developing embryoshasnotbeenaddressed. how itsactivity mayberegulated duringcellshapechangein robust MTarraysobserved inelongatingneuralepithelialcells,but 1991). MTs (Gunawardane etal.,2000;Job2003;Stearns protein thatisacomponentofthecomplex thatnucleates regulator ofMTassemblyis Meads andSchroer, 1995;RizzoloandJoshi, 1993).Onecentral beneath theapicalsurface (Bacallaoetal.,1989;Bre 1990; assembled fromadiffuse microtubule-organizing center(MTOC) In otherepithelialcelltypes,apicobasallyalignedMTs are Burnside, 1973;Karfunkel, 1971;Messier, 1978;Schroeder, 1970). cells changeshaperemainsunclear(BrunandGarson,1983; discrete populationsofMTsareassembledasembryonicepithelial (MTs) asthecausative cytoskeletal agentincellelongation,but how microscopy andinhibitorstudieshave implicatedmicrotubules apicobasal cellelongationremainalmostentirelyobscure.Electron least partiallydefined (seebelow), themoleculesgoverning Viamontes andKirk,1977). and vertebrates (e.g.Schroeder, 1970; Sweetonetal.,1991; constricting cellsinorganisms rangingfrom elongation. Anincreaseincelllengthisobserved in apically uniformly linked toanothercellshapechange,apicobasal of apicalconstrictionisthatthiscellshapechangealmost shaped cells,facilitating thebendingofcellsheets.Onepeculiarity (Fristrom, 1988).Thisprocessconverts columnarcellsintowedge- surface areawhilethebasalsurface remainsunchangedorexpands constriction, bywhichpolarizedepithelialcellsreducetheirapical multicellular organisms. Oneubiquitouscellshapechangeisapical Cell shapechangeisacentralmechanismformorphogenesisin INTRODUCTION Accepted 19January 2007 *Author forcorrespondence (e-mail:[email protected]) and MolecularBiology, UniversityofTexas, Austin,TX78712,USA. Department ofMolecularCellandDevelopmental Biology, andInstitute forCellular KEY WORDS:Shroom, gamma-tubulin,microtubule, neuraltubedefect,morphogenesis, behaviors bycoordinatingtheassemblyofbothmicrotubuleandactincytoskeletons. conserved featureofthisproteinfamily. Together, thedatademonstratethatShroom familyproteinsgovernepithelialcell apicobasal cellelongation.Finally, experimentswiththerelatedprotein,Shroom1,demonstratethat tubulin. Moreover, thischangein morphogenesis. We showthatShroom3issufficient andalsonecessarytoinducearedistributionofthemicrotubuleregulator have identifiedanovelrolefortheactin-bindingproteinShroom3asregulatorofmicrotubulecytoskeletonduringepithe coordination isachievedremainobscure,particularlyinthecontextofepithelialcellswithindevelopingvertebrateembryos. Cell shapechangesrequirethecoordinationofactinandmicrotubulecytoskeletons.Themolecularmechanismsbywhichsuch Chanjae Lee,HeatherM.ScherrandJohnB.Wallingford* microtubule architectureduringepithelialcellshapechange Shroom familyproteinsregulate Development 134,1431-1441(2007)doi:10.1242/dev.02828 Whereas themolecularcontrolofapicalconstrictionhasbeenat ␥ -tubulin isprobablyakey playerintheassemblyof ␥ -tubulin, aminus-endanchoring ␥ -tubulin distributionunderliestheassemblyofalignedarraysmicrotubulesthatdrive Volvox to arthropods rmaalbeepesdsqec as(Ss,w bandapartialcDNA obtaineda from available expressed sequencetags(ESTs), we genomicsequenceof Using primersbasedonthe length To facilitate studiesofShroom3function in Cloning of demonstrate thatthecontrolof neural tubeclosure.Experimentswiththerelatedprotein,Shroom1, elongation andapicalconstrictionintheneuralepitheliumduring is therebynecessaryandsufficient todrive bothapicobasalcell robust, parallelMTsthatdrive apicobasalcellelongation.Shroom3 regulator necessary andsufficient toinducearedistribution oftheMT architecture indeveloping epithelia.We show thatShroom3isboth identified (Hagensetal.,2006). proteins withinthefamily