Johnson SchoolofMedicine,Piscataway, NJ08854,USA. of Pharmacology, UniversityofMedicineandDentistryNewJersey-Robert Wood MD 20892-2790,USA. Genetics, NationalInstitutesofChildHealth andHumanDevelopment,Bethesda, amn Habas Raymond Dishevelled (Dvl)(Wallingford andHabas,2005).Thispathway processes, andismediatedbythePDZDEPdomains of pathway, regulates gastrulationcellmovements amongother catenin independentpathway ortheplanarcellpolarity (PCP) Wallingford etal.,2002). signaling pathway isrequiredfortheseprocesses(Keller, 2002; modification ofthecytoskeleton viathenon-canonicalWnt neural tubefoldingandclosureareincompletelyunderstood, but control convergent extension movements, blastoporeclosureand (Wallingford andHarland,2002).Themolecularmechanismsthat convergent extension movements provide theforceforthisprocess they apposeatthemidlineandfusetoformneural tube; primary neurulationthelateraledgesofneuralplateelevate until circumference oftheblastopore(Keller etal.,2003).Insubsequent mediated bya‘pursestring’mechanismthatconstrictsthe occurs asaresultofthesequentialcircumferentialshorteningthatis mediolateral surfaces (Wallingford etal.,2000).Blastoporeclosure results fromstabilizationofprotrusions(lamellopodia)onthe axis (Keller, 2002).Thedirectionalityofthesecellmovements (convergence) andanteroposteriorlengthening(extension) ofthe elongate, alignandintercalate,resultinginmediolateralnarrowing ‘convergent extension movements’ during whichcellspolarize, axis oftheembryothroughdirectedcellmigration,termed movements duringgastrulation.Thisprocessgeneratestheextended Establishment ofthevertebrate bodyplandepends oncell INTRODUCTION KEY WORDS:Profilin, vertebrate gastrulation. downstream ofDaam1thatcontrolsWnt-mediatedcytoskeletalreorganizationforaspecificmorphogeneticprocessduring did notaffect convergentextensionmovements,tissueseparationorneuralfoldclosure.Ourstudiesdefineamolecularpathway by non-canonicalWntsignaling.InhibitionordepletionofProfilin1invivospecificallyinhibitedblastoporeclosure fibers inresponsetoWntsignalingmammaliancells.Inaddition,depletionofProfilin1inhibitedstressfiberformationinduc required forcytoskeletalchanges.Profilin1interactedwiththeFH1domainofDaam1andwaslocalizedtoactinstres Dishevelled andtheRhoGTPaseforcytoskeletalmodulation.Here,wereportthatProfilin1isaneffector downstreamofDaam1 incompletely understood.We hadpreviouslyidentifiedtheForminhomologyproteinDaam1asanimportantlinkbetween gastrulation. However, themolecularmechanismsofhowWntsignalingregulatesmodificationactincytoskeletonremain Non-canonical Wntsignalingplaysimportantrolesduringvertebrateembryogenesisandisrequiredforcellmotility Akira Sato signaling andisrequiredforvertebrategastrulation Profilin isaneffector for Daam1innon-canonicalWnt Development 133,4219-4231(2006)doi:10.1242/dev.02590 Accepted 23August 2006 1 *Author forcorrespondence (e-mail:[email protected]) New Brunswick,NJ08903-2681,USA. Robert Wood JohnsonSchoolofMedicine,Piscataway, NJ08854,USA. Department ofBiochemistry, UniversityofMedicineandDentistryNewJersey- The non-canonicalWntsignalingpathway, alsotermedthe 1 , DeepakK.Khadka 4 The CancerInstituteofNewJersey, 195LittleAlbanyStreet, 1,4, Xenopus * , Daam1 1 , Wei Liu 3 Laboratory ofMolecular 1 , RituBharti 2 Department ␤ 1 - , Loren W. Runnels (Higgs, 2005;Kovar etal.,2006;Kovar andPollard,2004).One polymerize actinfilamentsand serves anactinnucleationfunction effectors toelicit cytoskeletal changes.TheFH2domaincan inhibition, followed bybindingoftheFH1andFH2domainsto Rho-GTP bindstotheGBDdomain, releasingtheproteinfromauto- (Alberts, 2002;Higgs,2005). It hasbeenproposedthatactivated Diaphanous auto-inhibitorydomain (DAD) totheaminoterminus inhibited state,whichismediatedbybindingoftheC-terminal is proposedthatFormin proteinsexist inthecytoplasm inanauto- (FH1) andFormin Homology2(FH2)domains(Alberts,2002).It termed theGTPase bindingdomain(GBD),Formin Homology1 and Alberts,2003).Formin proteinscontainthreemajordomains central playersincytoskeletal reorganization (Alberts,2002;Wallar 2003; Lietal.,1999;Yamanaka etal.,2002). GTPase, Rac,whichinturnstimulatesJNKactivity (Habasetal., DEP domainofDvlbut notDaam1andactivates theRhofamily al., 2003;Wallingford et al.,2002).Thesecondbranchrequiresthe mediates cytoskeletal reorganization (Marlow etal.,2002;Veeman et 2001), leadstotheactivation oftheRhoassociatedkinaseRock,and involves Daam1,which binds tothePDZdomainofDvl(Habasetal., Habas etal.,2001;Tahinci andSymes,2003).SignalingtoRho the activation ofthesmallGTPases RhoandRac(Habasetal.,2003; bifurcates downstream ofDvlintotwo parallel branchesthatleadto Fz independentlyfromLRP5/6(Heetal.,2004)andthepathway and Habas,2005).Inthispathway theWntsignalismediatedthrough (JNK), StrabismusandPrickle(HabasDawid, 2005; Wallingford pathway includeWnt11,Fz,Dvl,Daam1,Rho,Rac,Junkinase Liu, 2006).Additionalknown componentsofthenon-canonical can activate bothcanonicalandnon-canonicalsignaling(Cadigan and signaling (Heetal.,1997;Mikels andNusse,2006).Likewise, Wnt3a (Moon etal.,1997).However, Wnt5acanalsoactivate canonical deciphered but Wnt5awas firstidentifiedasaregulator ofgastrulation ligands fordistinctbranchesofthepathway remainspoorly et al.,2003;Wallingford andHabas,2005).ThespecificityofWnt cytoskeleton andappearstobeindependentoftranscription(Veeman regulates cellmovements throughmodificationoftheactin Daam1 isamemberoftheFormin family ofproteinsthatare 2 , IgorB.Dawid RESEARCH ARTICLE 3 and Xenopus but 4219 ed s

DEVELOPMENT (San Diego, CA). MATERIALS ANDMETHODS that controlsaspecificaspectofvertebrate gastrulation. our studiesreveal abranchpointinthenon-canonicalWntpathway in canonicalWntsignalingandmesodermspecification.Together, neural foldclosureareunaffected. Furthermore,Profilin1hasnorole blastopore closurebut convergent extension, tissueseparationand Overexpression ordepletionofProfilin1resultsininhibition the embryoatatimeandplaceconsistentwithroleingastrulation. mediated stressfiberformation. to Wntstimulation.DepletionofProfilin1inhibitsWnt-andDaam1- Daam1 andcolocalizeswithtoactinstressfibersinresponse Wnt signalingpathway. Profilin1bindstotheFH1domainof partner ofDaam1andafunctionalcomponentthenon-canonical function ofProfilin1duringembryogenesisremainspoorlydefined. date, nosignalingpathway isknown torequireProfilin1,andthe invivo remainsunknown. To required foramorphogeneticprocess 2004; Zigmond,2004).WhetherthisProfilin-Formin interactionis delivery andcappingfunction(Higgs,2005;Kovar andPollard, the FH1domainofFormin proteinsandserves anactinmonomer cell stage(Witke etal.,2001;Witke, 2004).Profilin1interactswith for cytokinesis andmouseknockout mutantsdieby thetwo- toeight- 2004). Amongthethreemammalianprofilins,Profilin1isessential cells foraxonalguidanceanddendriticspinemorphology(Witke, (Cooley etal.,1992;Verheyen andCooley, 1994)andinneuronal Profilin1 inoogenesis,spermatogenesis,bristleandeye formation Witke, 2004).Geneticstudiesin involved inactinpolymerization(Watanabe andHigashida,2004; Frazier