and negative-feedbackactivities. receptors, expressionofsolubleantagonists,aswellpositive- including thecontrolofligandprocessing,utilizationco- Nodal signalingistightlyregulatedbydiversemechanisms recently. Consistentwithitscrucialdevelopmentalfunctions, pluripotency andincarcinogenesishavebeenuncoveredmore this pathwayinthemaintenanceofembryonicstemcell the specificationofleft-rightasymmetry. Additionalrolesfor embryo duringtheinductionofmesodermandendoderm e-mail: [email protected] UMDNJ–Robert Wood JohnsonMedicalSchool,Piscataway, NJ08854,USA. Center forAdvancedBiotechnologyand MedicineandDepartmentofPediatrics, Importantly, NodalandtheTGF mediated byFoxH1 andMixer transcriptional activators (Fig.1). inhibitors oftheLeftysubclassTGF Furthermore, Nodalsignalingcanbeantagonizedbysoluble and areessentialforNodalsignaling(ShenSchier, 2000). membrane throughaglycosyl-phosphatidylinositol(GPI)linkage cysteine-rich extracellular proteinsthatareattached totheplasma pathway areco-receptorsoftheEGF-CFCfamily, whicharesmall transcriptional complexes inthenucleus.UniquetoNodal to theirinteractionwithSmad4andthesubsequentformationof receptors phosphorylatecytoplasmic Smad2and/orSmad3, leading and Shen,2000;Whitman,2001)(Table 1).Activated typeI Smad2/Smad3 branchoftheTGF serine-threonine kinasereceptors,andsignalthroughthe factor-beta (TGF Nodal pathway ligandsaremembersofthetransforminggrowth Key componentsoftheNodalsignalingpathway recent findings. mechanisms ofitsfunctionduringembryogenesis,withafocuson molecular propertiesoftheNodalpathway anddelineategeneral various species.Here,Idescribethecentralcomponentsand systems, anditsfunctionalconservation hasbeendemonstratedin molecular geneticstudiesinthefrog,zebrafish, chickandmouse . Itscentralimportancehasbeenestablishedusing primary bodyaxes, aswellfortheformationofmesoderm and particular, Nodalsignalingisessentialforthespecification ofthe andstagesofchordatedevelopment. In formation anddifferentiation thattake place duringthepre- The Nodalsignalingpathway isintegral toprocessesofpattern Introduction (TGF Nodal-related ligandsofthetransforminggrowthfactor-beta Michael M.Shen Nodal signaling:developmentalrolesandregulation (2007)doi:10.1242/dev.000166 Development 134,1023-1034 activity.) indistinguishable effects, Iwillreferto‘Activin/Nodal’ pathway inhibitors. (IncasesinwhichActivin andNodalarelikely tohave signaling isEGF-CFC-dependentandcanbeblocked by responses ingain-of-functionstudies,but differ inthatNodal ␤ ) superfamily playcentralrolesinpatterningtheearly ␤ ) superfamily thatbindtotypeIandII ␤ ligand Activin oftenelicitsimilar ␤ pathway (Schier, 2003;Schier ␤ factors (Table 1), andis independent Nodalactivity invivo. date, however, thereisnodefinitive evidence supportingreceptor- et al.,2004;Levine andBrivanlou, 2006;Onumaetal.,2005).To via mechanismsthatdonotinvolve heterodimerization(Haramoto as wellGdf3,canpotentiallyinhibitBMPsWntligands and Harland,2002;Yeo andWhitman,2001).Furthermore,Nodal, form signalingfactors thathave reducedordistinctactivities (Eimon other TGF responses, Nodalligandshave been shown toheterodimerizewith ligands couldreflecttheirabilitytoinducereceptor-mediated (Chen etal.,2006;Cheng2003). similarly toActivin, anddoesnotrequireEGF-CFCco-receptors squint [ zebrafish Nodal-related ligandsinfrogs(encodedbysix ligand isfoundinmouse,humanandchick,therearemultiple screens inmouseandzebrafish (Box1).WhereasasingleNodal Nodal ligandswereoriginallyidentified throughforward genetic Nodal andrelated ligands ciiyi io(neso ta. 06 onale l,2004). activity invivo (Anderssonetal.,2006;Jornvall etal., currently noevidence thatALK7isrequiredforNodalpathway independent manner(Reissmann etal.,2001);however, thereis also transduceNodalsignalingactivity, potentially inanEGF-CFC- Whitman, 2001).Theorphantype IreceptorALK7(Acvr1c)can complexes (Chen andShen,2004;Reissmannetal.,2001;Yeo and CFC co-receptors,despitetheirability tointeractwithALK4-ActRII Activin, Nodalligandslacksignalingactivity intheabsenceofEGF- 2001; Yan et al.,2002;Yeo andWhitman,2001).Incontrastto ActRII (ActRIIA;Acvr2a)orActRIIB(Acvr2b)(Reissmannet al., receptor ALK4(ActRIB/Acvr1b),togetherwiththetypeIIreceptors Nodal ligandssignalthroughthetypeIserine-threoninekinase Receptors andco-receptors By contrast,the can alsoantagonizeBMPsignaling(Levine andBrivanlou, 2006). Cheng etal.,2003);however, Gdf3functionmaybecomplex, as it Gdf3 incellcultureisEGF-CFC-dependent(Chenetal.,2006; (Vtg1) andmouseGdf1inmicroinjectedzebrafish embryosandby ( Nodal analyses have revealed partially overlapping functionsof Nodal pathway components(Chenetal.,2006),anddouble-mutant One suchligandis Nodal-like responsesinvivo (Chen etal.,2006;Cheng2003). ligands canutilizethecorecomponentsofthispathway andgenerate Nodal ligandsinamniotes. genes infish andfrogsappeartoperformthesamecorefunctionsas independently oftheNodalsignalingpathway, theNodal-related mutants for (Gdf1) andGdf3(Anderssonetal.,2006;Chen2006) between itsmammaliancounterpartsgrowth differentiation factor 1 2006), andwhoseconserved functionsappeartohave beensplit endoderm formationhasonlyrecentlybeenclarified (Birsoy etal., spw Although most,orindeedall,ofthebiologicalfunctionsNodal Interestingly, recentstudieshave shown thatadditionalTGF )] (Schier, 2003).Exceptfor (Andersson etal.,2006).Notably, signalingbyzebrafish Vg1 ( sqt ␤ ; cyclops ndr1 Gdf3 superfamily members,suchasBmp4andDerrière,to erfs nomto ewr)and – Zebrafish InformationNetwork) have phenotypesresemblingthose ofmutants ( cyc Xenopus ; ndr2 TGF Vg1, whoseroleinmesodermand – Zebrafish InformationNetwork), ␤ ligand Derrièreappearstosignal xNr3 whichmayfunction , PRIMER xNr genes) and southpaw Gdf1 . 1023 Null and ␤