andtoreflecttheorderinwhichthey were proteins wererenamedrecentlytoreflectthesimilaritiesof associated withtheactincytoskeleton (Hagensetal.,2006).These a memberofnew family ofproteins,all of whichhave been Soriano, 1999).Shroom3(previously simplyknown asShroom)is closure inbothmiceandfrogs(Haigoetal.,2003;Hildebrand Moreover, thisactin-bindingproteinisessentialforneuraltube hierarchy controllingthiscellbehavior (Haigoetal.,2003). suggesting thatthisproteinliesnearthetopofmolecular apical constrictionandactinaccumulationinnaïve cells, we foundthatthenovel proteinShroom3was sufficient todrive apical constrictionwerealsorequiredforcellelongation.Recently, (Rodriguez etal.,2003),weasked whethermoleculesgoverning Because theMTandactincytoskeletons areoftenjointlyregulated MATERIALS ANDMETHODS are coordinatedtocontrolcellshapeindeveloping embryos. identify anovel mechanismbywhichactinand MTcytoskeletons assembly ofactinandtubulin cytoskeletons. Theseresultsthus proteins govern distinctepithelialcellbehaviors bycoordinating et al.,2003),thedatapresentedheredemonstratethatShroomfamily Shroom3 issufficient todrive actin-basedapicalconstriction(Haigo feature ofthisproteinfamily. Inlightofourprevious finding that In thisstudy, weshow thatShroomfamily proteinsgovern MT Apicobasal cellelongationistightlylinked toapicalconstriction. ␥ -tubulin distributionand eou Shroom3 Xenopus ␥ Xenopus -tubulin, andthatthisactionunderliestheassemblyof Xenopus Shroom3 (see Fig.S4Ainthesupplementary material). ␥ -tubulin distribution isaconserved ␥ RESEARCH ARTICLE -tubulin regulationisa Xenopus Xenopus tropicalis , wehave clonedfull- We ␥ 1431 lial - and

DEVELOPMENT DAPI (4 stained withthephalloidinsolutionat4°Covernight. To visualizenuclei, (PBS plus0.1%Tween 20).Fixed embryoswererinsedwithPBTand containing Shroom3Lformin isoform (36%identical,48%similar).Noevidence was foundforaPDZ- Xenopus cDNA was obtainedbyPCRandwas clonedintoCS107,astandard sequence fromRACE, fourprimersetsweredesigned,andafull-length GAGTAGGAGGCATA-3 Murray’s solutionwere placedinaculturechamberwith acover-glass embryos inPBT were placedonaculturedishand embryosclearedin Wallingford (Davidson andWallingford, 2005).Phalloidin-stainedintact Imaging proceduresweregenerally asdescribedinDavidson and Imaging andimageanalysis mM NaBH autofluorescence ofyolkplatelets,theembryoswereincubatedwith100 To reduce TBS(155mMNaCl,10Tris-Cl, pH7.4). consecutively with for3hoursandrehydrated 67%methanol and hydrogenperoxide in 10% methanolandwerebleached Fixed embryosweredehydratedcompletelyin Immunohistochemistry andinsituhybridization to phalloidinsolution. were completelydriedbeforeuseandresuspendedin500 Two unitsofAlexa Fluor-green-phalloidin (MolecularProbes)inmethanol Actin filamentstaining Nieuwkoop andFaber (Nieuwkoop andFaber, 1994). (Davidson andWallingford, 2005).Embryoswerestagedaccordingto Embryos wereincubateduntilappropriatestagesandfixed inMEMFA was injectedintooneortwo dorsalblastomeresatthefour-cell stage. DNA, mRNA orantisensemorpholinooligonucleotide(Haigoetal.,2003) Capped mRNA was synthesizedusingmMessagemMachinekit(Ambion). Morpholino, DNAandmRNAinjection 5 were Gene-specific primersfor5 FirstChoice RLM-RACE Kit(Ambion). designed fromthispartialsequenceand5 sequence ofShroom3.To clonefull-length 1432 were obtainedwitheitherofthetwo different dilution (Abcam)orasinStearnsetal.(Stearnsal.,1991);identicalresults dilution, DM1A;Sigma),rabbitpolyclonalanti- inTBS). rinsed inTBST(0.1%Triton X-100 Haigo etal.,2003). constriction (seeFig.S4B-Einthesupplementarymaterial)also expression was sufficient toinduceapicalactinaccumulationand PDZ-containing XShroom3cDNA. Like themouseorthologs,Shroom3 Third, BLASTsearchesofavailable found inthe Shroom3 spliceform.Second,noPDZ-containingsequencecouldbe repeated attemptsusing5 containing Shroom3Lspliceformdoesnotexist in a PDZattheN-terminus.Threelinesofevidence suggestthatthePDZ- Shroom3 andShroom1(previously known asAPX)(Staubetal.,1992). 