andField,1997;Severson etal.,2002). Profilin, whichbindstheFormin mDia1(Evangelista etal.,2002; molecule known tobindtheFH1domainofFormin proteinsis 4220 positives, wereobtained. were screened,and12overlapping Profilin1fragments,inadditiontoother fragment ofDaam1(Fig.1A)asthe bait.3.9millionindependentclones A ratbraincDNA library(Clonetech) was screenedusingthec-Daam1 Yeast two-hybridscreen translational initiationsite,5 purified following themanufacturer’s instructions. oligonucleotides weresynthesizedusingtheDicerKit(Ambion) and request. cloned intopCS2+MTorpCS2+GFP. Detailsofplasmidsareavailable upon (isolated byaPCRapproachfrom Minnesota). RatProfilin1(isolatedfromourscreen)and pCS2+GFP vector (kindlyprovided byDrJeffrey Miller, University of at theNterminus)orpcDNA-HA (fortheHAtagataminoterminus),or digestion oraPCRapproach,andsubclonedinpCS2+MT(fortheMyc tag The humanDaam1andfragmentsofweregeneratedbyrestriction Plasmids andoligonucleotides (Eugene, OR).Anti- Phalloidin andOregon Green-PhalloidinwerefromMolecularProbes from Sigma.Alexa Fluoranti-mouseandanti-rabbitAbs,Texas RedX- and Cdc42werefromTransduction Laboratories,andagainstFlag(M2)was and Myc(N-262)werefromSantaCruzBiotechnology. mAbsagainstRac (10B5), Myc(9E10),andpolyclonalAbs(pAbs)againstRhoA(CAT119) Monoclonal antibodies(mAbs)againstHA(F-7),RhoA(26C4),Dvl2 Materials the negative control. was synthesized byGeneTools. AMO witharandomsequencewas usedas The XProfilin1Morpholinooligonucleotide(MO)complementarytothe The dsRNAi oligonucleotidesforProfilin1orcontrolGFP Here wereporttheidentificationofProfilin1asaninteracting Profilin isanevolutionarily conserved actinbindingproteinthatis RESEARCH ARTICLE ␤ -catenin antibodywas fromTransduction Laboratories Ј -TGTAGCCGTTCCAAGACATTGTTGT-3 Xenopus Drosophila Xenopus Stage 10.5cDNA library)were Profilin1 isexpressed in have uncovered rolesfor Xenopus Profilin1 Ј , manner sothatthescorerhadnoknowledge ofthesamplebeingscored. experiments wererepeatedatleastthreetimesandscoringwas doneinablind than10 fiberswas scoredasanincrease ordecreaserespectively. These fibers percellwas usedtoscore,thusany cellcontainingmorethanorless and Wnt-Daam1mediatedstressfiberinduction,abaselineof10 counted asapositive. For quantificationoftheeffects ofdepletionProfilin1 used andthemerged imageofDaam1orProfilin1ontothesefiberswas Daam1 orProfilin1tostressfibers,abaselineof10fiberspercellwas microscope (Oberkochen, Germany). For quantificationoflocalization microscope with100Xobjective lens(Melville,NY)oraZeissAxiovert 100 al., 2001).ImageswereobtainedusinganOlympusIX70fluorescent This was carriedoutasdescribedpreviously (Capellutoetal.,2002;Habas Immunocytochemistry by standardmethods. specific antibodywas affinity purifiedusingtheGST-Daam1 fusionprotein protein containingaminoacids967-1078ofhumanDaam1.TheDaam1 Anti-Daam1 antibodiesweregeneratedinrabbitsagainstaGSTfusion Antibody generation R&D systemsandusedataconcentrationof250ng/ml. manufacturers instructions.PurifiedWnt3aproteinwas purchasedfrom Wnt3a, Wnt5aandLcellconditionmediumwerepreparedaccordingtothe ATCC andculturedaccordingtothesuppliersinstructions.Serum-free Wnt3a-transfected, Wnt5a-transfectedorcontrolLcellswereobtainedfrom Wnt conditionedmedia 1-2 reagent (Qiagen),orDicertransfection(forRNAi experiments) with well plateweretransfectedusingthecalcium-phosphatemethod,Polyfect All werecarriedoutwithHEK293TcellsorNIH3T3cells.Cellsinasix- Transfections commercial Profilin1 antibody(Cytoskeletal Labs)thatisfunctional epitope-tagged Daam1.For theseexperiments, weutilizeda containing fragment(Fig.1B). to theFH1-containingfragment ofDaam1but nottotheFH2- separately (Fig.1A),welocalized theProfilin1interactingdomain fragments ofC-Daam1harboring theFH1orFH2domains contains theamino-terminaldomain(Fig.1B).Usingsmaller contains theFH1andFH2domains,but nottoN-Daam1, which found thatProfilin1bindstofulllengthDaam1andC-Daam1,which proteins expressed inmammalianHEK293Tcells(Fig.1A).We immunoprecipitation usingepitope-taggedwildtypeandmutant yeast, weexamined Profilin1interactionwithDaam1byco- of Profilin1asaC-Daam1-interactingprotein. Daam1 asbait.Fromthisscreen,weisolated12overlapping clones these processes,weperformedayeasttwo hybridscreen usingC- (Fig. 1A).Inordertoidentifydownstream effectors forDaam1 induce Rhoactivation andcytoskeletal changes(Habasetal.,2001) We hadshown thatafragmentofDaam1termedC-Daam1can Daam1 Identification ofProfilin1 asabindingpartnerfor RESULTS assays wereperformedasdescribed(Hukriedeetal.,2003). were performedasdescribed(ShihandKeller, 1992).Tissue separation described (Habasetal.,2003)using5ng/mlactivin. Keller explant assays RNAs. Convergent extension assaysinexplants wereperformedas Kato etal.,2002).Embryoinjectionsweredonewithinvitrotranscribed These wereperformedasdescribed(Habasetal.,2003;Habas2001; assays Embryo manipulations,RT-PCR, insituhybridizationand explant 1 inserts. ␮ We next examined whetherendogenous Profilin1interactswith To delineateinteractions betweenProfilin1andDaam1outsideof g plasmid.Transfected DNA amountswereequalizedviavectors without ␮ g ofeachindicatedplasmidor500ng-1 ␮ g annealedRNAi oligoplus Development 133(21)

DEVELOPMENT Profilin: aDaam1effector dramatic cytoskeletal reorganization ofCOSorBcellsinresponse media (CM)orWnt3aprotein. Previous studieshave revealed a cytoskeleton inNIH3T3 cells,usingWnt3aandWnt5aconditioned Rho activation. This suggeststhatProfilin1does notfunctioninDaam1-mediated did notinterferewithC-Daam1-mediated Rhoactivation (Fig.1D). Profilin1 didnot(Fig.1D).Inaddition,co-expression ofProfilin1 C-Daam1 expression inducedRhoactivation but expression of transfected withC-Daam1,Profilin1orboth.Asinprevious studies, and He,2006;Renetal.,1999)extracts ofmammaliancells Daam1-mediated RhoAactivation using theRhotekinassay(Habas (Habas etal.,2001),wetestedwhetherProfilin1functionsin C- As C-Daam1canmediateRhoactivation andcytoskeletal changes Rho activationdownstream ofDaam1 Profilin1 mediatescytoskeletal changesbutnot endogenous Profilin1(Fig.1C). FH2-containing constructofDaam1was foundtointeract with endogenous Profilin1(Fig.1C).AdditionallytheFH1-but notthe length Daam1andC-Daam1but notN-Daam1interactedwith immunocytochemistry. Inagreementwiththeabove results,full- for westernblottingbut notimmunoprecipitationor We next examined theeffects ofProfilin1ontheactin resulted innuclearaccumulationof of NIH3T3cellswithWnt3aCMbut notcontrolCMfor3hours effects onNIH3T3cellswerenotreported.We foundthattreatment to Wnt3astimulation(Endoetal.,2005;Qiang2003),but such Lastly, amutantconstructofDishevelled, formation but nonuclearaccumulationof CM. Thesestudiesrevealed arobust inductionofstressfiber Wnt3a protein(Fig.2A).We next examined theeffects ofWnt5a and nuclearaccumulationof of stressfibers(Fig.2A).Anidenticaleffect onstressfiberinduction formation ofstressfibers(Fig.2B)withoutinductionnuclear Tada andSmith,2000; Wallingford etal.,2000),couldinducethe solely involved innon-canonicalWntsignaling(Habasetal.,2001; (Fig. 2C),treatment withWnt3aCMresulted in colocalizationof localized inthecytoplasm ofNIH3T3cellstreatedwithcontrolCM stimulation inNIH3T3cells.Although GFP-Profilin1was diffusely localization ofGFP-Profilin1fusion proteininresponsetoWnt3a Roy andJacobson,2004; Witke, 2004).We examined the localization ofProfilin1totheactin cytoskeleton (Caoetal.,1992; stress fibers. respond tonon-canonicalWntsignalingwiththeinductionofactin catenin (notshown). TheseresultsdemonstratethatNIH3T3cells Previous studieshave revealed variable resultsontheeffects and described (Habasetal.,2003;Habas2001). and Rhoactivationassayswere carriedoutas activation inducedbyC-Daam1.Immunoprecipitation does notinduceRhoactivationorinterfere withRho Daam1 ortheFH2domainofDaam1.