DEVELOPMENT l,20) whereassolublehuman Criptoprotein(TDGFI)can al., 2002), (Chuetal.,2005;Yan et Nodal signalingincellcultureand invivo mouse Criptocanactasasecreted trans contexts, however, EGF-CFCproteins canhave distinctproperties: phenotypes whenoverexpressed (Zhangetal.,1998).Incertain 1997; Strahleetal.,1997),as wellasitsinabilitytoinduce embryos thataregeneratedby celltransplantation(Schieretal., permissive factor basedonitscell-autonomy inchimericzebrafish phenotype (Gritsmanetal.,1999) whereas theexpression ofActivin, but notNodal,canrescuethe phenocopy doublemutantsfortheNodalligands embryos thatlackbothmaternalandzygoticcontributions of Zebrafish (Dorey andHill,2006;ShenSchier, 2000)(Table1). XCR3 ( genes representasmallfamily, withtwo membersinmammals interactions (Yan etal.,2002;Yeo andWhitman,2001).EGF-CFC confer specificity forthetypeIreceptorALK4through protein Keyreferences Function Gene Pathway ligands Role Table 1.KeycomponentsoftheNodalsignalingpathway 1024 Inhibitors Receptors andco- rncito factors Transcription Smads Cripto eetr iaercpo 2001) kinasereceptor receptors EGF-CFC proteinsareessentialco-receptorsforNodalthat ), andasinglegeneinzebrafish, PRIMER and Cryptic ), threeinfrogs( Nodal Vg1 Gdf1 Gdf3 Lefty1, Lefty2 ActRII, ActRIIB ALK4 Cripto, Cryptic Cer1, Cer2 FoxH1 Smad4 Smad2, Smad3 Mixer Xnr5, Xnr6 Xnr1, Xnr2,Xnr4, southpaw cyclops, squint, XCR2, XCR3 (fish), one-eyed pinhead fo,fs,cik TGF (frog, fish,chick) mue TGF (mouse) mue TGF (mouse) mue hc) Nodal-related TGF (mouse, chick), FRL-1/XCR1, . Notably, fs) 2003;Loweetal.,2001;Norris2002;Rebagliati (fish), fo)al.,1993) (frog) fo)al.,1998) (frog) mue, G-F orcpos (Branford andYost, 2002;Dingetal.,1998;DoreyandHill, EGF-CFCco-receptors; (mouse), FRL1/XCR1 - one-eyed pinhead acting factor tomediate oep acts asacis TGF (Reissmannetal.,2001;Song1999;Yan etal.,2002; Type IIserine-threonine (Reissmannetal.,2001;Yan etal.,2002;Yeo andWhitman, Type Iserine-threonine ebrsDNfml (BertocchiniandStern,2002;Hashimotoetal.,2004;Marques /DAN family igdhlx (Germainetal.,2000;Hoodless2001;Kunwar (Chuetal.,2004) (Dunnetal.,2004;Vincent2003) Winged-helix Co-Smad Receptor-Smads oedmi rti (Germainetal.,2000;Hart2002;Kofron2004; Homeodomain protein iad 1994;Feldmanetal.,1998;Jones1995;Long ligands pathway through Nodal hog oa 2000) pathway through Nodal pathway through Nodal iaercposYeo andWhitman,2001) kinase receptors n G-F o 2001;Nakamuraetal.,2006;Perea-Gomez2002) 2004;Menoetal.,1999;1998; receptors and EGF-CFCco- with Nodalligands neatwt L42006;Feldmanetal.,2002;Gritsman1999;Onuma interact withALK4 ebr;itrc etal.,2004;Perea-Gomez2002;Piccolo1999) with Nodalligands members; interact rncito atr2003;Pogodaetal.,2000;SaijohSirotkin etal., ␤ ␤ ␤ ␤ , cyc iad inl (Birsoyetal.,2006;Cheng2003) ligand; signals iad inl (Anderssonetal.,2006;Cheng2003;Rankin ligand; signals iad inl (Chenetal.,2006) ligand; signals rtis neat (ChenandShen,2004;ChenSchier, 2002;Chengetal., proteins; interact XCR2 and - acting ,and ( oep oep sqt oep ) , ␤ Notably, Lefty genesareoftendownstream targets ofNodal these receptors(ChenandShen, 2004;Chengetal.,2004). indicating thatthey do not functionascompetitive inhibitorsof proteins have notbeenfoundtointeractwithALK4orActRIIB, (Chen andShen,2004;Cheng etal.,2004).Bycontrast,Lefty Nodal ligands,therebyblocking formationofreceptorcomplexes through theirinteractionswith EGF-CFCproteinsaswell members oftheTGF Nodal ligands.TheLeftyproteins,whicharehighlydiverged Multiple extracellular inhibitorscanmodulatetheactivity of Extracellular inhibitors 2005). to theactivation ofthecanonicalWnt/ axis in that thenon-canonicalWntligandWnt11specifies the dorsal-ventral 2002; Biancoetal.,2003).Furthermore,arecentstudyhasreported independent mannerinmammaryepithelialcells(Biancoet al., activate theRas/Raf/MAPKandPI3K/Akt pathways inaNodal- (Brennan etal.,2001;ChenandSchier, 2001;Conlonetal., Xenopus Kunwar etal.,2003) 1998b; Sampathetal.,1998;Vincent2003;Zhou et al.,2006;Schier1997;Yan etal.,1999;Zhang 2001) 2000; Watanabe andWhitman,1999;Yamamoto etal., embryos throughaninteractionwithFRL1,leading ␤ superfamily, antagonizeNodalsignaling ␤ -catenin pathway (Tao etal., Development 134(6)

DEVELOPMENT Nodal functioninvivo remainstobeclarified. the preciserelationshipofthese membrane-associatedinhibitorsto antagonists (Haffner etal.,2004;HarmsandChang,2003).However, (Tmeff1) andNicalin,have been proposedtorepresentpathway several membrane-associatedproteins, such asTomoregulin-1 et al.,2002).InadditiontosecretedinhibitorsofNodalsignaling, are primarilyNodalinhibitors(Marquesetal.,2004;Perea-Gomez Smad2 and/orSmad3, togetherwiththecommon mediator-Smad activity istransduced bythereceptor-associated Smads(R-Smads) Downstream ofALK4 andActRIIreceptors,Nodalpathway Signal transducersandtranscriptional regulators signaling in multifunctional abilityofCerberustoantagonizeBMPand Wnt interactions withNodalligands(Piccoloetal.,1999).Despite the proteins thatcanblockNodalsignalingthroughtheirdirect Meno etal.,1999). this pathway (BranfordandYost, 2002;Feldmanetal., signaling, forminganimportantnegative-feedback mechanismfor SchematicoutlineoftheNodalsignalingpathway. Fig. 1. Development 134(6) receptors toinhibittheirfunction.( 2006). ( can alsosignalthrough asimilarreceptor complex,althoughitiscurrently unknownwhethersuchsignalingisEGF-CFCdependen receptor inacomplexwithtypeIreceptor (ALK4)andtypeIIreceptor (ActRIIorActRIIB)dimers.Atleastinsomecontexts,u extracellularly bytheproprotein convertasesFurinandPace4.( with FoxH1orbytheSmadphosphatasePpm1A,whichpromotes thenuclearexportofSmad2andpossiblytargetsitforproteasomal to transcriptionalactivationthrough interactionswithARC105andtheMediatorcomplex.Pathwayactivitycanbeinhibitedbyi Smad4 (orSmad3-Smad4)complexesinteractwiththewinged-helixtranscriptionfactorFoxH1orMixerhomeoproteins ontargetpro intoendosomesandcanbetargetedby Dpr2forlysosomaldegradation.( complex undergoesinternalization phosphorylation ofSmad2(orSmad3).ActivatedorSmad3associateswithSmad4andtranslocatestothenucleus,whereas th Members oftheCerberusfamily arecysteine-rich extracellular C ) CerberusandLeftyproteins are solubleantagoniststhatcaninteractwithNodalligands;Leftyproteins canalsointeractwi Xenopus (Silva etal.,2003),themouseCerberusproteins A Proteasomal degradation membrane Plasma Nodal proprotein Furin D

) ReceptoractivationleadstothephosphorylationoftypeIreceptor bythetypeIIkinase,aswell Extracellular X Intracellular Pace4 Ppm1A Smad2 FoxH1 P Drap1 Smad2 Smad2 FoxH1 Gdf1 E prodomain B ( ) Mature Nodalligands,aswellGdf1andGdf3,canbindtoanEGF-CFCco- Smad4 A ) Nodalligandsare expressed ashomodimericproproteins, andcanbecleaved B Nodal D G-F EGF-CFC EGF-CFC

