models onPCPandconvergentextension. neuroepithelium, incontrasttoitsdrasticeffectonDvl2localizationthecochlea.Theseresultsarediscussedlightof convergent extensionintheneuroepitheliumandPCPcochleadoesnotdisruptDvl2membranedistribution localization, apre-requisiteforitsinvolvementinconvergentextension.Intriguing,the the PCPpathwayinfly.Inneuroepitheliumofneurulatingembryos,Dvl2showsDEPdomain-dependentmembrane allele infly,wefurtherdemonstratedahighdegreeofconservationbetweenDvlfunctionmammalianconvergentextension and (bacterial artificialchromosome)transgenesandintroducingdifferentdomaindeletionsapointmutationidentical to the interacts with lengthening andnarrowingoftheneuralplate,amorphogeneticprocessdefinedasconvergentextension. Institute, 2100West Third Street, LosAngeles,CA90057. elSegil Neil California InstituteofTechnology,California Pasadena,CA91125,USA. KEY WORDS:Mouse,Planarcellpolarity, Convergentextension,Neurulation Dishevelled polarity andpolarizedextensioninthecochleamouse.Hereweshow,usingmicewithnullmutationstwomammalian controlconvergentextension.WehaveshownpreviouslythatmammalianPCPhomologsregulatebothcell fly PCPpathway epithelium, perpendiculartotheapicobasalaxisofcell.In Drosophila The planarcellpolarity(PCP)pathwayisconservedthroughoutevolution,butitmediatesdistinctdevelopmentalprocesses.In Jianbo Wang neurulation pathway toregulateconvergentextensionduring Dishevelled genesmediateaconservedmammalianPCP Development 133,1767-1778(2006)doi:10.1242/dev.02347 Accepted 7March 2006 § † 1 determination (CadiganandNusse,1997).In anteroposterior (AP)polarityofthebodysegments andcellfate was firstdefinedin Veeman etal.,2003a; Wallingford etal.,2002).TheWntpathway 2002; Mlodzik,Myersetal.,Tree etal.,2002a; some commoncomponents(CadiganandNusse,1997;Keller, are theWntandplanarcellpolarity(PCP)pathways, whichshare of theevolved bodyplan.Two examples ofconserved pathways mechanisms areadaptedandmodifiedaccordingtothedemands embryogenesis. Duringevolution, theseconserved pathways and understanding offundamentalprocessesrequiredduring uncovered over thepastfew decades,leadingtoabasic mechanisms requiredduringeukaryoticdevelopment have been Many oftheconserved geneticpathways andcellbiological INTRODUCTION Jolla, CA92093-0627,USA. Neuroscience, SanDiego,9500GilmanDrive,MC0627, La UniversityofCalifornia, Street, Atlanta,GA30322,USA. Biology andOtolaryngology, SchoolofMedicine,EmoryUniversity, 615Michael Houston, TX77030,USA Gilman Drive,MC0627,LaJolla,CA92093-0627,USA. Biology, MedicalSchool, Norfolk,VA Virginia Eastern 23510,USA *Present address: Strelitz DiabetesInstitute, DepartmentofCellandMolecular Station C0930,UniversityofTexas, Austin,TX 78712,USA. Developmental Biology&InstituteforCellularandMolecularBiology, 1University Author forcorrespondence (e-mail:[email protected]) Department ofMolecularPhysiologyand Biophysics,BaylorCollegeofMedicine, SanDiego,9500 Department ofPediatricsandMedicine,UniversityCalifornia, , membersofthePCPpathwaylocalizeinapolarizedfashiontospecifycellularpolaritywithinplane 5 homologs, , ScottE.Fraser Loop-tail 1 , NatashaS.Hamblet Drosophila 5 Department ofCellandMolecularBiology, HouseEar Dvl1 , apointmutationinthemammalianPCPgene 3 Divison ofBiologyandBeckmanInstitute, and 3 , PingChen Dvl2 , whereitisrequiredforthe , thatduringneurulationahomologousmammalianPCPpathwayregulatesconcomitant 1, 2 *, SharayneMark Department ofCell 6 2 Molecular Celland , JohnB.Wallingford Xenopus 4 Department of , theWnt Xenopus 2 , MaryE.Dickinson (Mlodzik, 2002; Strutt,2002;Tree etal.,2002a;Veeman etal., the canonicalWntsignalingpathway aswellthePCPpathway Xenopus cell division (Gong etal.,2004). extension, atleastinpart, throughdeterminingtheorientationof zebrafish, thePCPpathway alsoappearstoregulate convergent intercalation (Jessenetal.,2002;Wallingford etal.,2000).In the polarityoflamellipodialprotrusionsthatdrive cell these animals,several PCPproteinshave beenshown tocontrol 2002; Wallingford etal., 2000).Duringconvergent extension in Takeuchi etal.,2003; Veeman etal.,2003b;Wallingford et al., Keller, 2002;Kinoshitaetal.,2003;Park andMoon,2002; Barbosa etal.,2003;Darken etal.,2002;GotoandKeller, 2002; concomitant narrowing ofthemediolateral(ML)axis(Carreira- extension, acoordinatedextension ofthe AP axiswith zebrafish, anhomologouspathway regulates convergent Drosophila Similarly, theconserved PCPpathway was firstuncovered in 2001; Wang andWynshaw-Boris, 2004;Yamaguchi, 2001). during gastrulationandorganogenesis (Huelsken andBirchmeier, pathway hasdiverse effects oncellproliferationandpatterning patterning (CadiganandNusse,1997).Inmammals,anexpanded pathway participatesinaxisdeterminationanddorsoventral (DV) 2002; Tree etal.,2002a;Veeman etal.,2003a).In orientation ofommatidialunitsthecompoundeye (Strutt, the epithelialcellsofwingandpreciselycoordinated oriented parallelarraysofcellularextensions calledtrichomes on the cell.Manifestationsofthispolarityincludesuniformly plane oftheepithelium,perpendiculartoapicobasalaxis The multifunctionalproteinDishevelled (Dshinfly, XDshin Vangl2 6 and zebrafish,severalhomologsofthecomponents and AnthonyWynshaw-Boris , andDvl1,Dvl2Dvl3inmammals) isinvolved inboth , duringneurulation.Bygenerating , whereitregulates thepolarityofacellwithin Loop-tail 3,† , Brendan C.Brinkman mutation thatdisruptsboth RESEARCH ARTICLE Dvl2 1,§ genetically Dvl2 4 , BAC Xenopus recent dsh1 1767 and

DEVELOPMENT stereocilia orientationdefectsin that atransgenicDvl2-EGFPproteincapableofrescuingthe manifestation ofPCP(Lewis andDavies, 2002).Indeed,wefound stereocilia onhaircellsinthecochleahasbeenproposedtobea sensory haircellsinthecochlea.Theuniformorientationof Dvl1 protein Armadillo/ Arrow/Lrp5/6 onthecellsurface. Asaconsequence,thecytoplasmic initiated fromWntbindingtoitsreceptorFrizzledandco-receptor the canonicalWntpathway, Dshisimportantintransmittingasignal 2003a; Wallingford etal.,2002;Wallingford andHabas,2005).In 1768 because, in mutants hasbeeninferredtobefailure ofconvergent extension, Wang etal.,2006).The causeofcraniorachischisisinthese Hamblet etal.,2002;Kibar2001;Murdoch defect termedcraniorachischisisinhumans(Curtinetal.,2003; neural tubefrommid-braintotailfails toclose,acongenital in auniquesevere neuraltubeclosuredefectinwhichtheentire disrupted in displayed asymmetricplasmamembranelocalizationthatis Crsh, Scy mechanism incoordinatingstereociliaorientation.Inaddition, 2006), suggestingaconserved PCPpathway astheunderlying Fzd6 inthesensoryhaircells,utriclesandcristae(Wang etal., a similar Loop tail 2003; Murdochetal.,2001;Wang etal.,2005;Wang etal.,2006). Hamblet etal.,2002;Kibar2001;Montcouquiol putative PCPpathway exists inmammals(Curtinetal.,2003; Veeman etal.,2003a). Strutt