( C-Daam1 ortheFH1domainofDaam1butnotN- Profilin1 isprecipitated withepitopetagged-Daam1, position asHA-Daam1(arrowhead). (C)Endogenous and anonspecificbandcomigratesatthesame migrates overlappingtheIgGbandandisnotresolved domain ofDaam1.NotethetaggedFH1 FH1 domainofDaam1butnotN-Daam1ortheFH2 (B) Profilin1 interactswithDaam1,C-Daam1andthe Precipitates were immunoblottedwithindicatedAbs. were immunoprecipitated (IP)withindicatedAbs. were cotransfectedintoHEK293Tcells,andlysates tagged-Daam1 orDaam1fragmentsandProfilin1 immunoprecipitation experiments.Plasmidsencoding indicating aminoacidpositions.( Profilin1 constructsshowingdomains,withnumbers Daam1. Fig. 1.Profilin bindstotheFH1domainof ( A ) SchematicdiagramoftheDaam1and ␤ -catenin was observed withpurified RESEARCH ARTICLE ␤ -catenin androbust induction ⌬ DIX-Dishevelled thatis ␤ B -catenin (Fig.2A). , C ) Co- D ) Profilin1 4221 ␤ -

DEVELOPMENT mammalian cells.Stainingwithaffinity purified signaling, weexplored thelocalizationofProfilin1andDaam1in To investigate theroleofProfilin1inmediatingnon-canonicalWnt cytoskeletal reorganization Profilin1 isrequired forWnt/Daam1mediated signaling thatmodulatestheactincytoskeleton. suggest thatProfilin1maybeacomponentofnon-canonicalWnt GFP-Profilin1 withactinstressfibers(Fig.2C,E).Thesestudies 4222 whether Daam1andProfilin1arecolocalizedinresponsetoWnt response was similartothatofProfilin1(Fig.2C),weexamined and toalesserextent totheplasmamembrane(Fig.2D,E).Asthis stimulation, Daam1relocalizedpredominantlytoactinstressfibers cytoplasm ofNIH3T3cells(Fig.2D).InresponsetoWnt3aCM polyclonal serashowed thatDaam1islocalized mainlyinthe RESEARCH ARTICLE ␣ -Daam1 more than60%withoutaffecting thelevels of mediated interference(RNAi) todepleteendogenousProfilin1by Daam1-mediated cytoskeletal changes.We employed dsRNA- cytoskeleton a commonmolecularpathway tomediateeffects ontheactin Wnt stimulationsuggestthatDaam1andProfilin1mayfunctionin endogenous Daam1(Fig.3A,B).Thiscolocalizationinresponseto stimulation andfoundthistobethecaseforbothtransfected stimulation, was markedly decreasedwhereasthenuclear Wnt3a or Profilin1 stressfiberinductioninresponsetoWnt3aCM,purified CM orDishevelled (Fig.3D).Importantlyincellsdepletedof Top-flash reporteractivation inNIH3T3cellsmediatedbyWnt3a (Fig. 3C).Transfection ofProfilin1orcontrolsiRNA didnotinhibit We next determinedwhetherProfilin1isrequiredforWnt-and ⌬ DIX-Dishevelled, but notinresponsetoserum respective panel. (white) are shownbeloweach magnifications ofboxedareas of eachbar. NoteinB,C,D cells evaluatedisshownatthetop the studiesofCandD.Number stimulation. ( after 3hoursWnt3aCM actin stress fibers(arrow) (green) of NIH3T3cellsbutislocalizedto localized diffusely inthecytoplasm ( 3 hoursWnt-3aCMstimulation. actin stress fibers(arrow) (red) after control CMbutassociateswith of NIH3T3cellsinthepresence of localized diffusely inthecytoplasm NIH3T3 cells.GFP-Profilin1 is localization ofGFP-Profilin1 in NIH3T3 cells.( stress fiberformation(green) in ⌬ by arrow. ( Position ofthenucleusisindicated stress fiberinductionisobserved. localization of to Wnt5aCM,nonuclear (green). After3hoursofexposure indicated byPhalloidinstaining nucleus andstress fiberinductionis ␤ CM orWnt3aprotein, endogenous hours ofexposure ofcellstoWnt3a conditioned media(CM).After3 and Wnt5abutnotcontrol ( response toWntstimulation. Profilin1 andDaam1in Fig. 2.Subcellularlocalizationof D A -catenin (red) islocalizedinthe DIX-Dishevelled (red) induces ) EndogenousDaam1(red) is ) NIH3T3cellsrespond toWnt3a B Development 133(21) ) Expression ofFlag- E ␤ C Quantificationof ) ␤ -catenin orDaam1 ) Subcellular -catenin isseenbut

DEVELOPMENT GFP siRNAreduces thelevel ofendogenousProfilin1 inNIH3T3cells. in AandB,magnificationsofthe boxed areas (white)are shownbeloweachrespective panel.( colocalizes withGFP-Profilin1 tostructures resembling actinstress fibers (arrow) inresponse to3hoursWnt3abutnotcontrol material). Attheblastulastage,Profilin1was observed pole ofthefertilizedegg (seeFig.S1Cinthesupplementary morphogenetic movements. Profilin1was expressed intheanimal developing embryoespeciallyinregions associatedwith hybridization revealed adynamicexpression profileinthe spatial patternofProfilin1geneexpression visualizedbyinsitu development (seeFig.S1Binthesupplementarymaterial).The showed thatProfilin1isexpressed maternallyandthroughout (see Fig.S1Ainthesupplementarymaterial).RT-PCR analysis XProfilin1 shares48.7%identitywiththeratandhumanproteins expression patternofProfilin1during To helpelucidatetheinvivo roleofProfilin1,weexamined the development Expression ofProfilin1 pattern during Daam1-mediated cytoskeletal changes. These studiesdemonstratethatProfilin1isrequiredforWnt-and formation was observed incellsdepletedofProfilin1 (Fig.5A,B). formation (Habasetal.,2001),asignificantreductionofstressfiber in cellstransfectedwithC-Daam1,whichinducesstressfiber accumulation of Profilin: aDaam1effector Fig. 3.Wntstimulationinduces acolocalizationofDaam1andProfilin1. Wnt3a CMandDishevelledbutnot control CMor siRNA doesnotinterfere withreporter activationbyeitherWntstimulation orDishevelledexpression. ␤ -catenin was unaffected (Fig.4A-C).Additionally Xenopus ⌬ DIX-Dishevelled inducesreporter activationinNIH3T3cells.Transfection ofcontrol orProfilin1 embryogenesis. Xenopus ␤ -catenin andDaam1are used asloadingcontrols. ( effects ofmisexpression ofProfilin1during To elucidatethefunctionofProfilin1invivo, weexamined the gastrulation Profilin1 overexpression interferes with 2004). pattern ofProfilin1overlaps withthatofDaam1(Nakayaetal., S1C inthesupplementarymaterial).We notethattheexpression expressed athigherlevels inthebrain,eyes andspinalcord(seeFig. and anteriorneuralplateduringlaterdevelopment was During theneurulastageProfilin1was expressed in theneuralfolds mesodermal cells(seeFig.S1Cinthesupplementarymaterial). circumferentially aroundtheblastoporelipandininvoluting structures includingtheheadandeyes werereducedand theneural significant effect (Fig.6A,E).InProfilin1-injected embryosanterior dependent mannerwhereasinjectionofLacZRNA hadno cell embryoresultedinsevere gastrulationdefectsinadosage injection ofProfilin1RNA intothedorsalmarginal zoneofthefour- concentration rangeof100pgto1ng(Fig.6A,E).Incontrast of thefour-cell embryohadnoeffect on Injection ofProfilin1RNA intothetwo ventral marginal blastomeres ( A ) Epitope-taggedDaam1(HA)or( C ) Transfection ofProfilin1 siRNA butnotacontrol RESEARCH ARTICLE B ) endogenousDaam1(red) Xenopus D ) Top-Flash assayreveals Xenopus CM stimulation.Note development ina development. Xenopus 4223

