Frizzled geneshave beenfoundinmetazoans(Adelletal.,2003; as Wntreceptors(Bhanotetal.,1996).Subsequently, atleast20 regulators oftissuepolarity(Adler, 1992),beforebeingrecognized Malbon, 2004).Frizzledgeneswerefirstidentifiedin are generallycoupledwithGproteins(Wang etal.,1996;Wang and proteins belongtoafamily ofseven-pass cell-surface receptorsthat process inseaurchinembryos. suggesting thatWntsignalingmightalsobeimportantin that have beenshown toplaycentral rolesingastrulationmovements, islimited.Nevertheless, inotherorganisms, Wntpathways control archenteroninvagination andelongationinseaurchin 2004); however, informationaboutthemolecularmechanismsthat primary (forareview, seeKominami andTakata, elongation). ItiswidelyacceptedthattheSMCsaretriggerof exerted bytheSMCsattipofarchenteron(secondary Finally, thearchenteronispulledtostomodealareabytraction display convergent-extension movements (primaryelongation). archenteron extends byrearrangementsofendodermal cellsthat form aprimitive archenteron(primaryinvagination). Next, thisshort cells andsecondarymesenchyme(SMCs),bendsinwards to First, thevegetal plate,whichiscomposedofspecifiedendodermal development ofthearchenteronthroughthreesuccessive phases. the ingressionofprimarymesenchymecells(PMC),followed bythe adhesion. Inseaurchin,gastrulationbegins atthevegetal polewith movements thatrequirechangesincellpolarity, motilityand three germlayersbecomeorganized throughmorphogenetic During embryogenesis,gastrulationisthecrucialstepwhen INTRODUCTION Key words: Seaurchin, Frizzled,Wnt,PCPpathway, Notchsignaling,,Primaryinvagination the resultssuggestthatFz5/8playsacrucialrolespecificallyinSMCstocontrolprimaryinvaginationduringseaurchingast Furthermore, amongthethreeknownWntpathways,Fz5/8appearstosignalviaplanarcellpolaritypathway.Takentogether, archenteron, maintenanceofendodermalmarkerexpressionandapicallocalizationNotchreceptorsincells. specification ofthemainembryonicterritories.Rather,itappearstoberequiredinSMCsforprimaryinvagination mesenchyme cells(SMCs).Loss-of-functionanalysesinwholeembryosandchimerasrevealthatFz5/8isnotinvolvedthe 60-cell stageintheanimaldomainandthenfrommesenchymeblastulastage,bothasubsetofsecondary Frizzled family,Fz5/8,duringseaurchinembryogenesis.Duringdevelopment,Fz5/8displaysrestrictedexpression,beginningat are transducedbyFrizzledproteins,thecognatereceptorsofWntligands.Thisstudyfocusesonroleamemberth Wnt signalingpathwaysplaykeyrolesinnumerousdevelopmentalprocessesbothvertebratesandinvertebrates.Theirsignals Jenifer Croce development initiate archenteroninvaginationduringseaurchin Frizzled5/8 isrequiredinsecondarymesenchymecellsto Development 133,547-557doi:10.1242/dev.02218 Accepted 23November 2005 2 1 *Author forcorrespondence (e-mail:[email protected]) Durham, NC27708,USA. Curie, Observatoire Océanologique,06230Villefranche-sur-Mer, France. Developmental, CellandMolecularBiology Group, Box91000,Duke University, Unité deBiologieduDéveloppement,UMR 7009,CNRS,UniversitéPierre etMarie Wnt ligandssignalbybindingtoFrizzledreceptors. 1,2 , LouiseDuloquin 1 , GuyLhomond Drosophila 1 , DavidR.McClay as pathway (forareview, seeWeidinger andMoon,2003). 2003), andthepathways frequentlyantagonizethe though they useDishevelled (Kühl etal.,2000b;Sheldahl characterized. Functional analyseshave demonstratedthat the whereas several componentsofthecanonicalpathway have been Wallingford etal.,2000;Wallingford etal.,2001). during gastrulation(Heisenberg etal.,2000;Kilian 2003; vertebrates asaregulator ofconvergent-extension movements determination (Shulmanetal., 1998) but isnow alsorecognizedin these pathways signalina modulating cellfate decisions(Torres etal.,1996).Furthermore, playing importantrolesincellpolarityandmigration,ratherthan characterized, thenon-canonicalpathways appearclearlytobe invertebrates (Cadigan,2002;Giles etal.,2003).Althoughlesswell- and frequentlycontrolscellfate specificationinbothvertebrates and understood. Thispathway includes pathway. Ofthese,the canonical pathway iscurrentlythebest intracellular Ca first identifiedin associated-kinase (ROCK) andJun-N-terminal-kinase (JNK).Itwas including RhoAandRac,thatactivate effectors suchasRho- al., 2002).ThePCPpathway functionsthroughsmallGTPases, (Kühl etal.,2000a;Kühl2000b;2001;Pandur et regulate cellmigrationduringgastrulationandheartdevelopment among otherrolestofunctionduringdorsoventral patterningandto II (CAMKII)(Sheldahletal.,2003).Thispathway hasbeen shown including proteinkinaseC(PKC)andcalmodulin-dependent Wnt/Ca signaling pathways: thecanonical(or 2004). cell adhesion,planarpolarityandapoptosis(HuangKlein, rearrangements, decisions andcontrolofproliferationtocytoskeletal fate receptors regulate diverse cellular processes, rangingfromcell Wang etal.,1996).Together withtheirWnt ligands,Frizzled The Wnt/Ca In seaurchin,littleisknown aboutthenon-canonicalpathways, Upon activation, Frizzledreceptorscansignalviathreedistinct 2+ (Wnt/Ca 2 and ChristianGache 2+ 2+ pathway actsbymeansofaGproteintostimulate release andactivate Ca 2+ Drosophila ) pathway andtheplanarcellpolarity(PCP) ␤ -catenin-independent manner, even for itsroleinplanarpolarity ␤ RESEARCH ARTICLE -catenin asakey intermediate 1, * ␤ 2+ -catenin) pathway, the -dependent enzymes, rulation. ␤ -catenin e the 547