etal.,2002;Strutt,Tree etal.,2002a;Tree etal.,2002b; 2004; Jenny etal.,2003;Jenny etal.,2005;Rawls andWolff, 2003; pathway inthefly(Axelrod, 2001;Bastocketal., 2003;Dasetal., required foranddependentuponproperestablishmentofthePCP localization ofDshandtheotherPCPmembersappearstobeboth PCP isnotwelldefined,anasymmetricplasmamembrane Although theprecisemechanismsthroughwhichDshdetermine al., 2005;Strutt,2002;Tree etal.,2002a;Veeman etal.,2003a). (Pk), Diego (Dgo)andFlamingo(Fmi)(Dasetal.,2004;Jenny et with adifferent setofproteinssuchasStrabismus (Stbm),Prickle Wynshaw-Boris, 2004).InthePCPpathway infly, Dshparticipates into thenucleustoactivate transcription(Heetal.,2004;Wang and of neuralplate,a morphogeneticprocessthatresembles convergent and regulates convergent extension inmammals. convergent extension defects, andifso,itisnotclearhow PCP neural tubedefectinthese PCP mutantmiceresultsfrom been investigated. Thereisnoexperimental evidence thatthe mammalian PCPpathway membersduringneurulation have not developmental mechanismsdisruptedbythe lossofconserved Greene, 2003;Wallingford andHarland,2002).However, the 2003; Darken etal.,2002;GotoandKeller, 2002;Uenoand usually resultsinsimilarneuraltubeclosuredefects(Coppet al., ofXDshandStbm(whichblocksconvergent extension) forms mammals (Wang etal.,2006). ( frizzled 6 pathway member ( al., 2003;Murdochet2001),while the flyPCPcomponent Crsh, Scy Recent geneticstudieshave alsoprovided evidence thata In thisstudy, weprovide evidence thatduring neurulation, Dvl2 –/– RESEARCH ARTICLE ; Dvl2 Vangl2 ( , are requiredforconcomitantlengthening andnarrowing ) aredistinctmutationsof Lp Fzd3 Lp/Lp Fzd6 ) isapointmutationof Xenopus –/– –/– mutants resultinmisorientationofstereocilia -dependent asymmetriclocalizationofFzd3and ) encodetwo oftheFrizzledreceptorsin fmi ; Fzd6 ␤ embryos (Wang etal.,2005).Othersreported -catenin becomesstabilizedandistransported Cri ta. 03.Fize ( (Curtin etal.,2003).Frizzled3 , overexpression ofwild-typeormutant Stbm –/– and (Kibar etal.,2001;Montcouquiol Lp Dvl1 , Crsh Celsr1 Ltap –/– Dvl1 ; , Dvl2 Scy or , ahomologofthePCP Crash –/– Vangl2 , –/– Fzd3 ; mutants allresult Dvl2 and –/– , ahomologof ; Fzd6 –/– Spin cycle Fzd3 mutants –/– ) and Dvl1 and Lp , MATERIALS ANDMETHODS convergent extension andPCP. transgene suggestsstringentconservation ofDvlfunctionin specifically abolishesthePCPpathway. Ouranalysisofthemutant carrying apointmutationidenticaltothe PCP andconvergent extension, weproduceda localization. To furthertestwhetherwecangeneticallydistinguish essential forDvl2functioninneurulationandplasmamembrane Wallingford andHarland,2001;Wallingford etal.,2000),was (Axelrod etal.,1998;BoutrosRothbacher2000; Dsh/Dvl functioninPCP/convergent extension infliesand contrast, theC-terminalDEPdomain,whichissolelyrequiredfor during neurulationorrecruitmenttotheplasmamembrane.By (Capelluto etal.,2002),was largely dispensableforitsfunction domain, whichisessentialforDvl2functionintheWntpathway (Wang etal.,2005).We foundthatpartoftheN-terminalDIX contrast toitsdrasticeffect onDvl2distribution inthecochlea have noeffect onDvl2localizationintheneuroepithelium, genes duringneurulationandincochleadevelopment, extension. Intriguingly, although neuroepithelium isconsistentwithitsfunctioninconvergent Dvl2-EGFP protein,wefoundthatsubcellularDvl2localizationin GraphPad InStatsoftware. analysis was performedbyusingthe Mann-Whitney U-testthrough embryos ofeachgenotypeatsomitestageweremeasured.Statistical view imageweretaken asthefinalLWR oftheembryo.Atleastthree width measurement.Averages oftheLWR derived fromventral anddorsal between thesetwo traceswas determinedbyImageProPlusandusedforthe and rightsideswas usedforthelength measurement,theaverage distance allantoic bud tothebaseofheadfold.Theaverage ofthetracesonleft On eachembryo,alinewas drawn alongthebodyedgefrombaseof and length-to-widthratiomeasurementsofthetrunkweremadeasfollows. ventral view ofeach embryowereacquiredandimportedintoImageProPlus, dsh1-EGFP extension. Usinga reactions thatcovered theentire genomic integrity andpotentialrearrangementbyusing11 overlapping PCR screen ofaBAC libraryfromGenomeSystems,was characterizedfor A BAC clone containingthe Generating on aLeicaMZFL naturally. EmbryoswerephotographedwithaSpotCCDcameramounted numbers, embryosweretransferredintoanemptydishandallowed toextend paraformaldehyde at4°Covernight andstoredinPBS.Aftercountingsomite sacs wereremoved forPCRgenotyping.Embryoswerefixed in4% Embryos derived fromappropriatecrossesweredissectedatE8.5andyolk embryos Length-to-width ratio(LWR) measurement inneurulating (Lee etal.,2001). pronuclear injection was performedasdescribedtocreate transgenicmice to Msubstitutionataminoacid446. ModifiedBACs werepurifiedand point mutationwas generatedbyreplacingAAG withATG tointroduceaK amino acids67-159and 442to736,respectively. deletion mutanttransgenesweregenerated byremoving sequenceencoding whereas intron 2forgenotypingpurpose(seeMousestrainsandgenotyping), cassette fusedin-frametothelastcodonof were performedasdescribed(Leeetal.,2001). LoxP sitesandEGFP-codingsequencein-frametothelastcodonof genome database.ModificationsoftheBAC, includinginsertionoftwo specific PCRprimersbasedonthesequenceinformationinmouse least two flankinggenesonthe3 Using BAC recombineeringtechniques, flanking gene Dvl2-EGFP2 BAC transgenes Dvl2-EGFP Acadvl III (acyl-Coenzyme Adehydrogenase, very longchain). Dvl2 dissection microscope.Imagesofbothdorsaland contained anadditionalLoxPsiteinsertedinthe3 and mutant BAC transgeneexpressing afunctional Dvl2 Ј Dvl2 and 5 genomic sequence,derived fromaPCR Lp genomic region. Theexistence ofat Ј ⌬ ⌬ ends wereconfirmedbydesigning DIX-EGFP genetically interactswithDvl Dvl2 ⌬ DIX-EGFP and aLoxPsiteinsertedinto Dvl2-EGFP1 dsh1 Development 133(9) , Dvl2 ⌬ ⌬ allele infly, which DEP-EGFP and BAC transgene Lp ⌬ had anEGFP DEP-EGFP dsh1-EGFP appears to Xenopus and Dvl2 Ј