DEVELOPMENT inhibit stress fiberinductioninducedbyserumstimulation.Nucleartranslocationofendogenous siRNA intoNIH3T3cellsinhibitsstress fiberinduction(green) by3hoursWnt3aCMorprotein stimulation.NotetheProfil respective panel.( fiber induction(green) inducedby 4224 Profilin1 antiseradidnotrecognizeendogenous the endogenousProfilin1protein(Fig.6B).Ascommercial loss-of-function approach,wedesignedanantisenseMOtodeplete (Wallingford etal.,2002). Profilin1 ingastrulationcellmovements and blastopore closure folds failed toclose.Thisphenotypeissuggestive ofarole Wnt3a protein stimulationisnotaffected byProfilin1 siRNA.( Fig. 4.Profilin1 isrequired forWnt3aand Dishevelled-mediated stress fiberinduction. manner (Fig.6D,E). Thephenotypeinduced byinjectionofthe MO resultedinembryoswithopen neuralfoldsinadosedependent dorsal cellshadlittleeffect but injectionof10-50ngtheProfilin1 development (Fig.6D,E).SimilarlyinjectionofcontrolMOintothe two cellsofthefour-cell embryohadlittleeffect on control MOatalevel of50ngintothemarginal zoneoftheventral using acontrolMO(Fig.6C,E). InjectionoftheProfilin1MOora of 25and50ngtheProfilin1MOnoeffect was observed translation ofMyc-Profilin1,but notofRhoandRac byinjection the Profilin1 MOtoinhibittranslationofaMyc-Profilin1construct in by westernblotanalysis(notshown), wetestedtheefficiency ofthe To studytheroleofProfilin1 inearly Xenopus RESEARCH ARTICLE embryo. We foundadose dependentsuppressionof C ) Quantificationoftheresults ofAandB.Numbercellsevaluatedisshownatthetopeachbar. ⌬ DIX-Dishevelled expression (red), magnificationsoftheboxedareas (white)are shownatthesideofeach Xenopus Xenopus development bya B Xenopus Profilin1 ) Transfection ofProfilin1 siRNAbutnotGFPintoNIH3T3cellsinhibitsstress canonical Wntsignaling. view thatProfilin1, like Daam1,functionsspecificallyinnon- Fig. S2Cinthesupplementary material). Theseresultssupportthe Dsh inductionof Furthermore, Profilin1or MOhadnoeffect onXwnt-8or this induction(seeFig.S2A,B inthesupplementarymaterial). of asecondaryaxis,andco-expression ofProfilin1hadnoeffect on Xwnt8 orDshventrally in (McMahon andMoon,1989;Sokol etal.,1991).Expression of signaling, weperformedsecondaryaxisinductionassays To investigate whetherProfilin1canregulate canonicalWnt Profilin1 andcanonical Wntsignaling gastrulation. Profilin1 MOandalsodemonstratethatisrequired for 6D,E). Theseexperiments demonstratethespecificityof MO bindingsitewas deleted,but notby100pgof LacZRNA (Fig. injection of100pga maximal doseof50ngProfilin1MOcouldberescuedby co- Xnr3 ( A ␤ ) Transfection ofProfilin1 siRNAbutnotGFP -catenin (red) inducedbyWnt3aCMorpurified and ⌬ N-Profilin1 RNA, aconstructinwhichthe Siamois Xenopus (Harland andGerhart,1997)(see embryos inducedtheformation Development 133(21) in1 siRNAdoesnot

DEVELOPMENT mesodermal marker normal mesodermanddorsalaxisformationasassayedbythe At stage10.5Profilin1andMO-injectedembryosshowed by co-injecting injected embryoswereabnormal,andthelattercouldberescued MO plus RNA, Profilin1MO,control Embryos wereinjecteddorsallyatthe4-cellstagewithProfilin1 mesendodermal andneuralmarkers byinsituhybridization. Xwnt8 mesodermal marker expression ofthepanmesodermalmarker injection ofProfilin1RNA norProfilin1 MOinhibitedthe gene expression inanimalcapexplants treatedwithactivin. Neither fate specification.We testedthispossibilitybyexamining marker affect gastrulationmightinvolve thedisruptionofmesodermalcell One mechanismbywhichProfilin1overexpression ordepletionmay Profilin1 andmesodermalcellfatespecification Profilin: aDaam1effector (Fig. 7).Atstage12suchembryosexpressed open blastopore(Fig.7). Profilin1 MO-injectedembryos, in thedeepanteriormesendodermwhereasProfilin1 and expression incontrolembryoswas observed intheprechordalplate large blastoporethatfailed toclose(Fig.7).Atstage13 induction. other inneuralectodermthatmaybeinducedviaplanar expression intheanteriormesodermthatfails toinvolute, andthe 7), onenext totheopenblastopore,whichprobablyreflects embryos, two separate stage 13.However, inProfilin1andMO-injected mesodermal andoverlying neuraltissuesincontrol embryosat of Profilin1doesnotinterferewith mesodermspecification. open blastoporebut lacks neuralplatemorphology(Fig.7). 2 at stage14.InProfilin1and MO-injectedembryos, expression was seen inabroaddorsalregion thatsurroundsthe We next examined theexpression andlocalizationof These experiments demonstrate thatoverexpression ordepletion (see Fig.S2Dinthesupplementarymaterial). ⌬ Sox-2 N-Profilin1 RNA. TheProfilin1and MO ⌬ is apan-neuralmarker that markstheneuralplate N-Profilin1 RNA asdescribedabove (Fig.6D,E). chordin Xbra Otx-2 Otx-2 , anddorsal , andventrolateral mesodermalmarker expression domainswereseen(Fig. Gsc was expressed anteriorlyinboth ⌬ N-Profilin1 RNA orProfilin1 expression remainednearthe Gsc brachyury and Xbra Otx2 surrounding a ( Xbra expression ), dorsal Sox- Gsc injected Xfz7, which was demonstrated tointerferewithtissue separation was observed (Fig.8E,F).Asapositive control we cultured theexplants on wildtypeanimalcaps.Noeffect ontissue explanted theaxialmesodermal region oftheinjectedembryos,and a tracerGFPRNA intothedorsalcellsof4-cellembryo, To examine tissueseparation,weinjectedProfilin1 RNA orMOand blastopore closure,neuralfold orany combinationthereof. in gastrulation,whichthusmightfunctiontissueseparation, or depletionofProfilin1nevertheless suggest aroleforthisprotein extension movements. Thephenotypesweobserved withexpression As ourresultsabove show, Profilin1doesnotregulate convergent Profilin1 regulates blastopore closure gastrulation. notrequiredforconvergent extension movements during is elongation (Fig.8C,D).TheseresultsdemonstratethatProfilin1 whereas dominantnegative Dsh,Xdd1,potentlyinhibited had noeffect onelongationoftheKeller explants (Fig.8C,D), 1985). Againweobserved thatexpression ordepletion ofProfilin1 undergoing convergent extension movements invivo (Keller etal., zone (Keller) explants, whichmorecloselyreflectthetissue Profilin1 inconvergent extension movements usingdorsalmarginal convergent extension movements. We thereforetestedtheroleof (Habas etal.,2003;Tahinci andSymes,2003)potentlyinhibit 2001), Rho(Habasetal.,2003;Tahinci andSymes,2003)Rac Fz7 (Djianeetal.,2000),Dsh(Sokol, 1996),Daam1(Habasetal., canonical Wntpathway includingWnt11(Tada andSmith,2000), effect onelongationoftheexplants (Fig.8A,B). resulted insevere gastrulationphenotypes(Fig.5A,D,E)hadno We observed that expression ordepletionofProfilin1atdosesthat elongation characteristicofgastrulation(SymesandSmith,1987). activin-treated animalexplants whichexhibit morphogenetic examined theeffects ofoverexpression ordepletionofProfilin1on Profilin1 functionsinconvergent extension movements, wefirst Profilin1 suggestaroleingastrulation.To delineatewhether The phenotypesobserved withoverexpression ordepletionof Profilin1 andconvergentextensionmovement This resultwas surprisingasallknown components ofthenon- RESEARCH ARTICLE the topofeachbar. cells evaluatedisshownat NIH3T3 cells.Numberof Daam1 expression in formation inducedbyC- GFP siRNAonstress fiber the effects onProfilin1 and panel. ( the sideofeachrespective areas (white)are shownat magnifications oftheboxed but notGFPsiRNA, expression ofProfilin1 siRNA effect isabrogated byco- stress fibers(green), andthis in NIH3T3cellsinduces Myc-tagged C-Daam1(red) induction. mediated stress fiber required forDaam1- Fig. 5.Profilin1 is B ) Quantificationof ( A ) Expression of 4225