DEVELOPMENT sequence KTXXXW was introducedbyPCRtogive pCS 261 to556frompCS P. lividus were culturedinartificialseawater at23°C. Treatments wereperformedon Marine Laboratory(Beauford,NC)andSeaLife(Tavernier, FL).Embryos Lytechinus variegatus (France). EmbryoswereculturedinMilliporefilteredseawater at18°C. Paracentrotus lividus , cultures andtreatments MATERIALS ANDMETHODS archenteron viasignalingthroughthePCPpathway. required intheSMCstocontrolprimaryinvagination ofthe Frizzled receptorFz5/8.Functionalanalysesshow thatFz5/8is functional datafortheseproteinshasbeenobtainedthusfar. protein (Ransicketal.,2002)–have beendescribed,althoughno Frizzled relatedprotein(Illiesetal.,2002)andapartialFrizzled-like 2004). Two proteinsrelatedtotheFrizzledfamily –asecreted- maternally nuclear progression ofendomesodermspecificationdownstream ofthe in thevegetal poleareaoftheearlyembryoandisrequiredfor role was notinvestigated (Ferkowicz etal.,1998).Wnt8isexpressed Wnt5 have beenidentifiedinseveral species,thoughtheirfunctional (Logan etal.,1999). independently ofanupstreamWntligandorFrizzledreceptor it appearsthatthispathway actsearlyandautonomously, et al.,2005;Vonica etal.,2000;Weitzel etal.,2004).Furthermore, vegetal axis(Emily-Fenouiletal.,1998;Logan1999;Range canonical pathway controlscellfate determinationalongtheanimal- 548 were clonedinpCS After PCRamplification,Fz(entire Fz5/8ORF)andFzE(codons1-230) Plasmid constructionsandinvitro transcription Zeiss confocallasermicroscope. a Zeissinverted microscope,whileimmunolabelingwereimagedwitha McClay, 1997).Bright-fieldandepifluorescenceimageswereacquiredwith Immunofluorescent analyseswerecarriedoutasdescribed(Sherwood and Antibody staining cell stagewith andvegetal halftransplantationswere performed atthe16-or32- Transplantation experiments al., 2003).Theprobewas theFz5/8full-length cDNA labeledwith Northern blotanalysiswas performedas describedbyCroceetal.(Croce Expression analysis cDNA encodinganORFof1668bp. stage cDNA library. Thelongestcloneisolatedwas identifiedasa5199bp P. lividus random screenbywhole-mountinsituhybridizationonacDNA libraryfrom A 2kbfragmentencodingaFrizzledreceptorproteinwas isolatedduringa Cloning ofFz5/8 Bisindolylmaleimide I(Calbiochem)dilutedfromstocksinH (Ghiglione etal.,1993)orwithY27632,JNKInhibitorI(L)-Form or pCS and usedat75 cDNA. Otherprobesweresynthesizedfrom et al.(Croceal.,2003).Fz5/8probecorrespondstothefull-length random primingusingthePrime-a-GeneLabelingSystem(Promega). 2001b) and and Gache,1990), (Croce etal.,2001a), Whole-mount insituhybridizationwas carriedoutasreportedbyCroce In thisstudy, wereportthe characterizationofaseaurchin Four seaurchinWntshave beenreportedtodate.Wnt1,Wnt4and 2+ FzE, respectively. pCS RESEARCH ARTICLE 60-cell stageembryos.Thisfragmentwas usedtoprobeagastrula embryos withLiClorZnCl LvDelta ␮ L. variegatus M, 50 Goosecoid 2+ (Sweet etal.,2002)genes. L36 2+ (Turner andWeintraub, 1994)togive pCS ␮ was collectedinthebayofVillefranche-sur-Mer ␤ animals wereobtainedfromtheDuke University Fz. ApointmutationchangingW intoGinthe M or3 -catenin signaling(Wikramanayake etal., (T. Lepage,unpublished), embryos asdescribed(Loganetal.,1999). 2+ , Coquillette FzTM1 was obtainedbydeletionofcodons ␮ M, respectively. 2 , asdescribedbyGhiglioneetal. , AA29 HE (Hatching enzyme)(Lepage (Croce etal.,2003),s Brachyury 2+ 2 (Croce etal., O orDMSO Fz-W521G. 2+ Fz and 32 P by ke-T ClustalX andthe tree wasdrawnwithTree View. proteins from seaurchin andotherorganismswere alignedusing members oftheFrizzledfamily. Amino acidsequencesofFrizzled oan lc,KXX oi.( domain; black,KTXXXWmotif. signal peptide;red, cysteine richdomain;green, transmembrane 1999). was usedasindicatedbySherwood andMcClay(Sherwood andMcClay, Emily-Fenouil etal.(Emily-Fenouilal.,1998).pBsNotchactivated form mMessage mMachinekit(Ambion).pCS (Tolwinski etal.,2003).The5 concentration of0.3 constructs wereinjectedupto2.5 Fig. 1.Fz5/8protein. sequence KTXXXW(Fig.1A). terminal cytoplasmic domain,whichcontainsthe conserved rich domain(CRD),seven transmembranedomainsandaC- organization oftheFrizzledreceptors:asignalpeptide,cysteine- Accession NumberAM084899)withthecharacteristicdomain isolated thatencodesa556aminoacidsprotein(GenBank unpublished), followed byacDNA libraryscreen,aFz5/8clonewas During arandomscreenbyinsituhybridization(T. Lepage, Cloning andsequenceanalysisofFz5/8 RESULTS After dilutionindoubledistilledH mRNA microinjection pCS activated formattheconcentrationsindicatedabove. of FzTM1mRNA withmRNA fromFz,Notchactivated formorRhoA activated form.Doubleinjectionswereperformedbysimultaneous injection GSK3 B A 2+ FzLRP5/6 was obtainedbyPCRasreportedTolwinski etal. ␤ ; 0.7 ␮ R 7 6 5 4 3 2 1 CRD S g/ ␮ l forNotchactivated form;and0.026 ␮ g/ ( A ␮ ) Schematicrepresentation ofFz5/8.Blue, l forFzE;0.55 Ј capped mRNAs weregeneratedwiththe ␮ B g/ ) Cladogramofrepresentative ␮ 2 l. OthersmRNAs wereusedatafinal O, Fz,Fz-W521GandFzLRP5/6 ␮ 2+ g/ GSK3 ␮ FZD7 Xenopus FZD7 Mouse FZD7 Human FZD2 Xenopus FZD2 Mouse FZD2 Human FZD1 Xenopus FZD1 Mouse FZD1 Human FZD6 Mouse FZD6 Human FZD3 Xenopus FZD3 Mouse FZD3 Human Fz5/8 SeaUrchin FZD5 Mouse FZD5 Human FZD5 Xenopus FZD8 Xenopus FZD8 Mouse FZD8 Human FZD4 Xenopus FZD4 Mouse FZD4 Human FZD10A Xenopus FZD10 Mouse FZD10 Human FZD9 Mouse FZD9 Human l forFzTM1;0.5 ␤ Development 133(3) has beendescribedby ␮ g/ ␮ l forRhoA ␮ g/ ␮ l for

DEVELOPMENT SMCs. Finally, throughoutgastrulationanduntil pluteusstage, results indicatethatFz5/8isexpressed inaninnersubsetofthe endodermal cellsandexternal tothesmallmicromeres,these secondary mesenchymecells (SMCs)areinternaltothe the two ringsseparated by two orthreerows ofcells.Asthe ring was concentricwith,and internalto,theBrachyuryring,with Gross andMcClay, 2001).Indouble-stainedembryos,theFz5/8 / boundaryatthisstage(Croceetal.,2001b; performed withBrachyury, whichisexpressed atthe determine theidentityofthesecells,anotherdoublelabeling was domain thatformsaringofcellscenteredonthevegetal pole.To blastula stage(MB),Fz5/8begins tobeexpressed inasecond (Angerer andAngerer, 2003;Takacs etal.,2004).Atmesenchyme which correspondstotheanimal(orapical)poledomain(ApD) that Fz5/8isexpressed intheanimal-mostregion oftheembryo, (PMC) (Croceetal.,2001a;Fuchikami2002),established specific marker ofthepresumptive primarymesenchymecell blastula stage(HB).Atthisstage,adoublelabelingwithske-T, a progressively reducedtoasmallregion oftheembryoathatched hemisphere. Duringcleavage, thisexpression domainwas oppositetothemicromeres,andthusanimal cell stage,Fz5/8expression was restrictedtoaregion ofthe Fz5/8 transcriptswereuniformlydistributed (Fig.2B).Atthe60- mount insituhybridization.Inegg andduringearly , cleavage andgastrulation. embryogenesis, withtwo majorphasesofexpression during Fz5/8 transcriptsaredetectableathigherlevels throughout kb) was detected(Fig.2A).Presentatlow levels inunfertilizedeggs, was performed.Asingle,appropriatelysizedFz5/8transcript(~5 To assessthetemporalexpression ofFz5/8,northernblotanalysis embryogenesis Fz5/8 expressionduringseaurchin pattern Frizzled 