P ALK4 Mediator ARC105 P Nodal ActRII Cerberus analyses inzebrafish indicatethatFoxH1 andMixer donotfully genes (Germainetal.,2000;Randall etal.,2004).However, genetic transcription complexes ontheenhancersofNodalpathway target interaction withSmad2/Smad3, leadingtotheformationofactive proteins containSmad-interaction motifsthatarerequiredfortheir FoxH1 andtheMixer subclassofhomeodomainproteins.These coupled withtheactivities ofthewinged-helixtranscriptionfactor (He etal.,2006). Smad2/3 tomediateTGF (Trim33 –MouseGenomeInformatics)tointeractwithactivated possibility isconsistentwiththeabilityofnuclearproteinTIF1 provide aco-Smadfunctioninearlymousedevelopment. Sucha mutants (Chuetal.,2004),suggestingthatotherproteins may less severe thanthatof Surprisingly, thephenotypeof can functionallyreplace gene substitutionexperiments inthemousehave shown that Smad3 have differing abilitiesto regulate target gene transcription, (co-Smad) Smad4(Massagueetal.,2005).AlthoughSmad2and Smad2

At thetranscriptionallevel, Nodalpathway functionistightly ActRII P ALK4 Nodal Smad4 Gdf3 Mixer P Cerberus C Smad2 Nodal Lefty EGF-CFC Nucleus Smad4 Receptor internalization Nodal ␤ Nodal Smad2 Lysosomal degradation signaling inhematopoieticprogenitors Dpr2 E ) Within thenucleus,activatedSmad2- ) Within Smad4 mutants or Nodal in vivo (Dunnetal.,2005). Lefty Lefty -null mutantsissignificantly ncleaved Nodalproprotein Smad2; Smad3 nteraction ofDrap1 t (Ben-Haimetal., PRIMER th EGF-CFCco- moters, leading e receptor degradation. double Smad3 1025 ␥

DEVELOPMENT 2001). are transcriptionallyrepressed(Dickmeisetal.,2001;Whitman, transcriptional activation inresponsetoNodalsignals,whereasafew suggesting that an excess ofleft-sidedNodal proteinhasundergone ectopic Nodalpathway activation intherightlateralplatemesoderm, Schier, 2001). Inthemouse, cells intheabsenceofasignaling relaymechanism(Chenand are unresponsive toSqt,activate responsesin distantwild-type andthus can traverse cells thatlacktheEGF-CFCco-receptorOep, transplantation experiments inzebrafish have shown thatSqtsignals Cyc cannot(ChenandSchier, 2001;Chen andSchier, 2002).Cell- function aslong-rangemesoderm-inducingsignalsinvivo, whereas In zebrafish, theNodalligandSqtaswellitsinhibitor Leftycan Long-range action Nodal Nodal responsesremaintobeidentified. 2003), indicatingthatadditionaltranscriptionfactors involved in account forNodal-mediatedtranscriptionalevents (Kunwar etal., 1026 fundamental forembryonictissuepatterning. interpreted have beenofparticularinterestbecausethey are The mechanismsbywhichsuchgradedsignalscanbegenerated and a developmental field ofresponsive cells(AsheandBriscoe,2006). signal thatactsover adistancetoelicitdose-dependent responsesin a Nodal ligandshave thepropertiesassociatedwithamorphogen: Nodal signals The generationandinterpretation ofgraded al., 2006;Rosset2006). component oftheSWI/SNFchromatinremodellingcomplex (Heet as wellBrg1 (Smarca4–MouseGenomeInformatics),a phosphorylated Smad2torecruitthep300histoneacetyltransferase to requirechromatinremodellingmediatedbytheabilityof complex (Katoetal.,2002).Transcriptional activation isalsolikely ARC105, asubunit oftheMediatortranscriptionalco-activator occur throughtheinteractionofSmad2/3-Smad4proteinswith Vincent etal.,2004).Inpart,transcriptionalactivation islikely to upstream left-sidespecific enhancer(LSE)(Saijohetal.,2005; 1999; Norrisetal.,2002;andRobertson,1999),byan asymmetric enhancer(ASE)locatedinitsfirst intron(Adachietal., particularly Activin(SchierandShen,2000). functions previously attributedtootherTGF Nodal pathwayactivityisresponsible formanyofthebiological et al.,1998).Muchsubsequentworkhasledtotherealization that gastrulation (Feldmanetal.,1998;Rebagliati1998b;Sampath the TGF corresponding locuswassubsequentlyshowntoencodeamemberof defective phenotype(Conlonetal.,1991;Conlon1994).The a retrovirally-induced mutantthatdisplayedanearlygastrulation- stem cells(Robertsonetal.,1986).Thisscreen ledtothediscoveryof from aretroviral insertionalmutagenesisscreen inmouseembryonic Unlike mostotherkeydevelopmentalregulators, Box 1.TheidentificationofNodalligands ( isolated loss-of-functionallelesintwoNodal-related genes, Rebagliati etal.,1998a).Inparallel,geneticscreens inthezebrafish species followedrapidly(Jonesetal.,1995;Levin (Zhou etal.,1993).Theidentificationof At present,mostknown targets oftheNodalpathway, suchas cyc and ) , PRIMER Nodal Lefty2 ␤ ligand superfamilythatisexpressed inthemammaliannode squint , itself ispositively autoregulated throughthe Pitx2 ( sqt ,anddemonstratedtheiressentialroles in ), , FoxA2 FoxA2 Lefty2 and hypomorphic mutantsdisplay Nodal Lhx1 ␤ -related ligandsinother superfamily members, (Table 1),undergo Nodal was isolated cyclops labile matureligand(Becketal.,2002;LeGood2005). convertases Furin(Spc1)orPace4 (Spc4/Pcsk6)thengeneratesa proprotein, anditssubsequentextracellular cleavage bytheproprotein ligands maycorrespondtothediffusion ofarelatively stable Good etal.,2005).Inparticular, thelong-rangemovement ofNodal ligand isreadilydegraded following itscellularinternalization(Le the Nodalproproteinisrelatively stable,whereastheprocessedmature range (Box2).Studiesincellcultureandzebrafish have shown that Nodal ligandprocessingareprimarydeterminantsoftheirsignaling through theextracellular matrix(Williams etal.,2004). instead, long-rangemovement ofXnr2appearstooccurbydiffusion no evidence oftranscytosis, argosomal transportorcytonemes; Xenopus GFP-tagged Xnr2proteinmovement fromXnr2-expressing embryos (Sakumaetal.,2002).Furthermore,thevisualizationof without amplification ofpathway activity, asthreefold differences cells appeartobeexquisitely sensitive toActivin/Nodal levels, even durations ofexposure, or both(Gritsmanetal.,2000).Responding due todiffering levels ofNodalpathway activity, ortodiffering 2004; Vincent etal.,2003). over longdistances(upto500 with thesefindings, GFP-labeledNodalorLefty2proteinscantravel long-range diffusion totherightside(Menoetal.,2001).Consistent in increasingdosesof progressively moresevere defectsinmesendodermformationobserved response toNodalpathway activity inmicehasbeensupportedbythe differentiation (Gritsmanetal.,2000).Asimilardose-dependent prechordal mesodermversus notochordduringaxialmesoderm vegetal axis(Douganetal.,2003),including the specification of are requiredforthepatterningofmesodermalonganimal- mutants infish have demonstratedthatdifferent levels ofNodalactivity 1999). Subsequentloss-of-functionanalysesof Xenopus as initiallyshown ingain-of-functionstudiesofActivin signalingin