm. formation inthemouse. mediates Nodalsignalingduringanterior-posterior andnode patterning Saijoh, Y. andHamada, H. Oxford UniversityPress. mesoderm inducerinXenopus. transient markerofendothelialcellprecursors. gene revealing peri-implantationasymmetryinthemouseembryoandanearly primitive streak formation. and Dodd,J. Biol. expression andexperimentalaxisdeterminationbythechickenVg1gene. Nature beta. vegetal hemisphere inXenopus eggscodesforagrowth factorrelated toTGF- 5634. default pathway. differentiation ofhumanembryonicstemcellsalongthe neuroectodermal Development signalling pathwaysinitiateprimitivestreak formationinthechickembryo. Dev. Biol. induction andreversalbymouseGdf1,aVg1-related ofleft-right pattern gene. polarity oftheearlymouseembryo. (1998). Smad2signalinginextraembryonictissuesdeterminesanterior-posterior Nodal signals. (2003). Cellfatedecisionswithinthemouseorganizerarebygraded governed visceral endoderm. (2004). Activecellmigrationdrivestheunilateralmovementsofanterior 169. growth, gastrulation,androstral headdevelopmentinmice. and Li,E. variations. mesoderm inducersintheearlyXenopusembryo. (2002). “Stemness”:transcriptionalprofiling ofembryonicandadultstem 6 Science Cell , 1476-1486. 403 19 227 51 (2003). Nodalsignalinginvertebratedevelopment. (1999). ThetypeIIactivinreceptors are essentialforegg cylinder Dev. Cell , 385-389. , 589-621. (2001). Nodalsignalinginearlyvertebrate embryos.Themesand , 861-867. 298 , 495-509. 128 (1997). Misexpression ofchickVg1inthemarginalzoneinduces Genes Dev. (1992). Development Dev. Biol. Dev. Biol. , 597-600. , 2915-2927. Development 1 (2000). Two novelnodal-related genesinitiateearly , 605-617. (2005). Two nodal-responsive enhancerscontrol. left-right In SituHybridization:APracticalApproach (2004). Emergingasymmetryandembryonicpatterning Genes Dev. Dev. Cell 17 260 Development 275 (2001). InteractionsbetweenWntandVg1 , 1646-1662. (2001). ThetranscriptionfactorFoxH1(FAST) (2000). Nodalsignallinginvertebratedevelopment. Dev. Cell 129 , 404-413. , 403-421. (1987). A maternal mRNAlocalizedtothe (1987). Amaternal Cell Dev. Dyn. 131 (1999). FAST-1 effector isakeymaternal of , 3455-3468. (1993). Processed Vg1protein isanaxial 3 74 Cell , 745-756. 15 , 1157-1164. 7 , 433-441. , 1242-1256. , 155-164. 92 124 Genes Dev. 232 , 797-808. (1996). Earlydevelopmental (2004). Nodalinhibits , 5127-5138. , 1031-1036. Development Development (2003). Dynamic Development (1998). 15 Development 133(2) (2000). Regulationofleft- , 2010-2022. (2000). Mesendoderm Nat. Genet. Hex Dev. Biol. 125 Annu. Rev. Cell 127 : ahomeobox 126 , 85-94. . London: , 5319-5329. Dev. Biol. , 5621- (2001). The 213 24 (2002). , 262- Curr. , 157- 261 ,

DEVELOPMENT Yan, Y.-T., Gritsman,K.,Ding,J.,Burdine, R.D.,Corrales,J.Price,S.M., Yamamoto, M.,Saijoh,Y., Perea-Gomez, A.,Shawlot,W., Behringer, R.R., Gdf3 actsinaNodal-likepathway 2537. for Talbot, W. S.,Schier, A.F. andShen,M. 392. formation oftheanteroposterior axisofthemouseembryo. Ang, S.L.,Hamada,H.andMeno,C. EGF-CFC genes invertebrateleft-rightaxisformation. (2004). Nodalantagonistsregulate (1999). Conservedrequirement Genes Dev. Nature 428 13 , 387- , 2527- Zhou, X.,Sasaki,H.,Lowe,L.,Hogan,B.L.andKuehn,M.R. Yan, Y. T., Liu,J.J.,Luo,Y., Chaosu,E.,Haltiwanger, R.S.,Abate-Shen,C. Yeo, C.andWhitman,M. Nature a novelTGF-beta-likegeneexpressed inthemousenodeduringgastrulation. dependent andCripto-independentmechanisms. Nodal signalingpathway. and Shen,M. 361 , 543-547. (2002). Dualroles ofCriptoasaligandandcoreceptor inthe (2001). NodalsignalstoSmadsthrough Cripto- Mol. Cell.Biol. 22 RESEARCH ARTICLE , 4439-4449. Mol. Cell 7 , 949-957. (1993). Nodalis 329

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