8(Fig.1B). later duringevolution tocreatetheparalogsFrizzled5and ortholog ofanancestralgeneFrizzled5/8thathasbeenduplicated those two subfamilies, suggestingthatFz5/8mightbethe Phylogenic analysisshows thatFz5/8branchesatthebaseof urchin proteinhasstrongsimilaritytoFrizzled5and8. sequences fromhuman,mouseand Fz5/8 required inSMCforprimaryinvagination The spatialexpression ofFz5/8was investigated bywhole- Alignment ofthepredictedaminoacidsequencewithFrizzled Xenopus showed thatthesea the top. mesenchyme blastula;Pr, prism.Lateral viewswiththeanimalpoleat hybridized withFz5/8antisenseprobe. HB,hatchedblastula;MB, 30 mMlithiumchloride.Allembryos were fixedatindicatedstagesand type formofGSK3 treatment with0.1mMzinc chlorideorbyoverexpression ofthewild- ( ofFz5/8 duringperturbeddevelopment. Fig. 3.Expression (Emily-Fenouil etal.,1998).AtHB,inbothZn treatment orbyoverexpression ofthewild-typeformGSK3 embryos thatcompletelylackendomesodermwereproducedbyzinc evaluated inanimalizedandvegetalized embryos.Animalized establishment oftheanimal-vegetal polarity, Fz5/8expression was To assesswhetherthespatialrestrictionsofFz5/8arelinked tothe patterning Fz5/8 expressionissensitivetoaxis pattern territory. in two restrictedareas,firstintheApDandthenSMC the archenteron.Thus,duringembryogenesis,Fz5/8isexpressed Fz5/8 transcriptsaredetectablebothintheApDandattipof stages (equivalent ofMBandgastrula)(notshown), establishingthat of theembryo(Fig.3A).Thisexpression patternpersisted atlater extended toward thevegetal pole,covering approximatelytwo-thirds GSK3 A Fz5/8expression inanimalized embryosobtainedeitherby ) ␤ -injected embryos,theanimalexpression domainofFz5/8 ␤ . ( B ) Fz5/8expression inembryos vegetalizedwith G, gastrula;Pr, prism;P, pluteus. MB, mesenchymeblastula;EG,earlygastrula; blastulastages;HB,hatchedblastula; B1-5, egg; 16,16-cellstage;60,60-cell with theanimalpoleattop.E,unfertilized with ske-TorBrachyuryprobes). Lateralviews (where Fz5/8probe wasusedsimultaneously antisense probe, exceptfordoublelabeling indicated stagesandhybridizedwithFz5/8 hybridization. Allembryoswere fixedat transcripts revealed bywhole-mountinsitu stages. ( total RNAisolatedfrom embryosatindicated ( of Fz5/8duringembryogenesis. Fig. 2.Spatiotemporalexpression pattern A Temporal blottingwith analysisbynorthern ) B ) SpatialdistributionofFz5/8 RESEARCH ARTICLE 2+ -treated and 549 ␤

DEVELOPMENT same cellularcompartment. membrane andwillthuscompetewithendogenousreceptorsinthe Unlike thesecretedversion, FzTM1proteinisretainedinthe same extracellular region plusthefirsttransmembranedomain. interacting withtheendogenousreceptor. FzTM1iscomposedofthe FzE proteinissecretedandbindstheWntligand,preventing itfrom of thereceptor, includingthepeptidesignalandCRDdomain. 1998; Kimetal.,2002).FzEcorrespondstotheextracellular region fz8a, shouldactinadominant-negative fashion (Deardorff etal., (Fig. 4A),thatbyanalogywithsimilartruncatedformsofxfz8and terminal truncatedformsofFz5/8,FzEandFzTM1,wereemployed To investigate thefunctionofFz5/8duringembryogenesis,two C- skeletogenesis Loss ofFz5/8functionaffects gastrulationand SMCs. previous conclusionthatatthevegetal poleFz5/8istranscribedin perturbations alongtheanimal-vegetal axis,andstrengthenthe distinct transcriptionregulatory modulesthatresponddifferently to suggest thatthetwo Fz5/8expression domainsareregulated by expression, itdidnotaffect vegetal expression. Together, thesedata vegetalization oftheembryoscompletelyabolishedFz5/8animal expressed inendodermbut ratherinSMCs.Thus,whereas et al.,2003)(notshown). Thisindicatesthusthat Fz5/8isnot display thewell-known LiClphenotype(Croceet al., 2001b;Croce morphological level, theseembryosareandatpluteusstagewill at prismstageexogastrulation isnoteasilydetectableatthe delaminating cellspresentatthevegetal pole(Fig.3B).Although surrounding thevegetal pole,andthen,atprismstage,in At MB,Fz5/8transcriptsweredetectableinaringofcells 3B). Bycontrast,thevegetal expression ofFz5/8was unaffected. expression ofFz5/8was undetectablefromHBto prism stage(Fig. mesodermal territories.Invegetalized embryos,theanimal expense oftheectoderm,but withoutapparentalterationofthe chloride treatment,whichinducesanincreaseinendodermatthe vegetal one. expression, expanding theanimalcomponentandsuppressing embryos. Thus,animalizationperturbedbothdomainsofFz5/8 Fz5/8 transcriptioninthevegetal hemispheredidnotoccurinthese 550 Vegetalization oftheembryoswas accomplishedbylithium RESEARCH ARTICLE the morphologicalpolarityisnotalonganimal-vegetal axis.To non-gastrulating spheres,but never inboth(Fig.5B),indicatingthat tracer was observed ineitherthethickorthinepitheliumof vegetal axis,asexpected (Fig.5B).InFzTM1-injectedembryos,the the left-rightaxes (about70%ofembryos),but notalongtheanimal- asymmetric eitheralongtheoral-aboral(about30%ofembryos)or one ofthetwo .Incontrolplutei,thetracerwas eggs, thenatthetwo-cell stage,alineagetracerwas introducedin distinguish thesepossibilities,FzTM1mRNA was injectedinto aboral axis,eachofwhichpossessanasymmetrycilia.To represented apolarizationabouttheanimal-vegetal axisortheoral- shown). Basedonmorphology, itwas unclearwhetherthis cilia (Fig.5A)(ciliamotilitywas observed bystroboscopy, not long non-motileciliaandathinepitheliumcovered withshortmotile a pronouncedectodermalpolarity, withathickepitheliumbearing At pluteusstage,FzTM1-injectedembryosaresphericalandpossess Polarity ofFzTM1-injectedlarvae required forgastrulationandskeletogenesis. these resultsstronglysuggestthatFz5/8signalingisspecifically resulted specificallyfromthelossofFz5/8signaling.Taken together, indicating thattheperturbationscausedbyFzTM1overexpression embryos containedfulltripartitearchenteronsandnormalskeletons, to larvae in95%oftheinjectedcases(Fig.4C). Theserescued injected withFzmRNA andtheresultingembryosdevelop normally mRNA encodingFzTM1,atlevels thatinhibitgastrulation,wereco- developmental defectswereobserved. Inexperimental injections, concentrations, untilcompromisingembryoviability, andno controls, mRNA encodingFzwas injectedatincreasinglyhigher wild-type Fz5/8(Fz)torescuetheFzTM1phenotypewas tested.In lack botharchenteronsandblastopores(Fig.4B5,B6). which pigmentcellsareinserted.They have few ornospiculesand thick epitheliumcovered bylongcilia,andathinepitheliumwithin At pluteusstage(Fig.4B4),injectedembryosaresphericalwitha PMCs withintheblastocoel,but thevegetal platefails toinvaginate. embryos have notgastrulated(Fig.4B2,B3).Theseembryoshave full-length archenterons(Fig.4B1),FzE-orFzTM1-injected defects (Fig.4B).Atlategastrulastagewhencontrolembryoshave To ascertainwhethertheseeffects werespecific,theabilityof Overexpression ofFzEandFzTM1causethesamemorphological injection ofFzTM1andFzina1:2ratio(right). injection ofFzTM1alone(middle),andco- ( FzTM1. B1-B3,gastrulastages;B4-B6,pluteus. associated withtheoverexpression ofFzEor transmembrane domain.