exemplified byNodal-mediatedspecification ofmesodermalidentity, signaling caninducedose-dependenceincellularresponses.This is As isthecasewithotherpotentdevelopmental signalingfactors, Nodal Dose-dependent responses et al.,2006;EimonandHarland,2002). proproteins thatcannotbecleavedcanretain activityinvivo(Ben-Haim ligands lacksignalingactivityasproproteins, mutantXnr2andNodal are regulated inasimilarfashion.Inaddition,althoughmostTGF currently unknownwhetherNodalligandsexistinalatentcomplexor in pre-gastrulation mouseembryos(Becketal.,2002).Moreover, itis trans-Golgi network,consistentwiththeirnon-cell-autonomousrole convertases FurinandPace4canoccurextracellularly, ratherthaninthe required torelease themature ligandfrom thelatentcomplex. mature ligandinabiologicallyinactivestate,andanactivationstepis network. Followingproprotein cleavage,dimericTGF dimers hasbeenthoughttooccurintracellularlyinthetrans-Golgi either monomersorrelatively labiledimers.Thecleavageofproprotein is absentinLeftyandGdf3proteins, suggestingthattheseproteins are an intrachaindisulfidebond;interestingly, thecysteineresidue involved TGF Box 2.Processing andsignalingabilityofNodalligands together withLatentTGF often remain non-covalentlyassociatedwiththemature dimericligand, Recent findings suggestthatthestabilityaswellefficiency of The dose-dependentresponsesof cellstoNodalsignalingmaybe In thecaseofNodal,however, processing bytheproprotein ␤ ligand proproteins undergodimerization,whichisfacilitatedby (Green andSmith,1990;Gurdonetal.,1994; animal capsintoadjacentnon-expressing capshasrevealed Smad2 ␤ binding proteins (LTBPs). Thismaintainsthe and Smad3 ␮ m) whenexpressed inchick mutant alleles(Dunnetal., Development 134(6) sqt and ␤ prodomains cyc double ␤

DEVELOPMENT (Fig. 2)(Chenand Schier, 2002; Saijohetal.,2000).Sucha reaction- generate astablegradedsignalacross aresponsive developmental field characteristics ofabiologicalreaction-diffusion system,whichcan The regulatory properties oftheNodalpathway stronglyresemblethe Reaction-diffusion mechanism from thenucleus(GrimmandGurdon, 2002). formation in to respondActivin/Nodal signalstowards theendofmesoderm binding ability(Iratnietal.,2002).Finally, thelossofcompetence embryos throughaninteractionwithFoxH1 thatinhibitsitsDNA- transcription factor Drap1candampenNodalsignalingin mouse embryos (Linetal.,2006).Atthetranscriptionallevel, thegeneral downregulate endogenousNodalsignaling activity inzebrafish can dephosphorylateactivated Smad2 andSmad3,can also shown thatthenuclearserine-threoninephosphatasePpm1A facilitating theirdegradation (Zhangetal.,2004).Recentwork has formation bybindingtoendocytosed ALK4/ALK5receptorsand Activin/Nodal pathway activity duringzebrafish regulator ofWntsignaling,functionsasanantagonist of Zebrafish InformationNetwork), whichwas initiallyidentified asa dependent responses.For example, Dpr2(Dapper2;Dact2– although itisnotyetunderstoodhow thesemightshapedose- pathway activity inNodal-responsive cellshave beendescribed, Many potentialmechanismsfordownregulating intracellular Intracellular downregulation ofpathwayactivity to beelucidated. activity areinterpretedasdistincttranscriptionalresponsesremain molecular mechanismsbywhichthesediffering levels ofpathway even afterligandwithdrawal (Bourillotetal.,2002).Atpresent,the receptor occupancy andSmad2activation, whichcanbemaintained overall pathway activity correspondstothemaximallevel of Smad2 concentration(ShimizuandGurdon,1999).Importantly, in receptoroccupancy resultinequivalent increasesinnuclear Yost, 2004).] [Adaptedfromleft-right patterning. Branford andYost (Branford and pathway activitymayfunctionduringmesendodermspecificationand prevail overalongerrange.Suchregulatory mechanismforNodal perceive littleornosignalingactivity, aslateralinhibitionbyLeftywill perceive highlevelsofsignalingactivity, whereas more distantcells than Nodalsignals.Cellsincloseproximity totheNodalsource thereby (red) isalsoinducedbytheNodalpathway, andhasagreater range and actatlong-range(bluearrows). Theexpression ofLeftyinhibitor source ofNodalsignals(atleft)thatundergopositiveautoregulation gradient (showninblue)across adevelopmentalfieldcanresult from a positional information. Reaction-diffusion mechanismforthegenerationof Fig. 2. Development 134(6) oa oreResponsive field Nodal source Nodal Lefty Xenopus is associatedwiththeexclusion ofSmad2 The generationofastableNodalsignaling Distance fromsource Kimelman, 2000), resultinginmesendoderm formationatthe the extraembryonic yolk syncytial layer(YSL)(Chenand induced byanasyetunidentified a dorsalidentity(Leeetal.,2001). onset ofNodalsignaling,withearlier andlongersignalingleadingto mesoderm patterningmightbedue todifferences inthetimingof (Fig. 3A)(Agiusetal.,2000;Kimelman,2006).Alternatively, D-V the marginal zone,withhigher levels resultingindorsalspecification Nodal signalthatinducesdose-dependentmesendodermformation in ligands inthevegetal region, leadingtoadorsal-ventral (D-V)graded activated maternally-encoded VegT transcriptionfactor cooperateswith canonical Wntsignalingpathway. In Nodal andVg1/Gdf3inthisprocess,theirinteractionswith the there appeartobespecies-specific differences intherelative rolesof Although Nodalpathway activity isessentialformesodermformation, Mesoderm inductionandpatterning particularly well-conserved (Box3). induction, neuralpatterningandleft-rightspecification appeartobe Nodal signalingandantagonisminmesodermendoderm ascribed tofunctionsoftheNodalpathway. However, therolesof Numerous biologicalactivities inearlyembryogenesishave been embryogenesis Central functionsofNodalsignalingin specification (ChenandSchier, 2002;Nakamuraetal.,2006). likely tofunctionduringmesodermpatterning,aswellinleft-right component ofsuchareaction-diffusion system.Thismechanismis that ofNodal(Sakumaetal.,2002),whichrepresentsacrucial diffusion ofLeftyinhibitorsintissueappearstobemoreefficient than autoregulatory loops(MeinhardtandGierer, 2000).Inparticular, the diffuse over alongdistance,coupled withpositive andnegative diffusion systemdependsontheabilityofligandsandantagoniststo 2004). Interestingly, therestricted expression of required formesodermformation(Dubocetal.,2004;Flowers establishing theoral-aboralaxisinseaurchins, althoughitisnot stages ofdevelopment,Nodalsignalingplaysacentralrole in expressed