DEVELOPMENT FV 1000confocalscanningmicroscope. fluoroscein-conjugated phalloidin.Imagewas acquiredusinganOlympus of Corti,whole-mountorgans ofCortiwerestainedwithrhodamineor Alexa Fluor568phalloidin.For analyzingplanarcellpolarityoftheorgan (A-21206, MolecularProbe,1:1000).Embryoswerecounterstainedwith (A-6455, MolecularProbe,1:1000)andadonkey anti-rabbitIgGantibody + 5%normalgoatserum,embryoswerestainedwithananti-GFPantibody were fixed in4%paraformaldehydeat4°Covernight. AfterblockinginPBS according tothemanufacturer’s protocol.For antibodystaining,embryos counterstained withAlexa Fluor568phalloidin(A-12380,MolecularProbe) in 4%paraformaldehydeat4°Cfor1hour. Embryoswerethen embryos orcochleasofappropriatestagesweredissectedandbrieflyfixed transgene fromtheendogenouswild-type Xenopus in neuraltubeclosure(Wallingford andHarland, 2002).In XDsh-mediated convergent extension morphogeneticmovement (Hamblet etal.,2002). ate hedgehog Wnt1 Gonzalez etal.,2002).We foundnormaldorsalexpression of severe neuraltubeclosuredefects(Copp etal.,2003;Ybot- defective DV patterning inthespinalcordisknown tocause hybridization todeterminedorsoventral (DV) patterningas closure in not shown). Theseresultssuggestthatthefailure ofneuraltube Dvl3 To visualizenative GFPsignalfrom Confocal microscopy for not beamplifiedinthisPCRreaction.Thesamewas usedtogenotype gave risetoaPCRproductof404bp.Thetargeted EGFP allele gave risetoaPCRproductof370bp,whilethe seconds, 72°Cfor30seconds;35cycles). Endogenouswild-type GTCCGAGACTCATGGG) forPCR(94°C20seconds,56°C intron 2wereused(E2F, TGCTTCAATGGAAGGGTTGTC; E3R,TTCT- described (Hambletetal.,2002).To differentiate the Genotyping ofthetargeted Harbor, ME)andgenotypedasdescribed(Murdochetal.,2001). Mice carrying Mouse strainsandgenotyping Convergent extensionduringneurulation Dvl1 RESULTS bifida, while globally disruptedin Wnt target gene,suggeststhatthecanonicalWntpathway was not patterning. Furthermore,thenormal expression of and sufficient byitselffornormalneuraltubeclosure.Bycontrast, most importantmammalianDvlgeneforneuraltubeclosureandis display neuraltubedefects.Thesefindingsindicatethat As previously reported,2-3%of lengthening andnarrowing ofneuralplate Dvl1 craniorachischisis (J.W., N.S.H.andA.W.-B., unpublished).However, GCCATGTTCACTGCTGTCTCTC). CACTGAGTGCCTCTTGC; E3R, TTCTGTCCGAGACTCATGGG; R3, alleles, athreeprimerPCRgenotypingstrategy was used(I1F, TTGCT- defect in closure, but contribute significantlywhen Recent studiesin In aneffort todeterminethecauseofcraniorachischisis ⌬ ⌬ Dvl3 DEP-EGFP –/– –/– DIX-EGFP , –/– transgenes, owing totheinsertionof34bpLoxPsiteinintero2, Pax3 (Lijam etal.,1997)and double mutants(J.W. andA.W.-B., unpublished)donot ; , convergent extension resultsincoordinated elongation Dvl2 are notsufficient bythemselves fornormalneuraltube ( shh Dvl1 Dvl1 Lp and Dvl1 ) inthespinalcordof –/– mutation was obtainedfromtheJacksonLaboratory(Bar and transgenes fromtheendogenous –/– –/– mutants disruptconcomitant Engrailed2 ; –/– ; dsh1-EGFP Dvl2 Xenopus ; Dvl2 Dvl1 Dvl2 Dvl2 –/– –/– –/– Dvl1 –/– –/– mutants isnotduetoabnormalDV ; ; Dvl2 have indicated anessentialroleof mutants, weperformedinsitu mutants displaycraniorachischisis Dvl3 transgenes. To genotypeanddifferenti- Dvl2 and ventral expression of Dvl3 and Dvl2-EGFP –/– –/– –/– Dvl1 Dvl2 –/– Dvl2 mutants. double mutantsalsodisplay single mutantsdisplayspina Dvl2 single mutantsor –/– , apairofprimersflanking alleles was performedas ; is completelymissing. Dvl2 Dvl2 or Dvl2-EGFP Dvl2 dsh1-EGFP –/– Dvl2-EGFP -null allelewould En2 mutants (data and Dvl2 , aknown Dvl2 Dvl1 or sonic is the mice, dsh1- Dvl1 BAC Dvl2 null –/– ; outside ofthenotochord(Fig.2A).In the notochordwas slenderandtherewereno pattern of signaling. Wnt pathway (Fig.2A,B)andthatreduceddoseofDvlin that thereducedLWR was notduetoafailure ofthecanonical in theanteriorprimitive streakappearedtobenormal,suggesting through, atleastinpart,activating brachyury( canonical Wntpathway iscrucialfortheelongationofAPaxis canonical Wntpathway inneuraltubeclosure.Inaddition,the neural tubedefectsexencephaly orspinabifida,implicatingthe 2004) allelesoftheWntco-receptor neural tubeclosureinthemouse. as thecauseofaxisshorteningin anterior primitive streak.Thelossof In stage, function ofthecanonicalWntpathway. Intheearlyheadfold of (Yamaguchi etal.,1999).Therefore,weanalyzedtheexpression Dvl2 the LWR in (Ybot-Gonzalez etal.,2002),therewas virtuallynoincreaseof closure 1atthe7-somitestageincontrolembryos(Fig.1C) stages, immediatelybeforethefirstappositionofneuralfoldsat analysis (Fig.1E).Morestrikingly, fromthefive- toseven-somite LWR fromtheearliesttimepointwherewecouldperformthis mutant embryos,wefoundconsistent,significant reductionof 2002). To determinewhether neurulation (Wallingford etal.,2002;Wallingford andHarland, a continuousincreaseofthelength-to-widthratio(LWR) during of theAPaxisandnarrowing oftheMLaxis,and isreflectedas of absence ofcelldivision (Beddington,1994),thedifferent pattern driven byconvergent extension-like cellmovements inthe lengthening ofthenotochordinmousewas argued tobe a few mutants, however, thenotochordwas wider, and therewerealso (Kibar etal.,2003; Kibaretal.,2001;Murdoch al.,2001).Whenwe harbors aloss-of-functionmutation inthemousePCPgene To testthevalidity ofthishypothesis,we examined convergent extension Lp/Lp midline. of PCP-dependentconvergence ofthenotochordcellstowards Dvl1 (Wallingford andHarland,2002) neural tubeclosuredefectsareobserved. Assuggested in adopted themethodsusedin defects consistentwithadisruptionofconvergent extension, we et al.,1999).However, in 1E). However, whenwesystematicallymeasured that theLWR continuouslyincreasedduringneurulation(Fig. (Fig. 1;Materialsandmethods).Incontrolembryos,wefound 2002) andmeasuredtheLWR ofneurulatingmouseembryos and isaffected in tissue (Wallingford etal.,2002),occursduringmouseneurulation which isdefinedasconcomitantlengtheningandnarrowing ofa In contrasttotheprimitive streak,weobserved adifferent Null (Pinsonetal.,2000)andhypomorphic(Kokubu etal., This resultreveals that,like T T Wnt3a staining inthe to confirmthatthereducedLWR was notduetothelossof –/– –/– T T ; embryos alsodisplaydisruption of mutants doesnotcausegloballossoffunctionWnt -positive cellsoutsideofthenotochord(Fig. 2B).As is expressed inthenotochord,nodeandprimitive streak. Dvl2 -null mice, T staining inthenotochord.Inwild-typeembryos, Dvl1 –/– mutants mayalsobeincompatiblewithnormal Dvl1 –/– Dvl1 ; Dvl2 T –/– expression was reportedtobelostinthe ; –/– Dvl2 –/– Dvl1 ; Dvl2 mutant embryos. Xenopus –/– –/– Dvl1 , mutants priortothetimeatwhich Xenopus the resultingwiderneuralplatein –/– ; Wnt3a RESEARCH ARTICLE Dvl2 mutants suggestsareduction –/– T Lrp6 (Wallingford andHarland, expression was interpreted ; –/– Dvl2 –/– , convergent extension, result inthelesssevere mutants, mutants (Yamaguchi –/– Dvl1 mutants display T Dvl1 -positive cells T Lp ) expression T –/– mutant that expression –/– ; Xenopus ; Dvl1 Dvl2 Dvl2 Vangl2 1769 –/– –/– –/– ;