( peptide; CRD,cysteinerichdomain;TM, encoded bycDNAconstructs.S,signal type ordominant-negativeformsofFz5/8 during embryogenesis. Fig. 4.PerturbationofFz5/8function C ) RescueofFzTM1phenotype.Control (left), Development 133(3) B ( A ) Phenotypes ) Structure wild-

DEVELOPMENT FzTM1-injected embryos, oralview(middle)andtransverse view(right). et al.,2004);green, counterstain. Control embryo,animalpoleview(left); immunostaining onFzTM1-injected larvae.Red,1e11antibody(Nakajima two blastomeres atthetwo-cell stage.( morphological polarityofFzTM1-injected larvaebylabelingoneofthe (right) imagesofFzTM1-injectedlarvae. ( to suggestthatitisanindirectconsequenceofFz5/8expression in At present,wehave noexplanation forthisobservation, otherthan 70% oral/aboralversus left/rightfate predictedby the firstcleavage. display oral/aboralentrainmentinsteadofobtainingthe30%versus the FzTM1expression isalsotoinduce100%oftheembryos larvae displayoral-aboralpolarity. Intriguinglyenough,aneffect of ciliated band.Theseobservations indicatethat FzTM1-injected thick epitheliumincludesneurons,itislikely toberelateda and axonsonlyinthethickepithelium.Thus,becauselarvae the 2004). InFzTM1-injectedlarvae, 1e11stainingrevealed nerve cells that arepresentonlyintheoralectoderm(Fig.5C)(Nakajimaetal., 1e11-stained nerve cellsthatareintheciliaryband,andtheiraxons were immunostainedfortheneuralmarker 1e11.Incontrollarvae, determine whetherthispolarityisoral-aboralorleft-right,embryos Fz5/8 required inSMCforprimaryinvagination Fig. 5.PolarityofFzTM1-injected larvae. C B ) Confocalimagesofneurons ) Determinationofthe ( A ) DIC(left)anddark-field function was blocked onlyintheanimal orthevegetal hemisphere experiments. Chimeric embryos weregeneratedsuchthatFz5/8 assessed Fz5/8functionin each domainbytransplantation As Fz5/8transcriptsarepresent inanimalandvegetal domains,we invagination Fz5/8 functionisrequired inSMCforarchenteron formation ofthearchenteron. of expression oflateendodermalmarkers associatedwiththe endoderm territory, but show thatitisrequiredforthemaintenance involved inthespecificationofmainterritories,including marker expression. Together, thesedatasuggestthat Fz5/8isnot specification, itappearstobenecessarymaintainendodermal 6T). Thus,althoughFz5/8isnotrequiredforendoderm 6P,T), andonlythe oral expression ofBrachyurywas detected(Fig. staining was observed inFzE-orFzTM1-injectedembryos(Fig. Logan etal.,1999).However, atlategastrulastage,novegetal by thematernalWnt/ and thereforewellafterendodermspecificationhadbeenlaunched Fz5/8 isexpressed inthevegetal hemispherebeginning onlyatMB to triggerendodermspecification.Theseresultswereexpected as loss offunction(Fig.6L,N,R),indicatingthatFz5/8isnotrequired expression ofbothL36andBrachyurywas notmodifiedbyFz5/8 Gross andMcClay, 2001)(Fig.6Q,S).AtMB,notsurprisinglythe the vegetal plateandthenaroundtheblastopore(Croceetal.,2001b; expressed inthestomodeumand,adynamicpattern, firstwithin the endoderm(Fig.6K,M,O).Duringsameperiod,Brachyuryis Brachyury, was tested.L36isexpressed throughoutgastrulationin Fz5/8 doesnotappeartobeinvolved inPMCspecification. 6I,J). Thus,althoughFz5/8loss-of-functionblocksskeletogenesis, organized incomparisonwiththePMCswithincontrolembryos(Fig. cells expressing ske-T thancontrols,andthesecellsalsofail tobecome (not shown). However, atgastrulastage,injectedembryoshave fewer expression ofske-T isnotalteredbyFzEorFzTM1overexpression (Croce etal.,2001a;Fuchikami2002).Inblastulae,the expressed inPMCsandtheirprecursorsfromblastulato gastrulastage pigment cellsinFzTM1-expressing larvae (Fig.4B). or differentiation, consistentwiththepresenceofSMC-derived Thus, Fz5/8doesnothave arequiredfunction inSMCspecification AA29-positive cellsthatarepresentwithintheblastocoel(Fig.6H). archenteron (Fig.6G).Atthesamestage,injectedembryosalsohave AA29 transcriptsarenormallylocalizedtotheSMCsattipof ectoderm specificationorpatterning(Fig.6A-F). FzTM1-injected embryos,indicatingthatFz5/8isnotrequiredfor Expression patternsofthosethreegeneswereunchangedinFzE-or (Angerer etal.,2001;Croce2003;Gross2003). and presumptive ectoderm(LepageandGache,1990),while Fz5/8 functionwas impaired(Fig.6).The territories was studiedbywhole-mountinsituhybridizationafter expression ofmolecularmarkers specificforthemain embryonic To determinewhetherFz5/8isrequiredforcellspecification,the maintenance ofendodermalmarkerexpression of themainterritoriesbutisrequired for Fz5/8 signalingisnotinvolvedinthespecification al., 1997). depends onsignalsfromthevegetal hemisphere(Wikramanayake et the vegetal hemisphere,asitisknown thatoral/aboralpolarity Finally, theexpression oftwo endodermalmarkers, L36and The T-box transcriptionfactor ske-T (T-brain) isspecifically For SMCsandtheirprecursors,AA29was used.At gastrulastage, coquillette (Tbx2/3) labeloralandaboralectoderm,respectively ␤ -catenin pathway (Davidson etal.,2002; RESEARCH ARTICLE HE gene labelsthe goosecoïd 551