ontherightside,notleft(Dubocetal.,2005).Atearlier et al.,2005).Unexpectedly, however, thiscassetteisspecifically highlighting itsevolutionarilyconservedrole indeuterostomes (Duboc expression cassetteisasymmetricallyexpressed inseaurchin embryos, Duboc andLepage,2006). in deuterostomes todefinetheregion ofthemouth(Cheaetal.,2005; specification hasledtothespeculationthatNodalpathwayarose prospective oralectodermanditsfundamentalrole inoral-aboralaxis anus (deuterostomes).] Inparticular, a opening results intheformationofmouth(prostostomes) orthe groupings ofbilateriananimals,anddiffer inwhethertheblastopore [Protostomes anddeuterostomes correspond tothetwomajor deuterostomes (Cheaetal.,2005;DubocandLepage,2006). restricted tovertebrates,butmaybemore broadly conservedin tunicates andechinoderms,indicatingthatthepathwayisnot Nodal andLeftyorthologshavebeenidentifiedincephalochordates, orthologs inthe protostomes, assuggestedbytheabsenceofNodal,EGF-CFC,orLefty Key componentsoftheNodalpathwayappeartobeabsentfrom Box 3.TheevolutionofNodalpathwayfunction The expression ofzygotic ␤ -catenin toactivate zygotictranscription ofXnrandVg1 Drosophila and sqt and C. elegans ␤ -catenin-dependent signal(s)in cyc in thezebrafish embryo is Nodal Xenopus genomes. Bycontrast, - Lefty PRIMER Nodal embryos, the - Pitx2 to the gene 1027

DEVELOPMENT Bmp4 signalsbacktotheepiblast toactivate convertases aswellBmp4(Ben-Haimetal.,2006).Subsequently, toinducetheexpression ofFurinandPace4 proprotein ligand cansignalfromtheepiblasttoadjacentextraembryonic prodomain cleavage mutanthasindicatedthatunprocessedNodal formation inthemouseembryo(Fig.3D).TheanalysisofaNodal and slow positive regulatory loopsisemployed forprimitive streak (Bertocchini etal.,2004;SkromneandStern,2001). subsequently induce primitive streakformationintheposterior marginal zone,and embryo, Vg1playsaprimaryroletogetherwithWnt8ctoinduce not alongtheD-Vaxis(Douganetal.,2003).Finally, inthechick responsible formesodermpatterningalongtheanimal-vegetal axis, zebrafish have suggestedthatlong-range gradedNodalsignalingis blastoderm margin (Fig.3B).Interestingly, geneticanalysesin Fig. 3.Modelsofregulatory pathwaysformesoderminduction. 1028 additional feedbackloopmaytakeplacethrough Criptoupregulation byBmp4andWnt3(Becketal.,2002;Morkel2003). induces itspositiveautoregulatory loop(fast-acting;bluearrow), aswellaslowerfeedbackloop(orangearrow) through Bm ectoderm, whichactivatesexpression ofitsproprotein convertasesFurinandPace4,aswellBmp4.Production oftheactivem adjacent epiblast.( arrows), ofthemesoderminmarginalzone.( andspecifythedorsal-ventralpatterning stabilization of VegT T-box transcriptionfactor(purplearrow). Corticalrotation dorsal afterfertilizationleadstotranslocationofmaternal to theactivityofotherfactorsasnoted.( expression are indicatedinblue;bluearrows indicate ro)expressed attheposteriormarginalzonecooperateswithposteriorly-expressed arrow) Graded Nodalsignaling(thinandthickarrows)ofmesoderm.( specifiestheanimal-vegetalpatterning (blue arrows) attheblastodermmarginare activatedbyanasyetunidentifiedsignal(s)thatemanatesfrom theextraembryonic loops, as 2003). Furthermore,Gdf3isalso likely tofunctioninthesefeedback the canonicalWntpathway (Ben-Haimetal.,2006;Morkel etal., can upregulate expression levels (Chen etal.,2006). mesoderm anddefinitive endodermthatcorrelatewithaltered A morecomplex regulatory circuitthatutilizes interlinked fast PRIMER Gdf3- Ventral A Frog ␤ Nodal -catenin (orangearrow) onthedorsalside.Thelevelsof D null mutantsdisplayvariable defectsinthe ) Inthemouseembryo(lateralview),Nodalproprotein (Nodal-Pp)expressed intheepiblastsignalstoextraembryonic and Nodal Xnr1, 2,4,5,6 Cripto Vegetal Animal VegT expression intheepiblast(Fig.3C) D Mouse Vg1 expression intheepiblast through β -cat A ) In Marginal zone Dorsal Xenopus Pace4 Wnt3 Furin/ Nodal-Pp Proximal Distal Nodal/Vg1 expression, which Bmp4 (lateral view),zygotic ZbaihCChick B Zebrafish Yolk syncytial layer Epiblast activity, orangearrows indicateWnt/ Extraembryonic Nodal ectoderm Depictions ofembryosatpre-gastrulation stages.Domainsof Xnr Cyc Animal Vegetal Vincent etal.,2003).In mesoderm formation(Agiusetal.,2000;Thisse levels ofNodalactivity thatarehigherthanthoserequiredfor Stainier, 2002),andmayrepresentadose-dependent responseto which ismediatedbyMixer homeoproteins(Lewis andTam, 2006; The formationoftheendodermalsorequiresNodalsignaling, Endoderm formation mutants forthemouse activity inanimalcapassays(Kofron etal.,2004).Similarly, null upregulation of down of down of related homeoproteins,andcanbeabolishedbymorpholinoknock- induced byoverexpression offourtheseven Mixer (Mix/Bix)- ligands. mesoderm-inducing genes,including thoseencodingNodal as high-level Nodal signalsbyinducingendoderm-specific genessuch mesendoderm progenitorsand specifyendoderminresponseto these studiessuggestthatMixer homeoproteinsareexpressed in generate excess axialmesoderm (Hartetal.,2002).Taken together, reduced definitive endoderm, but alsooverexpress as well as maternal as wellmaternal Xnr Sox17 transcripts (bluearrows) areencoded activatedbythematernally Sqt , whilesimultaneouslyrepressing theexpression ofseveral Wnt8c Mixer (Fast) Mixer B ) Inzebrafish(lateralview),zygotic (orange arrow) toinducestreak formationinthe ( Xnr1 Mix.3 Vg1 expands themesoderm,asshown by the Nodal and ␤ transcripts are higherdorsally(thickerblue ) (Kofron etal.,2004).Conversely, knock- C -catenin activity, andpurplearrows correspond ) Inthechickembryo(dorsalview), Cripto Bmp4 Xnr5 Mixl1 Wnt8c Xenopus Wnt3 andincreasesmesoderm-inducing , Anterior Posterior ( Vg1 Mml , endodermformationcanbe (Slow) oebxgn display homeoboxgene ) Wnt8c izing signalsandthe marginal zone Posterior Development 134(6) p4 andWnt3;an cyc yolk syncytiallayer. ature Nodalligand Epiblast and Nodal/Vg1 sqt Nodal transcripts Vg1 (blue and