DEVELOPMENT ( or Fig. 1. 1770 mutant embryos,wefoundthat,similarto Furthermore, whenwedeterminedtheLWR inneurulating be duetoadisruptionofconvergent extension (Fig.2C,D). cells outsideofthenotochord,furtherevidence thatthesedefectscould similar broadeningofthenotochord,aswellstranding analyzed Dvl1 Dvl2 effect neuraltubeclosureinthemouse. that themammalianPCPpathway regulates convergent extension to along theAPaxis(Fig.3B,C).Theseresultsprovide furtherevidence in thesamestagewild-typeembryos,neuraltubehasfusedextensively neural tubesoftenremainedunfusedatthe8-to9-somitestage,while LWR, displayed amoderatereductionofLWR. Accompanying thereduced nonetheless managetoclosetheirneuraltubesinmostcases,and closure 1(Fig.3A). Lp/Lp Dvl and Harland,2002;Wallingford etal.,2000),wedetermined whether et al.,2002;GotoandKeller, 2002;Park andMoon,2002;Wallingford PCP inflyandconvergent extension in As neurulation represents theaverageofatleastthree separate embryos.Theerror barsrepresent standard deviation.* survived, 50% oftheexpected and genotypedthesurviving offspring (Table 1).Outof48pupsthat observation that50%of (expected six,recovered three),consistentwithour previous E ) Length-to-widthratio(LWR) changesofcontrol and Dvl1 Dsh and +/– embryos displayedasignificantreductionoftheLWR priorto Lp/+ Dvl1 –/– and ; RESEARCH ARTICLE and Lp Dvl2 ; T Dvl2 genetically interactduringmouse neurulation.We crossed –/– expression inthe Stbm embryos displayeddelayedclosureofneuraltube:their Lp +/– ; +/+ Dvl2 ; genetically interactduring Lp/+ control embryos.( interact witheachotherandbothareinvolved inthe –/– Lp/+ mutants displayedreduced length-to-widthratioduringneurulation. triple heterozygoteswith Dvl2 mutants displaykinky orloopedtailsbut –/– Lp/Lp Dvl2 mutants dieatbirth becauseofheart A’-D’ –/– mouse embryos,weobserved a Xenopus homozygotes wererecovered ) Dvl1 Dvl1 –/– ; and zebrafish(Darken Dvl2 Dvl2 –/– ; Dvl2 –/– –/– Dvl1 homozygotes, mutants ofequivalentsomitestages.Floorplatesare expandedin –/– –/– T ; mutants, -positive Dvl2 Lp/Lp –/– mutant embryosatdifferent somitestageduringneurulation.Eachpoint that were effect ontheviabilityof However, reducingthedoseof that recover 50%oftheexpected not affect theviabilityof defects (Hambletetal.,2002).Reducingthedoseof closed neuraltubes.Bycontrast,all resembling displayed asevere reductionoftheLWR priorto closure1, Dvl2 Dvl1 stages, wefoundthat mutation. neurulation. Consistentwiththishypothesis, together inthemousetopromoteconvergent extension during Dvl1 In additiontotheneuraltubeclosuredefect,werecentlyfound that defect inthecochlea Association ofneuraltubeclosure defectwithPCP tube closure. conserved PCPpathway regulates bothhaircellpolarityand neural mutants identifiedsofar, phenotypes hasalsobeenreported intwo othermammalianPCP 2005; Lewis and Davies, 2002).Theassociationofthesetwo al., 2005),amanifestationofmammalian PCP(KleinandMlodzik, the uniformstereociliarybundle orientationinthecochlea(Wang et When wedissectedtheembryosfromthismatingatmidgestation Dvl2 –/– –/– –/– ; ; ; Dvl2 Dvl2 Lp/+ and Dvl2 Dvl1 –/– –/– Lp –/– showed craniorachischisis,aphenocopy ofthe mutants and and –/– ; genetically interactandsuggeststhey function ; Lp/+ Lp/Lp Dvl2 ( A-D Dvl2 Dvl1 , eitherwithorwithoutadditional –/– P Dvl2 mutants (Fig.3F,G). This resultindicates Lp ) Five-toeight-somitestage <0.05; ** and –/– Dvl2 +/– Lp and mutants, aswerecovered noneonates Dvl1 ; –/– Lp/Lp Dvl2 –/– by halfhadmuchmoredeleterious mutants aswewerestillableto Celsr ; +/– P Lp/+ <0.01. –/– ; mutants (Fig.3E). Dvl1 Dvl2 , stronglyimplyingthata and also displaydisruptionof +/– Dvl2 Dvl1 –/– Development 133(9) Dvl2 ; mutants (Table 1). Dvl2 –/– –/– ; –/– Dvl1 ; Dvl2 Lp/+ –/– Dvl1 mutants had ; –/– by halfdid Lp/+ –/– embryos mutants. ; Dvl2 Dvl1 and +/–

DEVELOPMENT crossed to129 129 Dvl1 embryos ( expressed inthenotochord, nodeandprimitivestreak incontrol a knowndownstream targetgeneofthecanonicalWntpathway, was normal polarity(datanotshown), whileallE18.5 were rescued,thestereociliary bundles incochleaalsodisplayed recovered atE18.5 orasadults,wheretheneuraltubedefects phenotype. Interestingly, inall modifiers fromC57B6background acttogethertorescuethis embryogenesis. Thisresultsuggeststhatmultipledominant recovered), indicatingsuccessfulneuraltube closureduring Convergent extensionduringneurulation stereociliary polarity defects( mutants thatshowed craniorachischisisalsodisplayed Dvl1 defects inamixed 129SvEv/C57B6background.WhenF1 mutants showed incompletelypenetrantneuraltubeclosure penetrant inaninbred129SvEvbackground, background variation. Althoughbothdefectswerecloseto100% headfold stage hybridizationanalysisof Fig. 2.Insitu age progeny genotyped,86 littermates ( movement towards thecentralmidline.Compared withwild-type of thenotochord (arrows), consistentwithadisruptionofcell was abnormallywidened. activity oftheWntpathway. However, in thenodeandprimitivestreak wasmaintained,suggestingnormal Lp/Lp notochord (D,arrows). We furtherfoundthat both defectscanbemodifiedbygenetic Dvl1 +/– –/– embryos ( ; ; Dvl2 Dvl2 Dvl1 –/– C ; ), Dvl2 –/– +/– –/– T D ; expression wasalsowidenedinthenotochord of Dvl1 ). Dvl1 Dvl2 mutants survived toadults(outof557weaning mice, derived fromanintercrossbetween inbred +/– T -positive cellswere alsoobservedoutsideofthe +/– –/– mice andwild-typeC57B6mice,wereback- –/– ; ). ( Dvl2 ; T B Dvl2 -positive cellswere alsoobservedoutside ) In –/– Dvl1 +/– n and =10) (seeWang etal.,2005).The Dvl1 Dvl1 mice, 13%oftheexpected F2 –/– Lp/Lp ; T Dvl2 expression inthenotochord –/– –/– Brachyury ; ; –/– mutant embryos. Dvl2 Dvl2 mutants, –/– –/– Dvl1 Dvl1 mutants, either ( T expected, 11 ) inearly T expression –/– –/– ; ; Dvl2 Dvl2 ( A ) T –/– –/– , of anumberPCPhomologsin antibody (Fig.5A,B).Inaddition, althoughmanipulatingtheactivity 5E,F), orfrompermanentlyfixed embryosstainedwithananti-GFP Dvl2-EGFP fromlive (Fig.5I,J)andbrieflyfixed embryos(Fig. observed inmultipletransgeniclinesusingconfocalmicroscopy of mutants (Fig.5B,F,J; data notshown). Consistentresultswere we observed thesamemembranedistribution patternin mutation didnotaffect themembranelocalizationofDvl2-EGFPas neuroepithelium (Fig.5A,E,I;datanotshown). Furthermore,the asymmetric patternofDvl2-EGFPdistribution onthe planeofthe through thecell.Nevertheless, wecouldnot findany evidence foran membrane asindividual opticalsectionsarescannedmorebasally clear thatbothDvlandactinwerelocalizedtothelateralplasma the entireseriesof close associationofDvl2-EGFPtotheapicalplasmamember. When underneath theapicalplasmamembrane,presumablyreflecting a 5A-D was theresultofcaptureasinglefocalplaneimmediately apparent distribution ofDvl2-EGFPandF-actininsomecellsFig. membrane intheneuroepithelium(Fig.5A,E,I).Themorediffuse Dvl2-EGFP transgenicproteinprimarilylocalizedtotheplasma In neurulatingembryosofdifferent somitestages,weobserved the fashion duringmouseneurulationusingthese (Lee etal.,2001). more consistentlytheendogenousgeneexpression pattern andlevel endogenous Dvl2,presumablyowing toitsabilityrecapitulate method toproducetransgenicproteincapableofreplacing transgenes, suggestingBAC transgenicapproachisareliable were