DEVELOPMENT marginal zoneofthe4-cellembryo,andphenotypeswere scored atthetadpolestage. ( phenotype asoverexpression ofProfilin1 RNA (see A).Thisphenotypeisreversed byinjectionof the endogenouslevelsofRhoandRacproteins. ( construct ( embryos havingopenneuralfoldsandreduced anteriorstructures. ( 4226 marginal zone ofthefour-cell embryo. Thesestudiesrevealed a time lapseimagingafterinjection withProfilin1RNA intothedorsal blastopore closureratherthanneural foldclosure. These datastronglysuggests a roleforProfilin1specificallyin majority ofinjectedembryosblastopore closurefailed (Fig.6A,D). injected embryosdisplayedadelay inblastoporeclosure,anda closure. Indeedweobserved thatProfilin1RNA orProfilin1MO- defect inblastoporeclosureratherthanadirecteffect onneuralfold posterior oftheembryossuggestingthatphenotypemayreflect a neural foldclosuredefectsintheseembryoswerelocalizedto the injection dorsallyormediallyhadnoeffect (notshown). However, the by co-expression ofthe not laterally(Fig.9B).Theeffects ofProfilin1MOcouldberescued Profilin1 MOdidaffect neuralfoldclosurewheninjectedmediallybut respectively (Fig.9A).Thesestudiesrevealed thatProfilin1RNA and These injectionsselectively target themedialorlateralneuralplate, into thedorsalmedialormarginal cellsofthe16-cellembryo. (Wallingford andHarland, 2002)byinjectingProfilin1RNA orMO this purposeweemployed thestrategy ofWallingford andHarland not regulate tissueseparationduringgastrulation. observations (Fig.8E,F).ThesestudiesindicatethatProfilindoes separation (Winklbauer etal.,2001),andcouldconfirmthese Fig. 6.XProfilin1 isrequired forgastrulation. E Quantitation ofthephenotypicresults from overexpression ordepletionofXProfilin1. Injectionswere performedintothedorsa ) We next examined theprocessofblastopore closuredirectlyusing We next examined theroleofProfilin1inneuralfoldclosure.For RESEARCH ARTICLE ⌬ N-Profilin1). ( ⌬ C N-Profilin1 RNA (Fig.9B),andcontrolMO ) InjectionoftheXProfilin1 MOinhibitstranslationofMyc-taggedProfilin1 inadosedependentwaybutdoesnotaffect D ( A ) DorsalinjectionofXProfilin1 MOinhibitsgastrulationandresults inasimilargastrulationdefect ) InjectionofProfilin1 RNAdorsallybutnotventrallyinhibitsgastrulationwiththeresulting B ) XProfilin1 Morpholino(MO)sequenceanddiagramoftheProfilin1 rescue of Daam1and colocalizes withDaam1toactin stressfibersin in non-canonicalWntsignaling. Profilin1bindstotheFH1domain In thisstudywedemonstratedthat Profilin1isaneffector ofDaam1 DISCUSSION signaling pathway during gastrulation. suggest thatProfilin1functions withDaam1inthenon-canonical increased substantially(Fig.9D).Taken togetherthesestudies Profilin1 MOwereco-injected,thenumberofaffected embryos minority oftheembryos(Fig.9D).However, whenbothDaam1and induce failure ofblastoporeclosureandgastrulationin onlya threshold levels ofDaam1MOorProfilin1 MO, whichindividually functions inthesamepathway. Embryoswereinjectedwithsub- blastopore closure,whichwould beexpected ifthetwo factors Profilin1 andDaam1totestforinteractive effects ongastrulationand Lastly, weperformedexperiments tosimultaneouslydeplete blastopore closure Loss ofProfilin1 andDaam1 synergisticallyinhibit Profilin1 (Fig.9C). closure andthisphenotypecouldberescuedbyco-injectionof Profilin1 usingMOrevealed asimilarfailure ofblastopore failure ofblastoporeclosure(Fig.9C).Depletionendogenous dramatic delayandinthemajorityofinjectedembryosacomplete ⌬ N-Profilin1 withtheXProfilin1 MO. Development 133(21) l orventral ⌬ N-

DEVELOPMENT Profilin: aDaam1effector blastopore closure duringembryogenesis. Profilin1 inWnt-andDaam1-mediated cytoskeletal changesandfor remained unknown (Witke, 2004).Herewereportafunctional rolefor their functionandmorphogenetic processregulated byProfilin1 filaments invitrobut the signalingpathways thatrequireProfilin1for proteins andProfilin1canstimulate thepolymerizationofactin activation andcytoskeletal changes. FH1, FH2,etc.)torecruitandregulate factors requiredforRho as ascaffolding proteinandutilizesindependentdomains(GBD, accomplishes theseeffects isnotknown. Itislikely thatDaam1acts required forgastrulationcellmovements but how Daam1 Dishevelled andtheRhoGTPase inmediatingcytoskeletal changes signaling (Habasetal.,2001).Daam1functionsasalinkbetween Daam1 isaFormin proteinthatisrequiredfornon-canonicalWnt Profilin1 isaneffector forDaam1 architecture. Wnt signalingtotheregulation ofthecellularcytoskeletal gastrulation. Thesefindingsilluminateamolecularpathway linking downstream ofDaam1forblastoporeclosureduring Daam1 overexpression. We furthershowed thatProfilin1isrequired inhibits stressfiberformationinducedbyWntstimulationand response toWntstimulation.DepletionofProfilin1specifically expression isseensurrounding theopenblastopore. expression domainsare obvious.Sox-2isexpressed intheneuralplateatst14(arrowheads), butinProfilin orXProfilin1 MO-in both mesodermalandoverlyingneuraltissuesincontrol embryosatst13butinProfilin orXProfilin1 MO-injectedembryos,twos blastopore (arrowheads), butinProfilin orXProfilin1 MO-injectedembryoremains trappedneartheopenblastopore. Otx-2isexpr large blastopore thatdoesnotcloseatst12.Gscexpression incontrol embryosatst13isobservedinanteriormesoendoderm pg). Injectedembryosshownormalexpression ofmesodermalXbra,anddorsalGscOtx2atst10.5,butexhibitanXbraexpres the control MO(50ng)aftergastrulationdefects.ThephenotypeinducedbytheXProfilin1 MOcanberescued byco-expression of normal mesoendodermandneuralinduction,butabnormaltissuelocalizationduetogastrulationdefects.Noeffects were observe Fig. 7.Profilin1 doesnotinterfere withmesoderminduction. Profilin1 was biochemically purifiedasoneofthefirstactinbinding Xenopus Embryos injecteddorsallywithProfilin1 (2ng)orXProfilin1 MO(50ng)show small GTPase Rhoactsdownstream ofDishevelled inthissignaling protrusions thatregulate cellmovements duringgastrulation,andthe is requiredinvivo intheformationandstabilizationoflamellopodial 2003; Shibamotoetal.,1998;Torres andNelson,2000). Dishevelled shape changesandregulates motility(Endoetal.,2005;Qiang cells, stimulationthroughthenon-canonical Wntpathway induces polarization andcytoskeletal reorganization. Inmammalian cultured The non-canonicalWntpathway playsimportantroles incell signaling Profilin1 isacomponent ofnon-canonicalWnt that bindtotheFH1domainformorphogenesis. likely thatthisactivity iscoordinatedwithfactors suchasProfilin polymerization ofactinfilamentsinvitro(Krebsetal.,2001)and it FH2 domainofFormins includingmDia1canstimulatethe 2003; Watanabe etal., 1997).Recentstudieshave shown thatthe proteins inmediatingactinpolymerization(Wallar andAlberts, previously andProfilinwas implicatedasaneffector forFormin the FH1domainofFormins suchasmDia1hasbeenreported domain ofDaam1(Fig.1B,C).AninteractionbetweenProfilinand physiological interaction(Fig.1C).Profilin1bindstotheFH1 between endogenousProfilin1andDaam1indicatinga immunoprecipitation (Fig.1B),andwedemonstratebinding Profilin1 interactswithDaam1asassayedbyco- RESEARCH ARTICLE jected embryos,Sox-2 far from theclosed eparate Otx-2 essed anteriorlyin d withinjectionsof sion surrounding a