DEVELOPMENT patterning. theaxialmesendodermproducesdpc); inturn, signalingfactors(blackarrows) thatare essentialforforebrain maintenanceand Conversely, Nodalsignalingisrequired forthegeneration ofaxialmesendoderm(orange)bytheanteriorprimitivestreak durin expression ofNodalantagonists( (Yamamoto etal.,2004).Nodalsignalingisalsorequired forthemovementofAVE (purplearrow) totheanteriorside(5.75 egg cylinderat5.5dpc;notethattheinitialappearanceofAVE isalready slightlyasymmetric,withabiastowards thepr throughout theepiblast[5.25dayspost-coitum(dpc)],andinducesformationofanteriorvisceralendoderm(AVE; red) atth 2006). Following initialevents thatestablish L-Rasymmetry, Nodal both locations(RayaandBelmonte, 2006;ShiratoriandHamada, the nodetoleftlateralplatemesoderm (LPM),andisrequiredin regulates thepropagation ofleft-sidedpositionalinformationfrom During left-right(L-R)axisspecification, Nodalpathway activity Left-right patterning 2000). transcriptional repressor regulators oftheNodalpathway, suchas holoprosencephaly patientshave identified geneticlocithatinclude Schier etal.,1997).Furthermore,analysesofhuman mutants orhypomorphic human holoprosencephaly, asobserved inzygotic Nodal pathway activity canresultinphenotypesthatresemble anterior forebrainterritories(Fig.4).Consequently, reductionsof for ventral patterningoftheneuraltubeandmaintenance anterior endoderm(Vincent etal., 2003),whicharenecessaryinturn are requiredforthegenerationofprechordalmesoderm and ligands toallow headformationin expressed intheanteriorendodermantagonizesNodal andWnt 2004; BertocchiniandStern,2002).Furthermore, another Nodalantagonistthatmaybe AVE) positionsandlimitstheprimitive streak,incooperationwith expressed intheextraembryonic (analogoustothemouse Gomez etal.,2002).Similarly, inthechickembryo, primitive streaksthatresultfromexcessive Nodalactivity (Perea- patterning andwhichprevent theformationofenlarged orduplicated Lefty1 andCerberus-1(Cer1),whichareessentialforanteriorneural (Rossant andTam, 2004).TheAVE producestheNodalantagonists key roleinanteriorspecification oftheadjacentepiblast(Fig.4) In themouseembryo,anteriorvisceralendoderm(AVE) playsa upon axialmesendodermgeneratedinresponsetoNodalsignaling. the subsequentmaintenanceandpatterningofneuraltissuedepends generation ofanteriorneuraltissuerequiresitsinhibition,whereas Nodal signalingplaysdualrolesinneuraldevelopment, asthe Neural patterning Nodal byNodalsignalingandantagonisminthemouseembryo. Fig. 4.Anteriorneuralpatterning Development 134(6) By contrast,highlevels ofNodalactivity intheposteriorepiblast and/or Gdf3 ; red shadingindicatesregions expressing theNodalantagonists Proximal .5dc55dc57 p .5dc7.5dpc 6.75dpc 5.75dpc 5.5dpc 5.25 dpc Distal TGIF Cripto endoderm Epiblast Visceral Lefty1 (de laCruzetal.,2002;Gripp mouse mutants(Chuetal.,2005; Expression ofNodalantagonists Expression ofNodalpathway ligands Xenopus , AVE Cer1 Lefty1 ) bytheAVE isessentialforthespecificationofanteriorneuralidentityinadjacentepiblast. (Piccolo etal.,1999). (Bertocchini etal., Cripto movement neirPosterior Anterior oep AVE Cerberus Cerberus zebrafish and the et al.,2004).Thesubsequentauto-activation of (Norris etal.,2002;Saijoh2000;2005;Vincent contain FoxH1-binding sitesthatareessentialfortheirfunction (ASE), whichbothdrive in particulartheleft-sideenhancer(LSE)andasymmetric well asintheLPM(Wall etal.,2000),and Intriguingly, mouse factor isinvolved inthetransferofleft-sidedinformation. expression intheLPM(Longetal.,2003),suggestingthat adifferent Nodal ligand 2002; Saijohetal.,2003).Inzebrafish, however, expression ofthe subsequent asymmetricgeneexpression intheLPM(Brennan etal., a processthatmightinvolve asymmetricCa activity isupregulated ontheleftsideofnode(Fig.5A),through of loop (Fig.5B).Nodalsignaling isalsoessentialfortheexpression downregulation ofNodalactivity by as theinductionof rapid spreadof interaction hasemerged from studiesof excluded. Inthemouse,evidence supporting adirectsignaling mechanism involving intermediary signalingfactors hasnotbeen long-range fromthenodetoLPM,althoughasignalrelay 2004). antagonize Nodalligands(Hashimotoetal.,2004;Marques Dand5 supported bystudiesofzebrafish 1999). AroleforNodalpathway functioninthenodeisfurther for theEGF-CFCgene a L-Rpatterningphenotypeindistinguishablefromthatofmutants examined, andleadstotissue-specific laterality decisions. Nodal pathway activity. Itisknown thattheasymmetricexpression of Yamamoto etal.,2003).Mathematical modelingshows thatthis and cansuppressectopicright-sided activity (Menoetal.,1998; that prevents theleakageofleft-sidedNodalsignalstorightside, Current modelssuggestthatNodaland/orGdf1proteinssignal at In themouse, Lefty1 AVE Activity ofNodalantagonists Activity ofNodalpathway ligands Lefty1 ,gnsthatencodeCer/DAN family membersthatcan genes ), occurs intheleftLPMofallvertebrate speciesthus far in theaxialmidline,whichcan act asamolecularbarrier and spaw Nodal Nodal Cer1. around Kupffer’s vesicle isnotessentialfor Gdf1 Primitive expression throughouttheleftLPM,aswell streak mesendoderm Shortly afterimplantation, Blue shadingindicatesregions expressing expression inthenodeisessentialfor Pitx2 Cryptic Nodal Axial is expressed intheperi-nodalregion as expression andthesubsequent (Rankin etal.,2000;Yan etal., expression intheleftLPM,and charon Lefty Nodal 2 viaanegative-feedback and mouse Node Gdf1- 2+ ospective anteriorside signaling andNotch g gastrulation(7.5 promoter elements, ventralneuraltube Nodal dpc),where the e distalendofthe Nodal null mutantshave PRIMER Cer2 is expressed results inthe ( Dante 1029 spaw ,

DEVELOPMENT transcripts arenotessentialfordorsalspecification. sqt 2005). Inparticular, maternal exist ateven earlier stages,astheasymmetricexpression ofa posteriorly (Yamamoto etal.,2004).Interestingly, A-Ppolaritymay side, whileallowing Nodalactivity todrive cell proliferation proliferation inthevisceralendoderm ontheprospective anterior proposed tomediatedirectional AVE movement byinhibitingcell 2004). Thisasymmetricexpression ofNodalantagonistshasbeen the prospective anteriorside(Takaoka etal.,2006;Yamamoto etal., distal visceralendodermdisplaysaslightlyasymmetricbiastoward phenotype ofmaternal-zygotic the significance ofthesefindings iscurrentlyuncertainbecausethe conserved developmental mechanism (Goreetal.,2005).However, mammalian species,includinghuman,raisingthepossibilityofa these sequencemotifsalsooccurin Xenopus utilized foractive transportofmRNA transcriptsin (Gore etal.,2005);theunderlyingmechanismmayresemblethose toheterologous similar localizationwhenfusedincis motifs inthe3 of leads toaventralized phenotype(Goreetal.,2005).Thelocalization eight-cell stages,whereasmorpholinoknock-down ofmaternal blastomeres inamajorityofzebrafish embryosatthefour-cell and (Mesnard