acquiredfromallsixindependentlinesderived fromthetwo endogenous Dvl2duringneurulation(Fig.4B).Consistentresults the encodedDvl2-EGFPfusionproteincouldalsosubstitutefor stereocilia orientationdefectsin the transgenewas alsoabletorescuethecochleaelongationand polarized membranedistribution intheorgan ofCorti.Furthermore, transgenic Dvl2-EGFPproteindisplayedaVangl2-dependent to thelastcodonof transgene thatcontainsanEGFP-codingsequenceinsertedin-frame recently generateda of planarcellpolarity(Axelrod, 2001;Bastocketal.,2003).We is bothdependentuponandimportantfortheproperestablishment 2001; Bastocketal.,2003).Thisasymmetricmembranedistribution are localizedasymmetricallyontheplasmamembrane(Axelrod, During flywingdevelopment, ithasbeenreportedthatDshproteins neurulation dependence ontheDEPdomainduring Plasma membranedistributionofDvl2andits underlies bothmammalianconvergent extension andPCP. extent, furtherarguing ahighlyconserved geneticcircuitry can bemodifiedbygeneticbackgroundvariation tothesame polarity in strict associationbetweenneuraltubeclosureandstereociliary did notreveal obvious differences ofcellpolarityinthe 2002; Veeman etal.,2003b;Wallingford etal.,2000),ouranalysis the cellorientationandelongation alongtheMLaxis(Jessenetal., contains two flankingLoxPsites,was alsoabletorescue additional development. substitute fortheendogenous al., 2005),indicatingthatthe Dvl2 We investigated whethertheDvl2proteinlocalizedinapolarized We furtherfoundthatthis –/– and Dvl1 Dvl2 Dvl2 –/– –/– BAC transgene( ; z ; Dvl2 stacks wereexamined (datanotshown), it was Lp/+ Dvl2 Dvl2 –/– mutants tofertileadults,suggestingthat bacterial artificialchromosome(BAC) mutants indicatesthatbothphenotypes (Wang etal.,2005)andfoundthat Dvl2 Dvl1 Xenopus Dvl2 Dvl2 RESEARCH ARTICLE BAC transgene,aswell as an Dvl2-EGFP2 –/– function duringinnerear BAC transgenecanfully ; Dvl2 and zebrafishcouldalter Dvl2 –/– mutants (Wang et , Fig.4A)that BAC transgenes. Dvl1 Lp/Lp 1771 –/– Lp ;

DEVELOPMENT Lp/Lp midline inwild-typeembryos( displayed intermediatereduction. Attheeight-somitestage,someofneuralfoldswere already apposedandstartedtofuse ( Dvl2 5E,F with5G,H).We notethatin 1772 neuroepithelium ofwild-typeand Fig. 3.Geneticinteractionbetween reduction ofLWR duringneurulation( neurulation. extension (Axelrod etal.,1998;Boutros 1998; Rothbacher different rolesintheWntpathway andthePCP/convergent confocal imaging. difficult toassessmediolateralpolarityinneuralcellswithstatic mediolaterally polarized(Eluletal.,1997).Thus,itmaybemore so atany given timeonlyafew cellswillbeelongatedandappear elongate onlyepisodicallyduringconvergent extension in mediolaterally (ShihandKeller, 1992).Bycontrast,neuralcells because asthecellsintercalate,they alignandelongate mesoderm undergoing convergent extension iseasytoobserve G Expe O Genot Neonate Table 1. ). Theerror barsrepresent s.d. b Two oftheconserved domainsinDsh/Dvl,DIXandDEP, play serve –/– c embryos ( y te ( pe F RESEARCH ARTICLE d ) displayednormalneuraltubeclosure atE10.5.Bycontrast,all d (8ttl 6 (48total) (8ttl 41 20033 3 0 0 12 6 10 14 (48total) ( A D ) Similarto ) ofthesamesomitestage.Similartoboth s derivedfrom genetic cro Dvl1 B ). Paralleltothereduction ofLWR, however, theneuralfolds were stillopenin –/– ; Dvl2 Xenopus Dvl2 E Lp/Lp ). From across between Dvl2 Dvl1 –/– +/– ; Lp/+ embryos, +/+ and ; embryos (compareFig. , MLcellpolarityinthe Lp ss Dvl2 between Lp/Lp and reduced length-to-widthratio(LWR) in Dvl1 +/– 6666666 ; Lp/+ +/– mutants displayedsignificantreduction ofLWR duringneurulation,while ; Dvl1 Xenopus Dvl1 Dvl1 –/– Dvl2 ; +/– Dvl1 Dvl2 +/– ; +/– ; Dvl2 , Dvl2 ; +/+ +/+ Dvl1 –/– ; and crossed into Wallingford andHarland, 2002;Wallingford etal.,2000).When (Rothbacher etal.,2000;Wallingford andHarland,2001; which displayedcraniorachischisis).Bycontrast,a Dvl1 completely failed torescuetheneurulationdefects(Fig.4C; eight 2000). In et al.,2000;Wallingford andHabas,2005;Wallingford etal., identical tothe same domainofDvl2,wegeneratedmutant al., 2000).To confirm thatmammalianneurulationrequiresthe domain ofXdsh(Wallingford andHarland,2001;Wallingford et requires theC-terminalDEPdomainbut nottheN-terminal DIX +/– +/– +/– ; ; Dvl1 Lp/+ ; Lp/+ Lp/Lp Dvl2 Dvl2 –/– Dvl1 +/– ; and +/– –/– ;

Dvl2 mutants, all female and ; +/+ Dvl2 +/– ; Lp/+ Xenopus ; Dvl2 +/– –/– ; ⌬

Lp and –/– Dvl1 ; DEP mutantin Dvl2 ⌬ /+ mice, allprogeny thatwere Dvl1 DEP-EGFP , mediolateralintercalationinthemesoderm Dvl2 Dvl2 –/– –/–

; Lp/+ Dvl2 Dvl2 +/+ Lp/Lp ; –/– ; –/– ; Lp/+ –/– ; Lp/+ Dvl2 –/– male mice and Dvl2 Lp/+ embryos displayedcraniorachischisis –/– Dvl1 Xenopus embryos alsodisplayedsignificant expected, sixrecovered, allof Dvl2 –/– ; Lp/+ embryos ( +/– background, ; –/– ; Lp/+ ( ⌬ Dvl2 DEP-EGFP Dvl2 Dvl2 Development 133(9) C Dvl1 embryos during ) andwidelyapartin at thedorsal –/– –/– ; +/+ +/+ BAC transgenes ; or Lp/+ ⌬ ⌬ Dvl1 DEP-EGFP DIX-EGFP embryos , Fig.4A) Dvl2 +/– Dvl1 ; –/– ; +/+ +/– ;

DEVELOPMENT Dvl2 EGFP wasprimarilylocalizedtothe membrane( (Fig. 4F;12 still fullyrescuetheneurulationdefectin transgene, inwhichpartoftheDIXdomainwas deleted,could Convergent extensionduringneurulation neurulation. convergent extensionandmembranedistributionduring Fig. 4.Domainrequirement ofDvl2foritsinvolvementin an additionalLoxPsiteinsertedinthe3 genotyping purpose(seeMaterialsandmethods), to thelastcodonof dsh1-EGFP all ofwhichdisplayednormalneuraltubeclosure).Asthe XDsh, transgenes, aswellendogenous mouseDvl1,Dvl2,Dvl3, alignment ofthewild-type a K point mutationwasgeneratedbyreplacing AAGwithATG tointroduce encoding aminoacids67-159and442-736,respectively. EGFP Dvl2-EGFP1 region thatisessentialforDvl2functioninthecanonicalWnt EGFP transgenes completelyfailedtorescue theneuraltubedefectsin neural tubeclosure defects in evenly distributed inthecytoplasm( and three mutant the twowild-type endogenous wild-type coenzyme Adehydrogenase, verylongchain). j –/– deletion mutanttransgeneswere generatedbyremoving sequence M substitutionataminoacid446.Lowerpanelshowedsequence Drosophila mutants ( transgene removed aVKEEISmotifintheN-terminal . Although , Dvl2-EGFP2 Dvl1 ( A ) Schematicdiagramofthegenomicstructure ofthe C Dsh andthe Dvl2 ). Confocalanalysisindicatedthat although Dvl2 –/– Dvl2 ; Dvl2-EGFP1 Dvl2 Dvl2 BAC transgenes: BAC transgenes, ( and aLoxPsiteinsertedintointron 2for B Dvl2-EGFP ) and allele, thetargeted –/– Dvl1 dsh1 ; ⌬ ⌬ DIX-EGFP had anEGFPcassettefusedin-frame –/– DIX-EGFP D mutation identifiedinfly. Although ; , Dvl2 and themodified E Ј ). ⌬ flanking gene Dvl2-EGFP1 DIX-EGFP –/– ( mutants, F Dvl1 ) canfullyrescue the ⌬ expected, 15recovered, Dvl2 G DIX-EGFP , H Dvl2-EGFP2 –/– , ), null allele( ⌬ and ⌬ ; DEP-EGFP ⌬ Acadvl