⌬ N-Profilin1 (50 4227

DEVELOPMENT pathway. morphological changesareindependentofthecanonicalWnt catenin wereobserved (Fig.4A),supportingthenotionthat cytoskeleton but noeffects ontheaccumulationofnuclear that inthesedepletionstudiesdramaticeffects ontheactin Wnt signaling(Fig.4A-CandFig.5A,B).Itisimportanttonote for Profilin1incytoskeletal changesmediatedby non-canonical Wnt, depletion ofProfilin1abrogatesstressfiberformationinducedby be regulated byaWnt-dependentmechanism.Furthermore, cells. Theseobservations suggestthattheactivity ofProfilin1may or Profilinhasbeenlocalizedtoactinstressfibersinmammalian 2C,D). To ourknowledge, thisisthefirsttimethataFormin protein stress fibersinresponsetoWntstimulationNIH3T3cells(Fig. changes, weshowed thatProfilin1andDaam1are localizedtoactin Daam1. addition totheRhopathway downstream ofDishevelled and suggesting thatProfilin1isrequiredforcytoskeletal changesin Profilin1 doesnotinduceorinhibitRhoactivation (Fig.1D), cascade (Endoetal.,2005;Wallingford etal.,2000).We showed that 4228 Characterizing therequirementforProfilin1incytoskeletal ⌬ DIX-Dishevelled orC-Daam1,demonstratingarequirement RESEARCH ARTICLE ␤ - material andFig.7),althoughgastrulationdefectsledtospatial manipulating Profilin1levels (seeFig.S1Dinthesupplementary expression levels ofneuralmarkers werenotaffected by tested wereunaffected bymanipulatingProfilin1. Likewise, specification, asexpression levels ofallmesodermalmarker genes depletion ofProfilin1didnotresultfromafailure ofmesodermal signaling. conclude thatProfilin1doesnotplayaroleincanonicalWnt (see Fig.S1A-Cinthesupplementarymaterial).We therefore inhibit target genesofthecanonicalpathway inanimalexplants Wnt- orDishevelled-induced secondaryaxisformationanddidnot Profilin1 didnotinduceasecondaryaxis,interferewith signaling (Habasetal.,2001)andwehave shown herethat have demonstratedthatDaam1isnotacomponentofcanonical and Dawid, 2005;Wallingford andHabas,2005).Previously we how Dishevelled channelssignaling intothesepathways (Habas Dishevelled andconsiderableeffort hasbeenexpended todecipher Wnt signalingbranchesintothreemainpathways downstream of signaling Profilin1 isnotrequired forcanonicalWnt The gastrulationdefectsobserved withoverexpression or as alongelongation. ratio of2orhigherwasscored elongation andalength/width below 2wasscored asashort explants, alength/widthratio as elongated.ForKeller ratio of2orhigherwasscored explant assays,alength/width separation assays.Foranimal ( assays in( the convergentextension ng)does.Quantitationof (1 whereas injectionofXfz7 tissue separationby1hr XProfilin1 MO)doesnotinhibit or depletionofProfilin1 (50ng ( movements. (Xdd1, 2ng)inhibitsthese dominant negativeDishevelled Keller explantswhereas treated withactivinor( extension inanimalexplants does notinhibitconvergent Profilin1 (50ngXProfilin1 MO) (2 ngRNA)ordepletionof extension. separation orconvergent required fortissue Fig. 8.Profilin1 isnot D E Overexpression (2ngRNA) ) Keller explantsand( ) Development 133(21) B ) animalexplants, ( A Overexpression ) C F ) in )

DEVELOPMENT signaling pathway regulates convergent extension movements have previously shown thataWnt-11/Fz/Xdsh/Daam1/Rho pathway (Tada andSmith,2000;Wallingford etal.,2000).We signaling, andDishevelled isanimportantcomponentofthis This morphogeneticprocessisregulated bynon-canonicalWnt axial extension andneuralfoldclosure(Wallingford etal.,2002). polarization andmigrationevents thatmediateblastoporeclosure, Vertebrate gastrulationinvolves adynamicseriesofcell Profilin1 isrequired forblastopore closure specification intheembryo. conclude thatProfilin1doesnotaffect mesodermal andneural Wnt signaling(Djianeetal.,2000;Habas2001).We therefore mesodermal specificationisunalteredbyinhibitionofnon-canonical results areconsistentwiththoseofprevious studies showing that mislocalization ofbothmesodermalandneuralmarkers. These Profilin: aDaam1effector closure exclusively intheposteriorregion oftheembryo(Fig.9B), Profilin1 indifferent regions andobserved effects onneuralfold targeted injectionstodirectoverexpression ordepletionof We furthertestedtheroleofProfilin1inneuralfoldclosureusing Profilin1 intissueseparationandobserved noeffects (Fig.8E,F). has noeffect onelongation(Fig.8A-D).We testedforaroleof activin orinKeller explants, expression ordepletionofProfilin1 morphogenetic processes.Inanimalcapexplants treatedwith Keller etal.,2003). morphogenetic processeshasremainedunclear(Keller, 2002; distinct effectors arespecificallyutilizedin thedifferent closure (Veeman etal.,2003;Wallingford etal.,2002).Whether convergent extension movements, tissueseparationandneuralfold implicated thenon-canonicalWntpathway inblastoporeclosure, during gastrulation(Habasetal.,2001),andmany studieshave We have examined theroleofProfilin1ineachthese signaling pathway (seeDiscussion). functions inthenon-canonicalWnt expression of phenotype canberescued byco- failure ofblastopore closure. This folds intheposterior, reflecting a fold closure butleadstoopenneural folds doesnotinhibitanteriorneural Profilin1 (50ngMO)intheneural Profilin1 RNA(2ng)ordepletionof ( were scored asintermediate tomild. shortened APaxisorbentbody or delayedblastopore closure anda Embryos withasmallopenblastopore were scored assevere embryos. reduced anteriorposterior(AP)axis an openblastopore andsignificantly and/or XDaam1MO,Embryoswith or co-injectionsofXProfilin1 MO phenotypes observedwithseparate gastrulation. Quantitationofthe XDaam1 synergisticallyinhibit ( injection of50pg MO phenotypeisrescued byco- of blastopore closure. TheXProfilin1 showing adelayandeventualfailure (2 ng)orXProfilin1 MO(50ng) embryos injectedwithProfilin1 RNA closure. ( Profilin1 hasnoeffect onneuralfold Lateral overexpression ordepletionof ( expression totheneuralfolds. the medialblastomeres targets neural foldswhereas injectioninto targets expression lateralofthe blastomeres atthe16-cellstage localized GFPinjectedintothelateral RNA (50pg)encodingmembrane- (Wallingford andHarland,2002). from Wallingford andHarland or lateraltotheneuralfolds,adapted to overexpress ordepleteProfilin1 in ( required forblastopore closure. Fig. 9.Profilin1 isspecifically E D B A ) AmodelforhowProfilin1 Targeted overexpression of ) ) DepletionofXProfilin1 and ) Schemaoftheinjectionapproach RESEARCH ARTICLE C ) Time lapseimagesof ) Time ⌬ N-Profilin1 (50pg). ⌬ N-Profilin1 RNA. 4229