DEVELOPMENT 552 specification and differentiation (Sherwood andMcClay, 1999; two arerequiredintheSMCs tomediate,viaDeltaactivation, SMC In theseaurchin,Notchisinvolved inthreedistinctevents. Thefirst pathway Relationship betweenFz5/8 andNotchsignaling elsewhere, andtherefore isnon-autonomous. indicating thattheskeletogenic defectoriginatesnotinthePMCs, but embryos thatwereindistinguishablefromcontrols(notshown), skeletogenic defectarisefromthePMCs.Such chimerasgave riseto produced withFzTM1presentonlyinmicromerestotestwhether the invagination andprevent skeletogenesis. Inaddition,chimeraswere normally expressed inSMCs,issufficient toblockarchenteron that inhibitionofFz5/8inthevegetal hemisphere, whereFz5/8is produced, skeletons werenot(Fig.7B12).Thus,thesefindings reveal larvae didnotproduceendodermaltissue,and,whilePMCswere (23/27; Fig.7B10,B4).Immunostainingdemonstratedthatthese phenotype asthatobserved withubiquitousFzTM1overexpression function isinhibitedinthevegetal hemisphere, producedthesame required intheanimalhalfforendodermalorskeletal development. tripartite archenteronsandskeletons (Fig.7B9).Thus,Fz5/8isnot markers furtherconfirmedthattheseembryosproducednormal (Fig. 7B8).Immunostainingwithendodermalandskeletogenic 7B7). Descendantsoftransplantedmesomeresformedtheectoderm gastrulated anddeveloped intonormalpluteuslarvae (20/24;Fig. pluteus stage(Fig.7B). normal embryo(Fig.7A).Resultingchimeraswereobserved at recombined withthecomplementaryanimalorvegetal halffroma animal andvegetal halves fromthoseembryoswereisolatedand eggs witharedlineagetracer. Then,between16and32-cellstage, (Fig. 7A).mRNA encodingFzTM1wereinjected intounfertilized By contrast,thereciprocalchimericembryos,inwhichFz5/8 When FzTM1was expressed exclusively inanimalhalves, embryos RESEARCH ARTICLE pathway and theplanarcellpolarity(PCP) pathway. To identify pathways: theWnt/ Frizzled proteinscantransduce Wntsignalsthroughthreedistinct pathway Fz5/8 signalsthrough thePCP/noncanonicalWnt to rescuearchenteroninvagination downstream of Fz5/8signaling. activation ofNotchpathway using theNactconstructisnotsufficient presence ofpigmentcellsintheFzTM1phenotype.Second,ectopic mediated SMCspecificationsignal,aswas suggested bythe Fz5/8 isexpressed inSMCs,itisnotrequiredforthefirstNotch- produced inexcess (Fig.8B).Theseresultsshow firstthatalthough with noendodermalstructures,althoughpigmentcellswere and Nactgave risetoembryosthatpresenttheFzTM1phenotype, (Sherwood andMcClay, 1999)(Fig.8B).Co-expression ofFzTM1 increase ofSMCderivatives celltypes,includingpigmentcells phenotype was tested.Whenoverexpressed alone,Nactinducesan activated formofNotchprotein(Nact) torescuetheFzTM1 lack ofinvagination inFzTM1-injectedembryos,theabilityofan mesencymal apicallocalizationofNotchinendodermal cells. of Notch(Fig.8A),indicatingthatFz5/8signalingisrequiredforthe 1997). Overexpression ofFzTM1eliminatedtheapicalexpression surface ofendodermalcells(Fig.8A)(Sherwood andMcClay, in SMCs,whereasthey areexpressed athighlevels attheapical and throughoutgastrulation,Notchproteinsarenolongerdetectable expression patternwithinbothSMCsandendodermalcells. AtMB was assessed. morphogenesis, therelationshipbetweenFz5/8andNotchsignaling 2001). AsFz5/8isexpressed inSMCsandisrequiredfor endoderm gastrulation (PetersonandMcClay, 2005;Sherwood andMcClay, Sweet etal.,2002),whereasthethirdoneisimportantfor To testwhetherthislossofapicalNotchwas responsibletothe During development, Notchreceptorspresentadynamic ␤ M,N,Q,R (viewedfrom thevegetalpole). views withtheanimalpoleattopexcept hatched blastula,(G-J,O,P,S,T) gastrula.Lateral (C,D,K-N,Q,R) mesenchymeblastula,(E,F) territories, asindicated.(A,B)Earlyblastula(B3), with probes forspecificmarkersofembryonic processed forwhole-mountinsituhybridization FzTM1 were fixedattheappropriated stagesand Fz5/8 signaling. phenotype associatedwiththedisruptionof Fig. 6.Molecularcharacterizationofthe -catenin orcanonicalpathway, theWnt/Ca ( A-T ) Embryosinjectedwith Development 133(3) 2+

DEVELOPMENT Fz5/8 required inSMCforprimaryinvagination (Hardin etal.,1992). (Wessel andMcClay, 1985)andwith1d5(PMCmarker)ingreen images ofthelarvaestainedwithEndoI(endodermantibody)inred the contributionoftransplantedhalves.(B3,B6,B9,B12)Confocal (B2,B5,B8,B11) Epifluorescence imagesofthesameembryos,showing animal half.(B1,B4,B7,B10)DICimagesofresulting larvae. vegetal half;B10-B12,injectedhalftransplantedwithcontrol lineage tracer;B7-B9,injectedanimalhalfrecombined withcontrol injected embryo;B4-B6,control embryoinjectedwithFzTM1andared embryo (white).( isolated andtransplantedtothecomplementaryhalfofanormal stage, animalandvegetalhalvesofFzTM1-injectedembryos(red) were and skeletonformation. Fig. 7.Requirement ofFz5/8signalinginSMCforarchenteron B ) Experimentalresults. B1-B3,control glycerol- ( A ) Experimentaldesign.Atthe16-cell canonical Wntsignal. again, thatFz5/8isnotusedintheseaurchinforreceptionof the potential canonicalWntreceptive activity hasnoeffect, indicating, Wnt pathway iffusedtomurineLRP5/6,mutationoftheFz5/8 Thus, althoughFz5/8canbeforcedtooperatethroughthecanonical the canonicalpathway (Heetal.,2004;Umbhauer2000). have dominant-negative effects onsignalingwhentheFzisused in urchin specification,orongastrulation.Inotherorganisms, these alone, orofthetruncatedLRP5/6construct,hadnoeffect onsea (Umbhauer etal.,2000).ExpressionofthemutatedFz5/8construct KTXXXW thatisessentialforactivation ofthecanonicalpathway 2000). Second,Fz5/8was alsomutatedattheconserved sequence dominant-negative effect onthe mouse Frizzledco-receptorLRP5/6was thenusedforitsspecific vegetalizing effect (notshown). Next, atruncatedformofthe embryos, asexpression oftheLRP5/6-Fz5/8proteinhad astrongly fusion constructwas madeandfoundtobeactive inseaurchin (Tolwinski etal.,2003).Accordingly, amurineLRP5/6-Fz5/8 Fz/LRP5 fusionisaconstitutively active canonicalsignal inhibition of specific tothecanonicalpathway wereused,ratherthanadirect canonical pathway, mutated formsoftwo different receptors normally andtointerfereonlywiththezygoticformof Thus, inordertoallow endomesodermspecificationtoarise the canonicalpathway, for whichnorolehasbeenyetdescribed. could bethereceptorusedforactivation ofalaterzygoticform no effect (notshown). However, thepossibilityremainedthatFz5/8 observation thatFzTM1,whenexpressed inmicromeresonly, has endomesoderm specification.Thisconclusionisreinforcedbythe therefore, isnotnecessaryforthecanonicalpathway thatinitiates endomesoderm specificationhadalreadyoccurred.Fz5/8, starting onlyatMB,whenbothnuclearizationof eliminated. Fz5/8mRNA isfirstdetectedinthevegetal hemisphere (Logan etal.,1999),althoughtodatethispossibilitycannotbe autonomous, suggestingthatnoFrizzledreceptorsareinvolved blastomeres. Thisaccumulationhasbeendescribedasbeingcell- accumulation ofitskey components The activation ofthismaternalsignalcausesthenuclear territory andtopatterntheembryoalonganimal-vegetal axis. early, beginning atthe16-cellstage,tospecifyendomesoderm been wellreportedinthepastdecade.Thispathway isrequired function phenotype. inhibitors weretestedfortheabilitytoreproduceFz5/8loss-of- which ofthesepathways actsdownstream ofFz5/8,specific In seaurchin,theroleofmaternalcanonicalpathway has ␤ -catenin. In Drosophila form (Nact). encoding FzTM1andNotchactivated injection orco-injectionofmRNA (G) stages.