DEP-EGFP was Dvl2 dsh1-EGFP DEP-EGFP dsh1-EGFP Dvl2-EGFP2 and Xenopus –/– (acyl- contained ⌬ ⌬ Dvl2 KO mutants DEP- DIX- and Dvl1 ⌬ DIX- BAC , –/– ), ; distribution duringstereociliary bundle formation.InE16.5 mutation mightdisruptPCP inmammals,weexamined its the plasmamembrane(Axelrod, 2001).To investigate how the PCP establishmentthroughabolishing DSH-EGFPlocalizationto orientation (Fig.7G,I).Bycontrast,inaP0 bundles ofsensoryhaircellsinthecochleashowed uniform cochlea. InP0(postnatalday0)controlpups,thestereociliary mammals, weanalyzedthestereociliarybundle orientation inthe 7J andFig.5D of innerhaircells,whichhasalsobeenfoundin Dvl2 Dvl2 resembling the orientation andalessrecognizableformofstereocilia(Fig.7H,J), EGFP 2005), weobserved ashorterandwidercochleain convergent extension underliescochleaelongation(Wang etal., convergent extension. Inaddition,consistentwithourproposalthat mutation completelydisruptedtheabilityofDvl2tofunctionduring which displayedcraniorachischisis),suggestingthatthe craniorachischisis (Fig.7A,D,eightexpected, sixrecovered, allof Dvl2 membrane (Fig. 6C,C detected, wild-type Dvl2-EGFPwas already localizedtothedistal neurulation defectin disrupted theabilityof A pointmutationidenticaltotheflydsh1allele during mammalianneurulation. of theDEPdomainintargeting Dvl2totheplasmamembrane consistent withexisting literatureandconfirmedtherequirement evenly distributed inthecytoplasm (Fig.4D).Thisresultis indistinguishable fromwild-typeDvl2-EGFP, was stillprimarilymembranelocalized(Fig.4G), protein duringneurulation,wefoundthatalthough due tothelossoffunctionWntsignaling. Dvl1 dsh1-EGFP EGFP recombineering, weintroducedanidenticalmutationinto (Axelrod etal.,1998;Boutros1998).Using BAC in aKtoMmissensemutationtheC-terminalDEPdomain affect convergent extension inmammals.The abolished thePCP(Axelrod etal.,1998;Boutros 1998), might pointmutation whether the and PCPestablishment.To furtheraddressthisissue,wetested difference inhow Dvlproteinsfunctionduringconvergent extension Wang etal.,2005).We wondered whetherthiscouldbeduetoa Lp/Lp showed anasymmetricmembranedistribution thatwas disruptedin neuroepithelium andthatintheorgan ofCorti,whereDvl2-EGFP However, thereweredifferences inDvl2-EGFPlocalizationthe involvement inneuralconvergent extension andneuraltubeclosure. localization ofDvl2-EGFPcouldbeapre-requirementforits The resultabove suggestedthatDEP-dependentplasmamembrane the neuraltubeclosuredefectin pathway (Capellutoetal.,2002),thisresultalsoconfirmedthat 7B,C) inneuroepitheliumduringneurulation. appreciably affect themembranelocalizationofDvl2-EGFP(Fig. In flywingdevelopment, the When weinvestigated thedistribution ofthemutantDvl2 To determinewhether –/– –/– –/– –/– mutants, reminiscentofflyPCPestablishment(Fig.6E)(see cochlea, weobserved adisruptionoftheuniformstereocilia ; Dvl2-EGFP ; Lp/+ BAC (Fig.4A)andproduced transgenicmice.All ; dsh1-EGFP ; Dvl2 embryos (Fig.6E,F).However, ineitherwild-typeor mutants (Wang etal.,2005), was alsoobserved (Fig. –/– Ј Dvl1 , arrows). background, thismutationdidnotappearto –/– embryos, beforestereociliarypolarity couldbe embryos recovered atE9.5orlaterdisplayed Ј ; ), similartothat inthemorematurehaircells Dvl2 dsh1 dsh1 –/– Dvl1 also affects thePCPestablishmentin dsh1 mutants. Occasionalmis-alignment Dvl2-BAC identified infly, whichspecifically Dvl1 –/– RESEARCH ARTICLE mutation was thoughttodisrupt ; Dvl2 –/– ; Dvl2 Dvl1 to rescue the –/– dsh1 –/– Dvl1 ⌬ –/– DEP-EGFP was mutants was not Dvl1 mutation results ; –/– Dvl2 ⌬ ; DIX-EGFP –/– Dvl2 –/– ; Dvl2 Dvl1 Dvl1 ; dsh1- –/– Dvl2- 1773 dsh1 dsh1 and –/– –/– –/– ; ; ;

DEVELOPMENT 1774 RESEARCH ARTICLE mesoderm. Lp/Lp mutant(L)embryosinculture. NE,neuroepithelium; SM,somatic (B,D,F,H,J,L; datanotshown).( in theneuroepithelium wasnotaltered inthe mesoderm atthefive-somitestage(E-H).OverallDvl2-EGFPdistribution actin distributionalsoappeared tobemore diffuse inthesomatic are scannedmore basallythrough thecell.Curiously, Dvl2-EGFPand localized tothelateralplasmamembraneasindividualopticalsections examined (datanotshown),itwasclearthatbothDvlandactinare ( the two-andfive-somitestagewere alsostainedwithPhalloidin during confocallasermicroscopy. andmutantembryosfrom Wild-type theapicalplasmamembrane focal planeimmediatelyunderneath of Dvl2-EGFPandactininsomecellsA,Bwasduetoscanningthe embryos inculture ( localization defects. and Dvl2proteinmayaccountforthepartialrescueofdsh1-EGFP E16.5 (datanotshown). Presumably, thepresenceofwild-typeDvl3 Dvl2-EGFP (Fig.6A,A appeared tobesomewhat punctatewhencomparedwithwild-type briefly fixedfive-somitestageembryo( membrane distribution ofdsh1-EGFPin distribution ofDvl2-EGFPin cytoplasm (Fig.6E).Nevertheless, boththerandomizedmembrane membrane localizationbut rarelyformedaggregates intheapical mutants, Dvl2-EGFPdisplayedrandomizedandreducedapical membrane localizationbut inadifferent fashion: inE16.5 at thedistalsideofhaircells(Fig.6B,B dsh1-EGFPproteinwas primarilylocalizedtomembrane cochlea, phenotypically wild-typeE18.5 largely rescuedbyincreasingthedoseofwild-typeDvl:in cells. Furthermore,thecytoplasmic dsh1-EGFPdistribution was cytoplasm but ratherrestrictedprimarilytothe distalsideofhair aggregates werenotevenly distributed throughouttheapical localization tothemembrane.Intriguingly, thedsh1-EGFP in mammalianPCPestablishmentthroughinterferingwithits cytoplasm, suggestingthatthe (Fig. 6D,D EGFP antibody ( and Lp/Lpmutanttwo-somitestageembryosstainedwithananti-EGFP neuroepithelium (A,E,I).Consistentresults were observedinwild-type EGFP wasprimarilylocalizedtotheplasmamembranein neurulating embryosshowedthat,inthewild-typebackground, Dvl2- laser-scanning confocalmicroscopy ofneuroepithelium from neuroepithelium duringneurulation. Fig. 5.Dvl2-EGFPwasprimarilylocalizedtothemembranein at E18.5(Fig.6A,A similar tothe PCP pathway regulates convergent extension in establishingPCPfly, suggestingthatamechanism highly and zebrafishrequiresseveral proteinswhosehomologsareinvolved Recent studieshave revealed thatconvergent extension in DISCUSSION hair cells,but notinneuroepithelium duringneurulation. Dvl2 membranelocalizationinmammalianPCPestablishment in Collectively, theseresultsdemonstratedtheimportanceofpolarized later development (Montcouquioletal.,2003; Wang etal.,2005). correlated withdisruptionoftheuniformstereociliarypolarityduring C , D It isinterestingtonotethatthe , G , H embryos, mostdsh1-EGFPlostitsmembranelocalization ) tolabelF-actin.Whentheentire seriesof A , B Ј , arrowheads) andformedaggregates intheapical ) orthrough direct visualizationofDvl2-GFPsignalfrom I , J ). Theapparently diffuse cytoplasmicdistribution Ј ). However, inE16.5 Ј ). Similardifferences werealsonoticedat K,L dsh1 ) DICimagesoflivewild-type(K)or Lp/Lp Lp Dvl1 E mutation affected Dvlfunction , F mutation alsoaffected Dvl2 Single opticalsectionsfrom ) orliveeight-somitestage Ј –/– mutants andthelossof ), althoughitslocalization Dvl1 ; Lp/Lp Dvl1 Dvl2 Development 133(9) –/– mutants z –/– ; +/– stacks were Dvl2 ; Dvl2 ; dsh1-EGFP –/– –/– Xenopus mutants ; dsh1- Lp/Lp