etal.,2006). (Rodriguez etal.,2005),aprocessthatisimpairedin inhibitory signalfromthedistantextraembryonic ectoderm may requiretheepiblasttoreachathresholdsizedilute an play anindirectpermissive roleintheseprocesses, asAVE formation null mutants(Chenetal.,2006).Finally, Nodalactivity mightalso well asforitsmovement, asbothprocessescanbe affected in Nodal pathway ligandGdf3isalsocrucialforAVE induction,as Lowe etal.,2001;Norris2002).Theactivity ofthemouse mutants, theAVE formsbut doesnottranslocate (Dingetal.,1998; al., 2002).Intheabsenceof evidence ofanA-Paxisisapparent(Brennanetal.,2001;Norris al., 1998).Intheabsenceof 12 hours(Rivera-Perez etal.,2003;Srinivas etal.,2004;Thomas cylinder, but thentranslocates totheprospective anteriorsidewithin initially formsinthemostdistalportionofpost-implantationegg formation anddirectionalmovement oftheAVE (Fig.4).TheAVE associated withanterior-posterior (A-P)axisspecification: the In themouse,Nodalsignalingisrequiredforatleasttwo events Anterior-posterior axisformation Nodal ligand 2005), arecentstudyhasproposedthatmaternaltranscriptsforthe identity infrogsandzebrafish (Kimelman,2006;SchierandTalbot, Although theactivation ofcanonicalWntsignalingspecifies dorsal transcripts Dorsal-ventral axisspecificationbymaternal evolutionarily conserved iscurrentlyunknown. continued toemerge. Theextent towhichthesefunctionsare Over recentyears,unexpected rolesforNodalsignalinghave Novel functionsoftheNodalpathway right side(Nakamuraetal.,2006). signals throughouttheleftLPM,whileinhibitingitsspreadto diffusion mechanismthatensurestheuniformpropagation ofNodal Nodal 1030 Prior toAVE movement, theexpression of sqt mutants (Aokietal.,2002),suggestingthatmaternal - transcripts todorsalblastomeresisconferredbysequence Lefty PRIMER embryos (Palacios andStJohnston,2001).Interestingly, - Pitx2 sqt Ј act asdorsaldeterminantsinzebrafish (Goreetal., untranslated region (UTR),whichcanconfera expression cassettegeneratesamodified reaction- sqt sqt Nodal Cripto transcripts arelocalizedindorsal mutants resemblesthatofzygotic , noAVE isformedandno or inhypomorphic Nodal Lefty1 genes inseveral Drosophila and Nodal lacZ Cer1 mutants mRNA Lefty1- Nodal Gdf3- in the and sqt sqt transgene isabolishedin is notrequiredforA-Paxisformation,expression ofthe are highlyexpressed inbothundifferentiated mouseandhuman ES stem (ES)cells.Indeed,allkey componentsoftheNodalpathway the maintenanceofundifferentiated humanandmouseembryonic Recent studieshave suggestedthatNodalsignalingisrequiredfor Maintenance ofundifferentiated EScells polarity inthemouse. suggesting thatNodalpathway functionisessentialforearly A-P implantation embryo(Takaoka etal.,2006).Although lacZ signals, andsuppresses ectopic midline expression of loop, andsubsequentlydownregulates positive-feedback loop. spreads through theleftlateralplatemesoderm(green) through a range action.( activate left-sided pathway activitythenpropagates totheleftlateralplatemesoderm on theleftsideofnode,andare antagonizedbyCer2(red). Nodal flow, Nodal(green arrow) and/orGdf1signals becomeelevated breaking around thenode,possiblyasaconsequenceofciliary-based inthemouseembryo. patterning SequentialfunctionofNodalsignalinginleft-right Fig. 5. B A transgene canbedetectedintheprimitive endoderm oftheperi- 8.25 dpc(3-8somitepairs) 8.0 dpc(0-2somitepairs) Lateral plate Lateral plate mesoderm mesoderm Nodal etRight Left etRight Left Nodal B ) Nodal Lefty2 Nodal Lefty1 auto-regulates itsownexpression, which Lefty2 expression, mostlikelythrough direct long- prevents thespread ofleft-sided FoxH1 is inducedthrough anegative-feedback Nodal Nodal Gdf1 Posterior Posterior Anterior Anterior Node Node mutants (Takaoka etal., 2006), ( A activation ontherightside. Lefty1 Nodal

) Followinginitialsymmetry Floor plate Floor plate nodal flow? Cer2 expression (red bar).Axial Development 134(6) Nodal Lateral plate Lateral plate mesoderm mesoderm Lefty1 Lefty1-lacZ Nodal itself

DEVELOPMENT provide futuretherapeutictargets. progression willpropelfurtherstudiesoftheirfunction,and may pathway componentsinstemcellpluripotency andcancer require additionalinvestigation. Finally, thepotentialrolesofNodal and/or synergistic functions ofthecanonicalWntpathway will Furthermore, thecross-talkbetweenthispathway andthe parallel activity inspecific tissuesand/ordevelopmental stages. undoubtedly employ precisegenetictoolstoremove pathway distinct pathway functionsinspaceandtime,futurestudieswill mechanisms remainunanswered.Given thecloseappositionof important questionsregarding itsfunctionsand molecular Despite two decadesofstudytheNodalpathway, numerous Conclusion activity. oncogenic effects of present, however, itremainsunknown whetherany ofthe an epithelial-mesenchymaltransition(Strizzietal.,2004).At Adachi, H.,Saijoh,Y., Mochida,K.,Ohishi,S.,Hashiguchi,H.,Hirao,A.and References Program. National InstitutesofHealthandbytheNewJerseyStemCellResearch manuscript. Work onNodal signalinginmylaboratoryissupportedbythe Kruithof-de Julio,AlexSchierandPatrickTam forhelpfulcommentsonthe length constraints.IamindebtedtoCoryAbate-Shen,RayHabas,Marianna I apologizetonumerous colleagues whoseworkcouldnotbecitedowingto analyses oftransgenicmicehave suggestedthat signaling mayfacilitate cancerprogressionremainunclear, but 2003; Sunetal.,2005).ThemechanismsbywhichNodal tumorigenesis inxenografts andtransgenicmice(Adkins etal., 2005), andwiththeabilityof Cripto 2006). Thesefindings areconsistentwiththeupregulation of decreases tumorigenicityinxenograft assays(Topczewska etal., is correlatedwithcancerprogression,whereaspathway inhibition particular, increasedexpression of pathway activity isupregulated inmany humancancers.In during laterdevelopment andadulthood,thereisevidence that Although mostgenesintheNodalpathway arerarelyexpressed Potential role incarcinogenesis 2006). et al.,2001;Camus2006;Ding1998;Mesnard pluripotency andprevent precociousneuraldifferentiation (Brennan findings thatNodalsignalinginvivo isrequiredtomaintainepiblast (James etal.,2005).However, theseobservations areconsistentwith suggesting possiblespecies-specific differences inNodalfunction treatment ofmouseEScellsdoesnotyieldthesameeffects, (James etal.,2005;Vallier etal.,2005).Bycontrast,similar ( renewal andlossofexpression ofthepluripotency regulators inhibition ofpathway activity