DEVELOPMENT required forblastoporeclosure.In an actinpolymerizationfactor, itcanmediatecytoskeletal changes recruited toaDishevelled/Daam1 complex (Fig.9E).AsProfilin1 is gastrulation inwhichDishevelled bindstoDaam1andProfilin1is We proposeamodelofnon-canonicalWntsignaling during Wnt signaling A modelforProfilin1 function innon-canonical activation inresponsetoWntstimulation. other effector moleculessuchastheRho-GEF, whichtriggersRho Daam1 toitsfunctionduringgastrulationaswelltheidentity of investigation ofthecontribution oftheindividual domainswithin the actincytoskeleton byDaam1andProfilin1willrequireadetailed (Matusek etal.,2006).Themechanismofthisdynamiccontrol over Drosophila Profilin1. Indeedarecentstudyhasrevealed aroleforDaam1in and weproposethisismediatedbytheactionofDaam1 and generation executed bytheactincytoskeleton (Keller etal.,2003) The ‘pursestring’mechanismofblastoporeclosurerequiresforce reorganization oftheactincytoskeleton duringblastoporeclosure. stimulation, thisDaam1/Profilin1complex isprobably required for abrogates inductionofactinstressfibersinresponsetoWnt Daam1 andProfilin1toactinstressfibersdepletionof during gastrulation.AsWntsignalinginducesacolocalizationof molecule Profilin1tomediateactinpolymerizationforcellmotility et al.,2001).We proposeherethatDaam1utilizestheeffector the actincytoskeleton forconvergent extension movements (Habas the Formin proteinDaam1mediatessignalingfromDishevelled to identified forthisprocess(Kwan andKirschner, 2005).Furthermore, changes tothemicrotubule cytoskeleton andaRho-GEFwas resolved. Recentstudieshave identifiedadynamicrequirementfor of themicrotubule network andcellularactincytoskeleton arepoorly However, whatfactors controltheactive assemblyanddisassembly which arecontrolledbyDishevelled (Wallingford etal.,2000). dependent onthestabilizationofprotrusionstermedlamellipodia, Wallingford etal.,2002).Additionallythesemovements are convergent extension movements (Keller, 2002;Keller etal.,2003; polarization ofthemigratingcellsformediolateralintercalationand changes tothecytoskeleton andtocellpolarity, involving It isclearthatcellmotilityduringgastrulationrequiresdynamic during gastrulation The molecularbasisforcytoskeletalchanges morphogenetic event. branch pointinthenon-canonicalpathway forthisspecific component specificallyrequiredforblastoporeclosureprovides a the actincytoskeleton. TheidentificationofProfilin1 asamolecular mediates asignalderived fromthenon-canonicalWntpathway to cytoskeleton (Keller etal.,1985),andwesuggestthatProfilin1 generation forblastoporeclosureprobablyinvolves theactin previously toberequiredforblastoporeclosure alone.Force component ofthenon-canonicalWntpathway hasbeenshown unaffected. although axialextension willbenormalifconvergent extension is result inafailure oftheneuralfoldtocloseinposterior observed insuchembryos.Afailure ofblastopore closurewill delayed blastoporeclosure(Fig.9C),whichcanexplain thedefects closure. Embryosoverexpressing ordepletedinProfilin1show indicative ofanindirecteffect throughinhibitionofblastopore 4230 modulation of the actincytoskeleton (Fig.9E).Wntsignaling leads toRhoandROCK activation thatindependently resultsin These resultsareintriguinginthecontext thatnosingle RESEARCH ARTICLE in actinpolymerizationduringtrachealdevelopment Xenopus gastrulation, Daam1also Keller, R., Davidson,L.A.andShook,D.R. Keller, R. Alberts, A.S. References http://dev.biologists.org/cgi/content/full/133/21/4219/DC1 Supplementary materialforthisarticleisavailableat Supplementary material Dimes andanNSFgrant(#0544061)toR.H. Heart Association,aBasilO’ConnorStarterScholarAward from theMarch of Cancer Research toA.S.;andbytheFoundationofUMDNJ,American from theUeharaMemorialFoundationandNewJerseyCommissionon of theNationalInstituteChildHealthandHumanDevelopment;bygrants for useoftheirmicroscopes. Thisworkissupportedinpartbyintramuralfunds reagents Wadsworth andweare gratefultoDrs.William andMichaelMatisse discussions. We thankDrsJeffrey Miller, SergeiSokolandMaszumiTada for critical comments,andmembersoftheDawidlaboratoryforstimulating We thankSunitaKramer, MichaelTsang andMichaelShenfordiscussion as branchesofthenon-canonicalWntpathway. combinations ofseparateoroverlapping signalsthataregenerated different aspectsofgastrulationmovements requiredifferent gastrulation movements (Habasetal.,2003).Ourresultssuggestthat which isrequiredindependentlyforexecution ofthefullarray through Dishevelled but notinvolving Daam1alsoactivates Rac, Hukriede, N.A.,Tsang, T. E.,Habas, R.,Khoo,P. L.,Steiner, K.,Weeks, D.L., Kato, Y., Habas, R.,Katsuyama,Y., Naar, A.M.andHe, X. Habas, R.andHe,X. Cooley, L.,Verheyen, E.andAyers, K. Capelluto, D.G.,Kutateladze,T. G.,Habas,R.,Finkielstein, C.V., He,X.and Cao, L.G.,Babcock,G.Rubenstein,P. A.andWang, Y. L. Cadigan, K.M.andLiu,Y. I. Habas, R.,Kato,Y. andHe,X. Habas, R.andDawid,I.B. Frazier, J.A.andField,C.M. Evangelista, M.,Pruyne,D.,Amberg,D.C.,Boone,C.andBretscher, A. Djiane, A.,Riou,J.,Umbhauer, M.,Boucaut,J.andShi,D. Endo, Y., Wolf, V., Muraiso,K.,Kamijo,Soon,L.,Uren, A.,Barshishat- Habas, R.,Dawid,I.B.andHe,X. Harland, R.andGerhart,J. Higgs, H.N. He, X.,Semenov, M.,Tamai, K.andZeng,X. He, X.,Saint-Jeannet,J.P., Wang, Y., Nathans,J.,Dawid,I.and Varmus, H. biomechanics ofgastrulation. movements. signalling. component oftheARC/Mediatorcomplex required forTGFbeta/Nodal 12 cell movementsduringgastrulation. Tam, P. P. andDawid,I.B. Methods Enzymol. the finalfrontier? 69 required forintercellular cytoplasmtransportduringDrosophila oogenesis. and vesicularmembranes. Overduin, M. 117 profilin andprofilactin onactinstructure andfunctioninlivingcells. Cell Sci. Cell vertebrate gastrulationandrequires anovelForminhomology protein Daam1. organizers? cell growth inyeast. (2002). Forminsdirect Arp2/3-independentactinfilamentassemblytopolarize 777-786. gastrulation inXenopuslaevis. frizzled 7intheregulation ofconvergentextensionmovementsduring RhoA activationandisspecificallyregulated bydishevelled-2. Kupper, M.andRubin,J.S. Wnt/Frizzled signalingisrequired forvertebrategastrulation. organizer. 295-309. Sci. Development proteins 5and6inWnt/beta-catenin signaling:arrows pointtheway. Wnt-5A. (1997). AmemberoftheFrizzledprotein familymediatingaxisinductionby , R796. , 173-184. , 1023-1029. 30 107 , 342-353. (2002). Shapingthevertebratebodyplan bypolarizedembryoniccell , 843-854. 119 Science Annu. Rev. CellDev. Biol. Nature (2005). Forminproteins: adomain-based approach. Curr. Biol. , 395-402. (2002). Diaphanous-related Forminhomologyproteins. Science 131 (2002). TheDIXdomaintargetsdishevelledtoactinstress fibres 275 , 1663-1677. J. Biol. 418 406 (2006). ActivationofRhoandRacbyWnt/frizzledsignaling. Nat. CellBiol. 298 , 1652-1654. , 641-646. 7 , 500-511. , R414-R417. , 1950-1954. 4 (2005). DishevelledandWntsignaling:isthenucleus , 2. Nature (1997). FormationandfunctionofSpemann’s (2006). Wntsignaling:complexityatthesurface. (2003). Conservedrequirement ofLim1functionfor (1997). Actincytoskeleton:are FHproteins local (2005). Wnt-3a-dependentcellmotilityinvolves Differentiation (2001). Wnt/FrizzledactivationofRhoregulates Development 419 (2003). CoactivationofRacandRhoby 4 13 , 32-41. Dev. Cell , 726-729. , 611-667. (1992). chickadeeencodesaprofilin (2003). Howweare shaped:the 127 71 4 (2004). LDLreceptor-related , 83-94. , 171-205. , 3091-3100. Development 133(21) (2002). A (2000). Roleof Genes Dev. J. Biol.Chem (1992). Effects of Trends Biochem. Curr. Biol. J. CellBiol. 17 . Cell , 280 J. ,