( mesenchyme blastula(MB)andgastrula and FzTM1-injectedembryosat Immunolocalization performedincontrol (Sherwood andMcClay, 1999). intracellular domainofthereceptor revealed byantibodiesagainstthe images ofNotchprotein localization and Notchsignaling. Fig. 8.RelationshipbetweenFz5/8 ␤ -catenin signaling(Tamaï etal., RESEARCH ARTICLE ␤ -catenin inthemostvegetal B ithasbeenshown thatan , ) Larvaeobtainedafter ( A ) Confocal ␤ -catenin and 553

DEVELOPMENT form ofLvRhoAprotein (actRhoA). injection orco-injectionofmRNA encoding FzTM1andanactivated added atHBseparatelyorincombination. ( (Bisindolylmaleimide I),ROCK(Y27632) orJNK[JNKinhibitorI(L-form)], shown). Second,asRhoAisanactivator ofbothJNKandROCK did notblockFz5/8expression ineithertheApDor SMCs (not situ analysesruledoutthishypothesis,asthecombinedtreatment combined effects wereduetorepressionofFz5/8transcription.In assessed indouble-inhibitedembryostoassesswhethertheir embryos. Thus,Fz5/8maysignalthroughthePCPpathway. lacking skeletons andblastopores(Fig.9A),similartoFz-inhibited Combined inhibitiongave risetoembryoswithpigmentcellsbut elongation but notblastoporeinvagination orskeletogenesis. Inhibitor I(L)form(Bonny etal.,2001)blocked archenteron without impairinggastrulation.Bycontrast,inhibitionwithJNK (Riento andRidley, 2003)aloneinterferedwithskeletogenesis effector wereused.Treatment withtheROCK inhibitor(Y27632) order tocompletelyinhibitthePCPpathway, drugsspecificforeach associated-kinase (ROCK) andJun-N-terminal-kinase(JNK).In pathway. (Fig. 9A).Thus,Fz5/8doesnotseemtofunctionviatheWnt/Ca organization ofthePMCs,althoughskeletogenesis was inhibited the development ofthetripartitearchenteronoringressionand Fz5/8 transcriptionbegins intheSMCs.Thisdrugdidnotperturb Koyanagi etal.,2005).Treatments werestartedatHB,shortlybefore I, ahighlyspecificPKCinhibitor(ChouandHoward, 2002; 554 ( Fig. 9.RelationshipbetweenFrizzled andthePCPpathway. A Representative phenotypes of larvaetreated withinhibitorsforPKC ) To reinforcetheseresults,firsttheexpression ofFz5/8was The PCPpathway involves two independenteffectors, Rho- The Wnt/Ca RESEARCH ARTICLE 2+ pathway was inhibitedwithBisindolylmaleimide B ) Larvaeobtainedafter 2+ erased the treated embryos(Fig.3)andaGSK3- lividus sensitive toenhancedvegetal signaling(Takacs etal.,2004),in ApD genes.Bycontrast,althoughin animalizing perturbationsextended theexpression domainofthe species. For both territory mayalsooccurindifferent ways amongseaurchin the Fz5/8expression domain.Alternatively, establishmentofthis cell stageandthenprogressively delimited duringcleavage, asis end ofcleavage, but ratherbebroadlydefinedasearlythe60- stage. Consideringthis,theApDmaynotemerge suddenlyatthe restricted totheanimalhemispheremuchearlier, atthe60-cell and Angerer, 2003).However, Fz5/8expression startsbeing previously thoughttobespecifiedattheendofcleavage (Angerer SpNK2.1 factor (Takacs etal.,2004).TheApDhasbeen 2003), andthenbytheuniquecaseofrestrictedexpression ofthe vegetalizing perturbations(forareview, seeAngererandAngerer, found intheoraloraboralpre-ectoderm,itsresistanceto first identifiedbythelackofexpression ofseveral markers mRNAs The ApDisaspecificareaoftheectodermterritorythathasbeen domain (ApD)(AngererandAngerer, 2003;Takacs etal.,2004). the blastula(Fig.2),anareapreviously definedastheanimal During cleavage, Fz5/8isexpressed intheanimal-mostregion of Fz5/8 intheanimaldomain changes inotherorganisms. PCP pathway, whichis involved incellpolarityandshape blastopore formation(seebelow). Finally, Fz5/8signalsthroughthe phenotypic defectsobserved areprobablyduetothe absenceof SMC lineagetocontrolprimaryinvagination, whiletheother animal domain,they clearlyindicatethatFz5/8isrequired inthe of-function analysescouldnotestablishaFz5/8functioninthe separate domains,theanimaldomainandSMCs.Althoughloss- urchin embryogenesis,Fz5/8displaysrestrictedexpression intwo related toacommonancestorofthesetwo families. Duringsea Fz5 andFz8aphylogeneticanalysissuggestsfurtherthatitis indicate thatthisproteinhasstrongsimilaritieswiththevertebrate the Frizzledreceptorfamily intheseaurchin.Sequencecomparisons In thisstudy, wehave characterizedthefirstidentifiedmemberof DISCUSSION in itsroleasaninitiatorofarchenteroninvagination. results stronglyindicatethatFz5/8signalsthroughthePCPpathway of actRhoAmRNA (W. S.Beane,unpublished).Together, these percentage ofcompleterescue(28%)isequivalent tothepenetrance normal tripartitearchenteronandaskeleton (Fig.9B).The complete; embryosdeveloped intonormalpluteuslarvae witha archenteron andsomespicules.In28%ofthecases,rescuewas cases, therescuewas partialandembryosproducedareduced phenotype in42%ofthecases( (Fig. 9B).Co-expression ofFzTM1andactRhoArescued such embryoswereindistinguishablefromcontrolsatpluteusstage invagination beforehatching (W. S.Beane,unpublished);however, of actRhoAaloneproducesembryosthatformaprecocious rescue theFzTM1-associatedphenotypewas tested.Overexpression (Kim andHan,2005),theabilityofactivated RhoA(actRhoA)to species. Despite that,thesymmetricalresponses ofFz5/8to establishment oritsregulation maynotbeidenticalinthetwo morphologically identicalinthe different seaurchinspecies,its Fenouil etal.,1998).Thus, even thoughtheApDappears (Takacs etal.,2004)and , ApDexpression ofFz5/8 completelydisappearedinLiCl- HE expression domain,including intheApD(Emily- Strongylocentrotus purpuratus Paracentrotus lividus n =129) (Fig.9B).In14%ofthe S. purpuratus ␤ overexpression completely Development 133(3) (Fz5/8, thisstudy), the ApDisnot (SpNK2.1) P.