DEVELOPMENT core PCPpathway componentbut notpartofthecanonical Wnt and interpretation derives fromourfindingsthatsimilardefectsinLWR pathway, but todefectsinthePCPpathway. Furthersupportforthis analysis on Dsh/Dvl arealsoessentialinthecanonicalWntpathway, ourinsitu resembling convergent extension in and narrowing oftheMLaxis,amorphogeneticprocessclosely mammalian Dvl2 Dvl2 Convergent extensionduringneurulation mutants. same setofcorePCPproteins. PCP co-exist inthemouseandmayhave co-evolved byusingthe demonstrate thatconvergent extension intheneuroepitheliumand 2002; Tada etal.,2002;Veeman etal.,2003a).Ourresultsfurther distinct downstream cellularanddevelopmental process(Mlodzik, that mayrepresentevolutionary adaptationineachpathway forthe 2003a). However, significantdifferences have alsobeenobserved (Mlodzik, 2002;Myersetal.,Tada etal.,2002;Veeman etal., with ourinterpretationthattheneural tubephenotypeofthe of Dvl1and2inWntsignaling.Theseresultsarealsoconsistent the remainingDvl3activity issufficient tocompensatefortheloss function ofWntsignalingduringneurulation,presumablybecause Using LWR measurementsand T –/– –/– localization werealsoobserved in mutant isnotsecondarytodefects inthecanonicalWnt embryos, wedemonstrateanessentialfunctionof Lp Dvl1 Dvl is amutationof the genes inacoordinatedlengtheningoftheAPaxis –/– ; Dvl2 –/– embryos didnotreveal globallossof Stbm T Xenopus in situofneurulating homolog Lp/Lp . Bycontrast,although Vangl2 and Lp/+; Dvl2 , whichisa Dvl1 Dvl1 –/– –/– –/– ; ; and that,asinthe force behindthisconvergent extension-like morphogenesisprocess findings suggestthatthemammalianPCPpathway isthe driving where elevating neuralfoldsappose atdorsalmidline.These found beforetheseven-somite stage,acrucial timeofneurulation closure defects.Ineachcase,significantreductionsinLWR were while Lp/Lp Dvl2 tube closure.Furthermore,threemousemutants, resembling convergent extension thatoccursduring coordinated lengtheningandnarrowing throughoutneurulation, solely tothedisruptionofPCPpathway. 2001; Murdochetal.,2001).Finally, thefact that pathway (Darken etal.,2002;GotoandKeller, 2002;Kibaretal., to doso,indicatedthattheneuraltubeclosuredefectin convergent extension in movement isinvolved inmammalian neurulation.Although important tonotethatourdata donotimplyasimilartypeofcell rather thantheresultofneural tube closuredefects.However, itis and Keller, 2000; Eluletal.,1997;Shih andKeller, 1992; was stillabletofullyrescuethelethalityof involvement inthecanonical Wntpathway (Capellutoetal.,2002) transgenes withadeletionoftheVKEEISmotifessentialforDvl2 Our analysesofLWR suggestthatneuralplateundergoes –/– ⌬ and DEP-EGFP was notduetothelossoffunctionWntsignaling,but Lp/+; Dvl2 Xenopus and Dvl1 with wild-typeDvl2-EGFP(A,A’). Bycontrast,in punctate (asteriskinBandB’)whencompared distal membraneappeared tobemore background, dsh1-EGFPlocalizationalongthe At E18.5inanotherwisewild-type bundles (red) outlinedwithphalloidinstaining. and haircellmembranestereociliary (green, A’,C’,E’) ordsh1-EGFP (green, B’,D’) (B,D) signal.( the nativeDvl2-EGFP(A,C,E)ordsh1-EGFP whole-mount organofCortiatE18.5,showing scanning attheapicalsurfaceofhaircellsin EGFP intheorganofCorti. Fig. 6.LocalizationofDvl2-EGFPanddsh1- Dvl1 distribution alongthedistalmembranein type Dvl2-EGFPcouldmaintainaneven of innerhaircells(arrows inD’).Althoughwild- dsh1-EGFP E,E’) in longer restricted tothedistalside(arrows in membrane localizationwasreduced andno observed (arrowheads inD,D’). lost andaccumulationinthecytoplasmwas EGFP localizationonthemembranewasmostly –/– Xenopus dsh1-EGFP , alldisplayedsimilarsevere neuraltube –/– –/– , adisruptionofthisprocessisthe cause ; ; Lp/Lp Dvl2 Dvl2 is driven bycellintercalation (Elul mutants alsodisplaymis-alignment –/– –/– A’-E’ mutants atthisstage. RESEARCH ARTICLE background atE16.5(C,C’),its background atE16.5,dsh1- transgenes completelyfailed ) are theoverlayofDvl2-GFP Dvl1 –/– ⌬ ; DIX-EGFP Dvl1 ( Dvl2 A-E Xenopus Dvl1 ) Confocal –/– –/– –/– ; ; mutants, Dvl1 Dvl2 Dvl2 neural 1775 BAC –/– –/– –/– ; ; ,

DEVELOPMENT Dvl1 extension andPCPestablishmentco-exist inthreemousemutants: mammals bydemonstratingthatdisruptionofneuralconvergent and zebrafish.Ourresultsextend andstrengthenthis notionin the PCPpathway inflyregulates convergent extension in extension remainstobedetermined. 2002). Theexact cellbehavior behindmammalian neuralconvergent (Gong etal.,2004;Solnica-Krezel1995;Wallingford etal., through directedmigrationororientedcelldivision inzebrafish Wallingford andHarland,2001),itcanalsobeaccomplished 1776 mutations ofthemammalianPCPhomologs 2005). Basedonthesimilarneuraltubeclosuredefect,weinferthat during neurulation. Bycontrast,althoughVangl2 isknown tobind to mediateneuraltubeclosureand presumably, convergent extension for Dvl2-EGFPlocalizationtothe plasmamembraneanditsability Xenopus extension (Park et al.,2005).Similartothefindingsinflyand neuroepithelium, aprerequisite foritsinvolvement inconvergent EGFP isprimarilylocalized totheplasmamembranein capable ofreplacingendogenousDvl2function,wefoundthatDvl2- extent. neural tubeclosureandstereociliarypolaritydefectstothesame supported bythatfact thatgeneticbackgroundvariation modifies convergent extension andestablishmentofPCP. Thisnotionisalso Dsh/Dvl functionsthroughvery conserved mechanismsin convergent extension andstereociliarypolaritysuggestedthat Boutros etal.,1998)alsoabolishesthefunctionofDvl2toregulate that specificallydisruptsthePCPpathway (Axelrod etal.,1998; Our findingthatthemissensepointmutation defect duringneurulation(Curtinetal.,2003;Wang etal.,2006). which causePCPdefectsintheear, alsoleadtoconvergent extension It hasbeenargued thatahighlyconserved pathway analogousto Using BAC transgenesthatexpress Dvl2-EGFPfusionproteins –/– ; RESEARCH ARTICLE , wefoundthattheC-terminal DEP domainwas required Dvl2 –/– , Lp/Lp and Lp/+; Dvl2 –/– (see alsoWang etal., dsh1 Celsr1 identified infly and Xenopus Fzd3/6 , possibility isthatanother and sensoryhaircellsundergoing PCPestablishment? One localization inneuroepithelium undergoing convergent extension to Dvlandthe protein thatallows forthemembrane localizationofDvl2in and through highlyconserved mechanisms,thenwhywould the neuroepithelium hasnotbeenreported(Wang etal.,2006). localization intheinnerear, but theirdistribution inthe been shown thatFzd3andFzd6display