DEVELOPMENT Li, L.,Yuan, H.,Xie,W., Mao,J.,Caruso,A.M.,McMahon,A.,Sussman,D.J. Sokol, S.,Christian,J.L.,Moon,R.T. andMelton,D. A. Marlow, F., Topczewski, J.,Sepich,D.andSolnica-Krezel, L. Kwan, K.M.andKirschner, M.W. Krebs, A.,Rothkegel,M.,Klar, M.andJockusch,B. Kovar, D.R.,Harris,E.S.,Mahaffy, R.,Higgs,H.N.andPollard, T. D. Kovar, D.R.andPollard, T. D. Keller, R.E.,Danilchik,M.,Gimlich,andShih,J. Profilin: aDaam1effector Shibamoto, S.,Higano,K.,Takada, R.,Ito,F., Takeichi, M.andTakada, S. Shih, J.andKeller, R. Severson, A.F., Baillie,D.L.andBowerman,B. McMahon, A.P. andMoon,R.T. Matusek, T., Djiane,A.,Jankovics,F., Brunner, D.,Mlodzik,M.and Moon, R.T., Brown, J.D.andTorres, M. Roy, P. andJacobson,K. Qiang, Y. W., Endo,Y., Rubin,J.S.andRudikoff, S. Nakaya, M.A.,Habas,R.,Biris,K.,Dunty, W. C.,Jr, Kato,Y., He, X.and Mikels, A.J.andNusse,R. Ren, X.D.,Kiosses,W. B.andSchwartz,M.A. Chem. Regulation ofLEF-1andc-JunN-terminalkinaseinmammaliancells. and Wu, D. Development controls cellmorphologyduringconvergentextensionofXenopuslaevis. Control oftheassemblyATP- andADP-actinbyforminsprofilin. Suppl Exp.Morphol. Embryol. mechanism ofconvergentextensionduringgastrulationXenopuslaevis. assembly ofcorticalmicrofilaments inC.elegans. protein andaprofilin are required forcytokinesisandArp2/3-independent RNA inducesacompletebodyaxisinXenopusembryos. mesoderm ofXenopuslaevis. convergence andextensionmovements. Rho kinase2actsdownstream ofWnt11tomediatecellpolarityandeffective actin cytoskeleton. of functionaldomainsmDia1,alinkbetweenthesmallGTPaseRhoand 423-435. Acad. Sci.USA barbed endsinassociationwithforminsproduces piconewtonforces. Cells (1998). Cytoskeletalreorganization bysolubleWnt-3aprotein signalling. of invasivebreast cancercells. 966. throughpattern organizingtheactincytoskeleton. J. Mihaly, behavior duringvertebratedevelopment. e115. beta-catenin-TCF signalingdependingonreceptor context. Cell oncogene int-1inXenopusembryosleadstoduplicationoftheembryonicaxis. 578-585. GTP-binding protein Rhobycelladhesionandthecytoskeleton. cell neoplasia. Daam genesinmouseandXenopus. Yamaguchi, T. P. 58 3 , 659-670. , 1075-1084. 274 , 129-134. (2006). TheDrosophila forminDAAMregulates thetrachealcuticle (1999). Dishevelledproteins leadtotwosignalingpathways. 132 Oncogene 101 , 4599-4610. (2004). Identificationandcomparativeexpression analysesof J. CellSci. , 14725-14730. (1992). Patterns ofcellmotilityintheorganizeranddorsal (1992). Patterns (2004). Overexpression ofprofilin reduces themigration 22 (2006). PurifiedWnt5aprotein activatesorinhibits , 1536-1545. 114 . (2004). Insertionalassemblyofactinfilament Development Cell Motil.Cytoskeleton 89 (1989). Ectopicexpression oftheproto- , 3663-3672. , 185-209. (2005). Amicrotubule-binding Rho-GEF Gene Expr. Patterns Curr. Biol. (1997). WNTs modulatecellfateand Trends Genet. 116 , 915-930. (1999). Regulationofthesmall (2002). AForminHomology Curr. Biol. 12 (1985). Thefunctionand Development (2003). WntsignalinginB- , 876-884. 57 13 (2001). Characterization Cell , 84-95. 5 (1991). InjectedWnt , 157-162. , 97-105. 12 67 PLoS Biol. (2002). Zebrafish , 2066-2075. , 741-752. MOJ. EMBO 133 J. Biol. Proc. Natl. , 957- Cell (2006). 4 Genes 18 , J. 124 , , Witke, W. Watanabe, N.and Higashida,C. Winklbauer, R.,Medina,A.,Swain,R.K.andSteinbeisser, H. Watanabe, N.,Madaule, P., Reid,T., Ishizaki,T., Watanabe, G.,Kakizuka,A., Wallingford, J.B.andHabas,R. Wallar, B.J.andAlberts,A.S. Wallingford, J.B.,Fraser, S.E.andHarland,R.M. Wallingford, J.B.,Rowning,B.A.,Vogeli, K.M.,Rothbacher, U.,Fraser, S.E. Wallingford, J.B.andHarland,R.M. Verheyen, E.M.andCooley, L. Veeman, M.T., Axelrod, J.D.andMoon,R.T. Torres, M.A.andNelson,W. J. Tada, M.andSmith,J.C. Zigmond, S.H. Tahinci, E.andSymes,K. Symes, K.andSmith,J.C. Sokol, S.Y. Witke, W., Sutherland,J.D.,Sharpe,A.,Arai,M.andKwiatkowski,D. Yamanaka, H.,Moriguchi,T., Masuyama, N.,Kusakabe,M.,Hanafusa,H., 856-860. 7 signallingcontrols tissueseparationduringXenopusgastrulation. actin filaments. GTPase andisaligandforprofilin. mammalian homologofDrosophila diaphanous,isatargetprotein forRhosmall Saito, Y., Nakao,K.,Jockusch,B.M.andNarumiya, S. development. extension: themolecularcontrol ofpolarizedcellmovementduringembryonic Development Dishevelled: anenigmaticproteincellfateandpolarity. governing 5815-5825. Dishevelled-dependent convergentextensionofthemidline. the cytoskeleton. 728. gastrulation. and Harland,R.M. dependent processes duringDrosophila development. 5 Functions andmechanismsofbeta-catenin-independentWntsignaling. Biol. dishevelled andactinduringWnt-inducedmesenchymalmorphogenesis. Cell Biol. 318-335. canonical Wntpathway. regulation ofgastrulationmovementsviaDishevelled,butnotthrough the 349. marker ofmesoderminductionin processes. required forconvergentextensionduringXenopusgastrulation. development. development. (2001). Profilin Iisessentialforcellsurvivalanddivisioninearlymouse vertebrates. canonical Wntpathwaytoregulate convergentextensionmovementsin Takada, R.,Takada, S.andNishida,E. , 367-377. 149 (2004). Therole ofprofilin complexesincellmotilityand othercellular , 1433-1442. 16 (1996). AnalysisofDishevelledsignallingpathwaysduringXenopus Trends CellBiol. , 99-105. EMBO Rep. Nature 132 (2004). Formin-inducednucleationofactinfilaments. Dev. Cell Curr. Biol. Proc. Natl.Acad.Sci.USA Exp. CellRes. Trends CellBiol. , 4421-4436. 405 (2000). Dishevelledcontrols cellpolarityduringXenopus 2 3 , 81-85. 6 Development , 695-706. (2000). Xwnt11isatargetofXenopusBrachyury: , 69-75. (2003). DistinctfunctionsofRhoandRacare , 1456-1467. 14 (1987). Gastrulationmovementsprovide anearly 301 , 461-469. (2003). Theformins:activescaffolds thatremodel (2000). Colocalizationandredistribution of (1994). Profilin mutationsdisruptmultipleactin- (2005). Thedevelopmentalbiologyof (2004). Formins:processive cappersofgrowing , 16-22. 13 EMBO J. Xenopus laevis , 435-446. (2002). Neuraltubeclosure requires 127 98 (2002). JNKfunctionsinthenon- RESEARCH ARTICLE , 2227-2238. , 3832-3836. 16 , 3044-3056. (2003). Asecondcanon. . (2002). Convergent Development Development (1997). p140mDia,a Development (2001). Frizzled- Dev. Biol. 101 Nature Curr. Opin. 120 , 339- Dev. Cell , 717- J. Cell 4231 129 413 259 , , ,

DEVELOPMENT