DEVELOPMENT this studydoes notclarifythefunctionalrelationship between boundary position(Sherwood andMcClay, 2001).Thus, although to mediateSMCformationand toregulate ectoderm-endoderm previous datathat show thatNotchactsviatwo distinctprocesses of pigmentcellswas produced. Theseresultsareconsistentwith sufficient torescuearchenteronformation,even though anexcess activation ofNotchsignalingdownstream ofFzTM1 was not which itdoessohasnotyetbeencharacterized.However, ectopic McClay, 2005;Sherwood andMcClay, 2001),themechanismby signaling isclearlyimportantforgastrulation(Peterson and normally continuingexpression ofBrachyury. AlthoughNotch normally isexpressed, expression ofFzTM1 eliminatesthe hatched blastula,inendoderm;however, atMB,whenFz5/8 distribution. Similarly, Brachyuryisexpressed, beginning at (Fig. 8),indicatingthatFz5/8isrequiredforthisparticular appearance ofapicalNotchproteinisabsentfromMBonwards level onapicalmembranes.InFzTM1-injected embryos,this endodermal cellswhere,startingatMB,they arelocalizedatahigh to thisstudy. However, Notchreceptorsarealsoexpressed on differentiation, andthisoccursindependentlyofFz5/8,according required inSMCstomediateSMCspecificationand normal SMCderivative (Fig.4).Notchsignalingisknown tobe staining (Fig.6),andthey correctlygive risetopigmentcells,a their epithelial-mesenchymetransition,asrevealed byAA29 injected embryos,SMCsarenormallyspecified,correctlyexecute because thatspecificationprecedesFz5/8expression; inFzTM1- However, lossoffunctionFz5/8hasnoeffect onSMCcellfate Fz5/8 expression isnotexpanded inLiCl-treatedembryos(Fig.3). in theendodermatendoderm-SMCboundary. Furthermore, Fz5/8 isentirelyinsidetheBrachyuryring,whichknown tobe by doubleinsitucomparisonswithBrachyuryexpression (Fig.2). This restrictedvegetal expression intheSMCterritoryissupported Starting atMB,Fz5/8isexpressed inasubsetoftheSMC territory. Fz5/8 inthevegetalhemisphere pathway. most probablyviathedistribution ofothercomponentsits embryo musthave away torestrictthefunctionofreceptor, overexpression Fz5/8ispresentthroughouttheembryo.Thus, displayed anormalphenotype.Thiswas despitethefact thatwith embryos co-injectedwithFzTM1andFzmRNAs similarly overexpression ofFz5/8alonehasnophenotypeandtherescued pathway couldalsobelimitedattheanimalpole.Furthermore, Alternatively Fz5/8ligandorotheressentialcomponentsof redundancy betweenFz5/8andothersignalsinthisarea. subtle tobeobserved withcurrentlyavailable tools,orthereissome elimination ofFz5/8.Potentially, theroleofFz5/8inApDistoo signaling. Thus,nofunctionoftheApDwas revealed by indicating thatestablishmentofaxisdoesnotrequireFz5/8 and 1E11isexpressed inneuronstheoralterritory(Figs5,6), continued tooccurattheboundaryoforalandaboralterritories, embryos, oral-aboralpolarizationwas present,neurogenesis perturb neurogenesis(Fig.5C).Furthermore,inthesuppressed function throughouttheembryo,orinanimalhalves alone,didnot neurons develop (Nakajimaetal.,2004).SuppressionofFz5/8 then becomesthepartoforalhoodlarva inwhichthe During embryogenesis,theApDfirstextends atuftoflongcilia, controlled bymaternalnuclear 60-cell stage,suggestthatduringearlycleavage Fz5/8maybe restriction ofitsexpression totheanimalpolearea,startingat vegetalization andanimalization,togetherwiththeprogressive Fz5/8 required inSMCforprimaryinvagination The functionalroleofFz5/8intheApDremainsenigmatic. ␤ -catenin signaling. specific inhibitorsfortheWnt/Ca does notsignalthroughtheWntcanonicalpathway. 1991). Thus,togetherthoseobservations stronglysuggestthatFz5/8 blastula stage(Livingston andWilt, 1992;Nocente-McGrathetal., vegetal platespecificationorinvagination whenitisaddedafter when appliedearlyenhancesendodermformation,hasnoeffect on development ofthearchenteron.Finally, LiCltreatment,which potential zygoticformofthepathway, hasnoimpactonthe (He etal.,2004;Umbhauer2000),andusedtoblockthe negative effects ofwhichareeachspecifictothecanonicalpathway mutants ofFz5/8ortheco-receptorLRP5/6,dominant- are absentinanimalizedembryos.Third,overexpression ofthe FzTM1-injected embryoshave bothPMCsand pigmentcells,which animalization obtainedbydownregulation ofthecanonicalpathway. activation. Second,theFzTM1phenotypeisdifferent froman responsible ofthischaracteristicevent ofthecanonicalWntpathway (Logan etal.,1999),indicatingthatFz5/8isunlikely tobe expressed inSMCs, accumulation (Loganetal.,1999).Furthermore,whenFz5/8is occurs inSMCatMB,many hoursafterthefirst maternal orthezygoticcanonicalpathway. First,Fz5/8expression Several piecesofevidence indicatethatFz5/8doesnotuseeitherthe 2003). Atthecellular level, thePCPpathway controls cellspolarity 1998; Hardenetal.,1995;Strutt etal.,1997;Tahinci andSymes, (Barrett etal.,1997;Boutros al.,1998;Hacker andPerrimon, to controlcellpolarityand shapechangesduringgastrulation activated byFz5/8.Inotherorganisms, thePCPpathway isknown initiation ofgastrulationoperates throughthePCPpathway andis to rescuetheFzTM1phenotype furthersupportsthehypothesisthat embryogenesis. Theabilityofanactivated formofRhoA(actRhoA) independently requiredindistinctimportantevents of embryo thetwo downstream componentsofthePCPpathway are ROCK inhibitiondisruptsskeletogenesis, showing thatinseaurchin skeleton. However, skeletogenesis was notaffected. Bycontrast, affects oral-aboralpolaritycausingformationofaradialized inhibition ofJNKactivity suppressesarchenteronelongationand by