in convergent extension, but notsufficient. Like Dvl2,ithasrecently distribution ofDvl2-EGFPmightbenecessaryforits involvement Collectively, thesedatasuggestedthatplasmamembrane change ofdsh1-EGFPdistribution intheneuroepithelium. extension duringneurulation,wecouldnotdetectany significant dsh1 concomitantly, itsabilitytoestablishPCP. Bycontrast, althoughthe in thecochleasensoryhaircells same pointmutationabolishedDvl2-EGFPmembranelocalization localization ofDishevelled (Axelrod, 2001).Here,wefoundthatthe establishment. Infly, the convergent extension andsensoryhaircellsduringPCP also revealed adisparitybetweenneuroepitheliumundergoing displayed an contrast toourprevious findingsincochleawhereDvl2-EGFP dependent uponVangl2 function.Thisobservation isindirect membrane localizationofDvl2inneuroepitheliumisnotcrucially neuroepithelium ofneurulating 2004), wedidnotfindany changeofDvl2-EGFPdistribution inthe 2005). Similarly, ourcurrentresultswiththe resembling theestablishmentofPCPinflywingcells(Wang etal., correlates withthelocalizationandorientationofstereocilia, If Dvlproteinsfunctioninconvergent extension andPCP dsh1 mutation alsodisruptedDvl2-EGFPfunctioninconvergent mutation have different effects onDvl2-EGFP Lp -dependent asymmetricmembranedistribution that Lp recovered atp0from in theorganofCortiap0 region (I)showinguniformstereociliary orientation Dvl2 Dvl2 ( analysis ofphalloidinstaining.( membrane intheneuroepithelium. ( EGFP wasprimarilylocalizedtotheplasma mutants. ( stereociliary polarityphenotypesin craniorachischisis, cochlearelongationand Fig. 7. extension defectinneurulation not abletocompensatefortheconvergent E9.5 andE16.5,suggestingthatdsh1-EGFPwas embryos (rightinA)displayedcraniorachischisisat Dvl2 observed. alignment intheouterhaircellscouldalsobe were mis-oriented(arrows) insomehaircells.Mis- in also failedtorescue thecochleaelongationdefect ( be duetodisruptionofconvergentextension. H G mutation weakens thebinding(Torban etal., , , Dvl1 J I Confocalscanateitherthebase(G)ormedial ) However, intheorganofCortia ) –/– –/– –/– ; dsh1-EGFP ; dsh1-EGFP –/– dsh1-EGFP embryos. dsh1 ; B Dvl2 Vangl ) Confocalanalysisindicatedthatdsh1- point mutationdisruptsmembrane –/– Lp/Lp mutants thatwasalsothoughtto homolog, ( mouse, thestereociliary bundles mice, indicatingthat A failed torescue the Dvl1 , D Dvl1 mutants, indicatingthatthe ) Lp- Dvl1 –/– –/– dependent asymmetric ; dsh1-EGFP ; Development 133(9) Dvl2 –/– Dvl1 Dvl2 Vangl1 ; E Dvl1 Dvl2 , F –/– +/– ) Cochlea –/– ; –/– C –/– and Dvl2 , codesfora embryos and ) Confocal ; ; dsh1-EGFP Dvl2 Dvl1 dsh1-EGFP Dvl1 Dvl1 +/+ transgene –/– mouse. –/– –/– –/– ; ; ; Lp

DEVELOPMENT both convergent extension andPCPdetermination. understanding ofhow thecorePCPproteinsco-evolved toregulate function oftheWnt/Ca proteins suchasDshandPk(Veeman etal.,2003a).Analyzingthe determination inflybut nonethelessrequiresthefunction ofPCP Wnt/Ca that convergent extension in the cellularandmolecularlevel. Inthisregard, itisinterestingtonote but alsothedifferences betweenPCPandconvergent extension at in mammals,itwillbeimportanttofindoutnotonlythesimilarities, does notrequirethefunctionofVangl2, norconferpolarity. neuroepithelium mayrepresentonlyasteadystatedistribution that 2004). Inthisscenario,membranelocalizationofDvlinthe intercalation maybedeterminedbytheAPpolarity(Ninomiyaetal., convergent extension, whilethealignmentandorientationofcell proposal thatPCPsignalingserves asapermissive factor in (Wallingford etal.,2000).Thisview isalsoconsistentwiththe extension alsoleadstolessstablemembraneprotrusions the observation thatin Wallingford andHarland,2002).Supportingevidence comesfrom cell-cell adhesionandgeneratestraction(Ulrichetal.,2005; mobile tomediatetemporarycytoskeleton assemblythatunderlies the localizationofDvlandotherPCPcomponentsisrelatively in cellsundergoing dynamiccellrearrangementandintercalation, location ofstereociliaryassemblywithinthehaircells.Bycontrast, distribution ofDvlandotherPCPcomponentsdictatestheprecise establishment inthewell-alignedsensoryhaircells,polarized demand distinctmodesofaction.For example, duringpolarity establishment andconvergent extension, different cellularprocesses convergent extension (Park etal.,2005). membrane localizationofDshinmesodermalcellsundergoing Similar studiesin that couldbecorrelatedwitheithertheAPorMLaxisofembryo. preliminary resultsshowed nopolarizeddistribution ofDvl2-EGFP of individual Dvl2-EGFPcellssurroundedbynon-GFP cells.Our Dvl2-EGFP2 resolve this,weareusingdifferent imaging techniquesarenotsensitive enoughtorecognizethem.To that dependuponVangl2 andafunctionalPCPpathway, but our convergent extension, Dvl2-EGFPisdistributed atspecificlocations developing mammalianneuraltubeorinnerear. Vangl1 similar biochemicalfunction(JessenandSolnica-Krezel,2004). Vangl2 have largely non-overlapping patternsofexpression but neuroepithelium but nottheinnerear. Inzebrafish,Vangl1 and Convergent extensionduringneurulation Axelrod, J.D. References Woodruff Foundation toP.C. GM074104), A.W.B. (R01HD43173-02)andP.C. (R01DC005213);andthe J.B.W.; andbygrantsfrom theNationalInstituteofHealthto J.B.W. (R01 J.W.; aBurroughs Wellcome FundCareer Award intheBiomedicalSciencesto Clifford andEvelynCherryFellowshipanAHApostdoctoralfellowshipto P30 NS047101).ThisworkissupportedbyaPeteLopiccolaFellowship, Core fortransgenicservice, andtheUCSD/NINDSNeuroscience Core (NINDS We thankEllaKothariand Jun ZhaointheUCSDCancerCenterTransgenic Axelrod, J.D.,Miller, J.R.,Shulman,M.,Moon, R. T. andPerrimon,N. Frizzled planarcellpolaritysignaling. the planar cell polarity and Wingless signalingpathways. the planarcellpolarity andWingless (1998). Differential recruitment ofDishevelled provides signalingspecificity in 2622. To understandmorethoroughlythefunctionofPCPpathway A thirdpossibilityisthatalthoughDvlfunctionssimilarlyinPCP The secondpossibilityisthatinneuroepitheliumundergoing 2+ expression hasnotbeencarefullyexamined inthe signaling, apathway thathasnotbeenimplicatedinPCP (2001). Unipolarmembraneassociation ofDishevelledmediates transgenes togeneratechimericembryosthatconsist Xenopus Xenopus 2+ pathway inthemousemayleadtoabetter also didnotreveal consistentpolarized Xenopus , XDsh Genes Dev. Cre mutant thatblocksconvergent and zebrafishalsoinvolves transgenes andthefloxed 15 , 1182-1187. Genes Dev. 12 , 2610- Boutros, M.,Paricio,N.,Strutt,D.I.andMlodzik,M. Carreira-Barbosa, F., Concha,M.L.,Takeuchi, M.,Ueno,N.,Wilson,S.W. Capelluto, D.G.,Kutateladze,T. G.,Habas,R.,Finkielstein,C.V., He,X.and Cadigan, K.M.andNusse,R. Beddington, R.S. Copp, A.J.,Greene, N.D.andMurdoch, J.N. Huelsken, J.andBirchmeier, W. 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