2000), withdigoxygenin-labeled,antisense full-lengthprobesto Murray’s Clearsolution(benzylbenzoate:benzylalcoho1,2:1). Embryoswereclearedin was addedinsecondaryantibodysolution. To stainnuclei,propidium iodideorDAPI diluted 1:250inFBSsolution. orAlexa Fluor-555 IgG(MolecularProbes) rabbit IgG(MolecularProbes) goatanti- (MolecularProbes),Alexa Fluor-488 immunoglobulin G(IgG) Primary antibodies weredetectedwithAlexa Fluor-488 goatanti-mouse and5%dimethylsulfoxide(DMSO)]. FBS containing 10% clone9E10; Abcam). Laboratories) andmonoclonalanti-Mycantibody(1:300dilution, shown)], polyclonalrabbitZO-1antibody(1:200dilution;Zymed Shroom3 Primary antibodiesusedweremonoclonalanti- In situhybridizationwas performedaccordingtoSive etal.(Sive etal., solution[TBS Antibodies weredilutedinfetalbovine serum(FBS) Ј RESEARCH ARTICLE ␮ -ACTCTTCTGAGATTCCACGCTGT-3 vector usedfortranscriptionofcappedmRNA. g/ml, Sigma)orpropidiumiodide(20 4 nTSfr4hoursatroomtemperatureorovernight at4°Cand in TBSfor4 encodes aproteinwithhighsimilaritytomouseShroom3S tropicalis genome within>100kbupstreamofShroom3. Ј ae nafl-eghXenopusShroom3 full-length . Basedona Ј RACE failed toidentifyaPDZ-containing Xenopus Xenopus . Like Shroom3S,Shroom3lacks Xenopus Shroom3 Ј ␥ RACE was performedusing -tubulin antibodies (datanot ␥ ESTs failed toidentifya ␮ -tubulin antibodies[1:200 ␣ g/ml, Sigma)was added Ј -tubulin antibody(1:400 and 5 Xenopus Ј , primerswere -TCACTGC - ␮ Xenopus l ofPBT Ј . First, RACE the changeinneural epithelialcellshapeduringneural tubeclosure. lsr sae 14-19; closure (stages (red; cellheightin surface measured from cross-sections) andapicobasalcellelongation changes inapicalconstriction(blue; ratioofapicalsurfacetobasal elongate, butconstrictonlyalittle (arrowhead). ( regions constrictandelongate (arrow), cellsinintermediateregions (grey). ( illustrating theneuralepithelialcells(red) andothersurrounding tissues epithelial cellfrom A.( neurulating confocal opticaltransversesectionsthrough theanteriorneuralplateof neural epitheliumduringtubeclosure. Fig. 1.Apicalconstrictionandapicobasalcellelongationinthe inhibitors. Aftercentrifugation,10 deoxycholate, 2mMEDTA, 50mMTris, pH8.0)containingprotease Embryos werelysedinRIPA buffer (150mMNaCl,1%NP-40,0.5% Immunoblotting Photoshop. study have beenenhancedusingtheUnsharpMaskfilter inAdobe was measuredwithImageProPlussoftware. Imagesusedthroughoutthis position weremeasuredwithLSM5Pascal software andcellsurface area captured onastereomicroscope(LeicaMZ16FA). Cellheightandnuclear microscope. Theimagesforbrightfield andfluorescenceview were (Davidson andWallingford, 2005). thick (250-300 bottom. For cross-sections,embryoswereembeddedin2%agarose,and dilution, clone9E10;Abcam),rabbitpolyclonalanti-actinantibody(1:2000 Primary antibodiesusedweremonoclonalanti-Mycantibody(1:2000 PAGE andwesternblotassayswereperformedusingstandardprotocols. The imagingwas performedwithaZeissLSM5Pascal confocal b Ј Aschematicdiagramofthesection in a ) Xenopus ␮ m) sectionswerecutwithaVibratome 1000system ␮ m) ofneuroepithelial cells during neuraltube embryo. ( B n =10 cellsperstage). ( AschematicdiagramofthesectioninA, ) a Ј High-magnificationviewofaneural ) ␮ g supernatantwas analyzedbySDS- D Schematicillustrating ) Development 134(7) C ) Graphillustrating Ј ( A . Cellsinhingepoint Projection of )