thedualinhibitionofJNKactivity andROCK inhibition.Alone, the likely mechanism(Fig.9).TheFzTM1phenotypeismimicked canonical pathway, thePCP pathway andtheWnt/Ca Frizzled proteinscantransducethreedistinctWntpathways: the Fz5/8 andthePCPpathway of theSMCsappearstooccurnormally, thisseemslessprobable. emitted directlybytheSMCstoPMCs.However, asthebehavior defect canalsobecausedbyalackofFz5/8-dependentsignal the archenteronplaysaroleinskeletogenesis. Alternatively, this around itusingthehostPMCs(Beninketal.,1997),suggestingthat archenteron couldinduceanectopicbilaterallysymmetricskeleton of invaginated cells.Aprevious studyreportedthatatransplanted that thelackofskeletogenesis isasecondaryeffect oftheabsence animal halfdidnotaffect skeletogenesis (Fig.7).Thus,wesuggest ectoderm asFzTM1expression exclusively inmicromeresorthe nonspecific inhibitionofotherFrizzledsignalinginPMCsor (Figs 6,7).TheFzTM1dominant-negative effect isnotdueto FzTM1-injected embryos,PMCsingressanddifferentiate normally Again, Fz5/8isnotexpressed inPMCortheirprecursors,and an event forwhichnoupstreammediatorisknown todate. for theapicallocalizationofNotchproteinsinendodermalcells, Fz5/8 andNotch,itestablishesthatisrequired,indirectly, the Wnt/Ca Comparison oftheFzTM1phenotypewiththoseobtained Another defectoftheFz5/8lossfunctionislackspicules. 2+ pathway asacandidate,andleftthePCPpathway as ␤ -catenin isnolongernuclearinthatterritory 2+ or thePCPpathway eliminated RESEARCH ARTICLE ␤ -catenin nuclear 2+ pathway. 555

DEVELOPMENT providing additionalcandidatesfortheligandofFz5/8. genome willprovide thecompletesetofseaurchinWntgenes, Nevertheless, completionofthesequencing oftheseaurchin expression patternthatabut oroverlap Fz5/8expression. Wnt proteinsreportedinseaurchinhave aspatialandtemporal identifying thecellularsourceofsignal.Atpresent,none the for archenteroninvagination. Ofparticularinterestwillbe further understandingoftheinductive signalingprocessresponsible cells (Inghametal.,1991). Frizzled signalsynthesizeHedgehogwhichactsontheneighboring in segmentation polarityin a paracrinesignalthatactsdirectlyonendodermalcells,asitdoes injected embryos,weproposethatFz5/8pathway activates inSMCs explain thelossoflateendodermalmarkers expression inFzTM1- molecular signalstothemorphogeneticmachinery. Furthermore,to via thePCPpathway. Fz5/8 would thusserve toconnectinductive by regulating SMCadhesion,shapeandpolaritybyactivating RhoA Fz5/8 would thereforecontroltheinitiationofprimaryinvagination morphogenetic movements responsibleforbottlecellformation. cells. Thus,wehypothesizethatFz5/8serves asamodulator ofthe the unlabeledsmallmicromeres,andsoarelikely tobethebottle is expressed inasmallringofoneortwo layersofcellssurrounding primary invagination (KimberlyandHardin,1998).AtMB,Fz5/8 invaginate andablationstudiesindicatethatthey arecrucialfor (Kimberly andHardin,1998).Thebottlecellsarethefirstto elongated cellsthatwillformthelipofearlyblastopore between theeightsmallmicromerederivatives andatierof bottle shape.Thesecellsformaone-ortwo-cell wideringlocated of thevegetal platemodifiestheirshapetoacquiretheso-called At theonsetofinvagination, a subsetSMCslocatednearthecenter archenteron morphogenesis,following endoderm specification. seems mostlikely thatFz5/8isrequiredinSMCstocontrol FzTM1-injected embryossupportsthisconclusion(Fig.6).Thus,it commitment; normalexpression ofendodermalmarkers atMBin therefore unlikely tobeinvolved eitherinendodermspecificationor 1997). AsFz5/8isexpressed inSMCsbeginning atMB,itis mesenchyme blastulastage(ChenandWessel, 1996;Godinetal., while thecommitmentperiodisbetweenlateblastulaandearly shown thatendodermspecificationoccursearly during cleavage, the absenceofablastoporeandarchenteron.Previous studieshave A dramaticalterationresultingfromdisruptionofFz5/8functionis invagination Fz5/8 isrequired inSMCsforarchenteron properties thancellfate decisions. signal throughthePCPpathway, itismorelikely tocontrolcell Marlow etal.,2002;Winter et al.,2001).Thus,asFz5/8appearsto through effects oncytoskeletal organization (Wallingford, 2005; 556 Adell, T., Nefkens, I.andMuller, W. E. References HD14483. by theMinistère delaRecherche (ACI),andbyNIHgrantsGM61464 Cancer, bytheFondation pourlaRecherche Médicale, byaHargittFellowship, Marie Curie(ParisVI),bygrantsfrom theAssociationpourlaRecherche surle manuscript. ThisworkwassupportedbytheCNRS,UniversityPierre et Bradham, ThierryLepageandWendy Beaneforcriticalevaluationofthe Burke, ChuckEttensohnandXiHeforplasmidsantibodies;Cyndi screen duringwhichFz5/8 was isolated.We alsothankWendy Beane,Robert We thankThierryLepagefor initiatingthecollaborativeinsituhybridization the receptor intheepithelium/pinacoderm. demosponge Suberites domuncula:identification,expression andlocalizationof Identification oftheWntligandthatbindstoFz5/8willaidin Fz5/8 isrequiredforinvagination andisexpressed intheSMCs. RESEARCH ARTICLE Drosophila (2003). Polarityfactor‘Frizzled’inthe FEBS Lett. for whichcellsreceiving 554 , 363-368. Boutros, M.,Paricio,N.,Strutt,D.I.andMlodzik,M. Bonny, C.,Oberson,A.,Negri,S.,Sauser, C.andSchorderet, D.F. Ferkowicz, M.J.,Stander, M.C.andRaff, R.A. Emily-Fenouil, F., Ghiglione,C.,Lhomond,G.,Lepage,T. andGache,C. Chen, S.W. andWessel, G.M. Cadigan, K.M. Bhanot, P., Brink,M.,Samos,C.H.,Hsieh,J.C.,Wang, Y., Macke,J.P., Adler, P. N. 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