leadstodecreasedstemcellself- surface ofembryoidbodies(Vallier etal.,2004).Conversely, the simultaneously promotingvisceralendodermdifferentiation atthe culture), andmaintainscellsintheundifferentiated state,while bodies (formedfromthree-dimensionalaggregates ofEScellsin human EScellsinhibitsmesodermdifferentiation withinembryoid cells (Brandenberger etal.,2004).Overexpression ofNodalin Development 134(6) Adkins, H.B.,Bianco, C.,Schiffer, P., S.G.,Rayhorn, Zafari,M.,Cheung,A. POU5F1 Genes Dev. by aleftside-specificenhancerwithsequence similaritytoalefty-2enhancer. Hamada, H. E., Orozco, O.,Olson,D.,DeLuca,A.,Chen, L.etal. that isobserved inmany epithelialcancers(Strizzietal., – HumanGeneNomenclatureDatabase)and 13 (1999). Determinationofleft/rightasymmetric expression ofnodal , 1589-1600. Cripto are dependentonNodalpathway Cripto Nodal overexpression topromote in malignantmelanoma Cripto (2003). Antibody can induce NANOG OCT4 Chen, Y. andSchier, A.F. Brandenberger, R.,Wei, H.,Zhang,S.,Lei,Murage,J.,Fisk,G.Li,Y., Cheng, S.K.,Olale,F., Bennett,J.T., Brivanlou,A.H.andSchier, A.F. Chen, Y. andSchier, A.F. Chen, S.andKimelman,D. Chen, C.,Ware, S.M.,Sato,A.,Houston-Hawkins, D.E.,Habas,R.,Matzuk, Chen, C.andShen,M. Chea, H.K.,Wright, C.V. andSwalla, B.J. Bourillot, P. Y., Garrett, N.andGurdon, J.B. Branford, W. W. andYost, H.J. Chu, G.C.,Dunn,N. R.,Anderson,D.C.,Oxburgh,L.and Robertson,E.J. Cheng, S.K.,Olale,F., Brivanlou,A.H.and Schier, A.F. Aoki, T. O.,Mathieu,J.,Saint-Etienne,L.,Rebagliati,M.R.,Peyrieras,N.and Camus, A.,Perea-Gomez, A., Moreau, A.andCollignon,J. Brennan, J.,Norris,D.P. andRobertson,E.J. Ben-Haim, N.,Lu,C.,Guzman-Ayala, M.,Pescatore, L.,Mesnard, D., Beck, S.,LeGood,J.A.,Guzman,M.,Haim,N.B.,Roy, K.,Beermann,F. and Ashe, H.L.andBriscoe,J. Bertocchini, F., Skromne, I.,Wolpert,C.D. L.andStern, Bianco, C.,Adkins,H.B.,Wechselberger, C.,Seno,M.,Normanno,N.,De Branford, W. W. andYost, H.J. Agius, E.,Oelgeschlager, M.,Wessely, O.,Kemp,C.andDeRobertis,E.M. Andersson, O., Reissmann, E., Jornvall, H.andIbanez,C.F.Andersson, O.,Reissmann,E.,Jornvall, Bertocchini, F. C.D. andStern, Brennan, J.,Lu,C.C.,Norris,D.P., Rodriguez,T. A.,Beddington,R.S.and Birsoy, B.,Kofron, M.,Schaible,K.,Wylie,C.andHeasman,J. Bianco, C.,Strizzi,L.,Rehman,A.,Normanno,N.,Wechselberger, C.,Sun,Y., squint-mediated nodalsignaling. Nodal signaling. differentiation. elucidates signalingnetworksthatcontrol humanEScellgrowth and Xu, C.,Fang,R.,Guegler, K.etal. 2167-2180. gradient isinterpreted bycontinuoustransductionflow. GDF1. EGF-CFC proteins are essentialcoreceptors for theTGF-betasignalsVg1and morphogen. embryo. Nodal signalingpromotes precocious neuraldifferentiation inthemouse left-rightasymmetry.governs layer patterning inzebrafish. layer patterning Development acts inaNodalsignalingpathwaythepre-gastrulation mouseembryo. M. M.,Shen,andBrown, C.W. evolution ofdeuterostome gastrulation. Curr. Biol. wnt-responsive organizergeneexpression isessentialfor normalgastrulation. 20. 215-226. subset ofTGFbetasignalsbyantagonizing EGF-CFCcoreceptors. Invest. blockade oftheCriptoCFCdomainsuppresses tumorcellgrowth invivo. mouse embryo. Robertson, E.J. by TARAM-A, aTGFbeta-related typeIreceptor. Rosa, F. M. Biol. interaction betweenGdf1andNodalduringanterioraxisdevelopment. source ofitsconvertasesandBMP4. Nodal precursor actingviaactivinreceptors inducesmesodermbymaintaininga Bischofberger, M.,Naef,F., Robertson,E.J.andConstam,D.B. during gastrulation. Constam, D.B. Development Development acting sequentiallyduringprimitivestreak formationinthechickembryo. of embryonicpolarityinaregulative system:evidenceforendogenousinhibitors 744. mammary epithelialcells. activates Nodal-andALK4-dependent-independentsignalingpathwaysin Luca, A.,Sun,Y., Khan,N.,Kenney, N., Ebert, A.etal. of antagonismdecoded. an essentialsignalingmoleculeinXenopusdevelopment. Development (2000). EndodermalNodal-related signalsandmesoderminductioninXenopus. positions theprimitivestreak byantagonizingnodalsignaling. and c-Src. ALK4-independent signalingpathwayactivatedbyCripto-1through Glypican-1 Khan, N.,Hirota, M.,Adkins,H.,Williams,K.etal. 293 Genes Dev. 112 , 370-381. Dev. Biol 12 Cancer Res. , 575-587. (2002). Regulationofnodalsignallingandmesendodermformation , 2136-2141. Nature 133 133 131 127 Nat. Biotechnol. Nature Curr. Biol. (2002). Extraembryonicproteases regulate Nodalsignalling . (2001). Nodal signalling in the epiblast patterns theearly (2001). Nodalsignallingintheepiblastpatterns , 319-329. , 385-394. , 3381-3390. , 1173-1183. 295 17 Nat. CellBiol. 411 , 31-36. , 743-755. 63 411 , 607-610. (2002). Leftyproteins are long-rangeinhibitorsof (2001). ThezebrafishNodalsignalSquintfunctionsasa Curr. Biol. , 1192-1197. Mol. Cell.Biol. (2004). Two modesbywhichLeftyproteins inhibit (2006). Theinterpretation ofmorphogengradients. 14 (2000). Therole oftheyolk syncytial layeringerm , 965-969. Development , 618-624. Genes Dev. (2002). Thehypoblastofthechickembryo (2002). Lefty-dependentinhibitionofnodal-and (2004). Nodalsignaling:CrypticLeftymechanism 22 Curr. Biol. 4 , 707-716. 14 , 981-985. (2004). Transcriptome characterization Dev. Cell , R341-R343. Dev. Dyn. 22 (2006). TheVg1-related protein Gdf3 16 127 (2005). Nodalsignalingandthe , 2586-2597. 12 , 2339-2344. (2002). 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DEVELOPMENT Gripp, K.W., Wotton, D., Edwards, M.C.,Roessler, E.,Ades,L.,Meinecke,P., Gritsman, K.,Zhang,J.,Cheng,S.,Heckscher, E.,Talbot, W. S.andSchier, A. Grimm, O.H.andGurdon, J.B. Green, J.B.andSmith,C. Gore, A.V., Maegawa,S.,Cheong, A.,Gilligan,P. C.,Weinberg, E.S.and Germain, S.,Howell,M.,Esslemont,G.M.andHill,C.S. Feldman, B.,Concha,M.L.,Saude,Parsons,J.,Adams,R.Wilson,S. Chu, J.,Ding,Jeays-Ward, K.,Price,S.M.,Placzek,M.andShen, 1032 Dougan, S.T., Warga, R.M.,Kane,D.A.,Schier, A.F. andTalbot, W. S. Gritsman, K.,Talbot, W. S.andSchier, A. F. Dorey, K.andHill,C.S. Ding, J.,Yang, L.,Yan, Y. T., Chen,A.,Desai,N.,Wynshaw-Boris,A.and Dickmeis, T., Aanstad,P., Clark,M.,Fischer, N.,Herwig,R.,Mourrain,P., Flowers, V. L.,Courteau,G.R.,Poustka,A.J.,Weng, W. andVenuti, J.M. de laCruz,J.M.,Bamford, R.N.,Burdine, R.D.,Roessler, E.,Barkovich,A.J., Conlon, F. L.,Lyons, K.M.,Takaesu, N.,Barth,K.S.,Kispert,A.,Herrmann, Conlon, F. L.,Barth,K.S.andRobertson,E.J. 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