DEVELOPMENT 1A,a this neuralepithelium;certaincellsconstrictdramatically(Fig. tube closure.Thedegree ofapicalconstrictionisdiscontinuouswithin undergo dramaticelongationandapicalconstrictionduringneural In developing during neuraltubeclosure and apicobasalcellelongationintheneuralplate Shroom3 isrequired forbothapicalconstriction RESULTS SuperSignal West PicoChemiluminescent Substrate(Pierce). dilution; Abcam).Horseradishperoxidase(HRP)signalsweredetectedby dilution; Abcam)andrabbitpolyclonalanti- Shroom proteinsmicrotubule govern architecture disrupts neuralplatebendingwheninjectedinto MO resultsintranslationofanon-functionalShroom3proteinand effectively blockspropersplicingofShroom3;introductionthis function usinganantisensemorpholinooligonucleotide(MO)that involved inthecontrolof cellelongation.We disruptedShroom3 cells (Haigoetal.,2003),weasked whetherthisproteinwas also 22 doubled theirheight,fromapproximately12 onset ofneurulation(stages13-14),cellstheneuralplatealmost in cellheightcouldoccurindependentlyofapicalconstriction.Atthe neuroepithelial cellsover time(Fig.1C,D).We observed thatchanges stages revealed aprogressive elongationandconstrictionof material). Examinationofcross-sectionsatsuccessive developmental sections withthesurface views (seeFig.S1inthesupplementary of theseconstrictedcellsismosteasilyseenbycomparingcross- anteroposterior arraysofcells,calledhingepoints,andtheorganization little (Fig.1b (Haigo etal.,2003). Because Shroom3inducesapicalconstrictioninneuralepithelial ␮ m, duringatimewhennoapicalconstrictionoccurred(Fig.1C). Ј ,B,b Ј , arrow), whereasothersincreasetheirheight,but constrict Ј , arrowhead). Thehighlyconstrictedcellsareordered in Xenopus embryos, superficial neuralepithelialcells ␥ -tubulin antibody(1:4000 ␮ m toapproximately Xenopus embryos the dominant-negative fragmentShroom3 an alternative method.We showed previously thatexpression of Shroom3 morphants(Fig.2C). Apical surface areasinbothregions weresignificantly increasedin intermediate regions, wherelessconstrictionoccurs(Fig.2D,E). cells intherobustly constrictinghingepointregions andalsointhe in theneuralplatebymeasuringapicalsurface areaofneural remained columnar(Fig.2A,a characteristic wedgeshape,whereascellsontheinjectedside apparent. Cellsontheuninjectedsideofembryostook cells wereattheonsetofneurulation. 2B). Bymid-neurulation,thesecellsareonlyaslongwild-type Shroom3 functionwas approximatelyhalfthatofcontrolcells(Fig. elongation ofneuralplatecells,suchthatthelengthcellslacking function byinjectionoftheShroom3-MOblocked apicobasal on theuninjectedsideoftheseembryos.DisruptionShroom3 of theembryo(Fig.2A,a develop withobvious defectsincellelongationontheinjectedside embryos injectedunilaterallywithShroom3MO(morphants) its roleinapicalconstriction. apicobasal cellelongationinneuralepithelialcellsadditionto material). TheseresultsdemonstratethatShroom3isrequiredfor comparable totheShroom3-MO(Fig.S2c and cellelongationthroughouttheneuralplatetoadegree In addition,expression ofDN-Shrm3reducedapicalconstriction cell elongation,similartothatobserved inShroom3 morphants. in neuralepithelialcells,weobserved cleardefectsin apicobasal vitro assay(Haigoetal.,2003).Whenweexpressed DN-Shrm3 potently disruptstheactivity ofco-expressed Shroom3inan To confirm ourfindings, wedisruptedShroom3functionusing A failure ofapicalconstrictioninShroom3morphantswas also Confocal imagingofcellswithintheneuralplaterevealed that with Shroom3-MO. material). Embryohasbeeninjected ononeside of thepanel(seealsoFig.S1in supplementary indicated byred outlinesand labeledatthebottom shapes. Cellsdefinedasthehpand intregions are embryo stainedwithphalloidintovisualizecell areas. ( stage 18embryotovisualizeapicalcellsurface supplementary material.( intermediate regions are definedinFig.S1the 18 (mean±s.e.m.; intermediate (int)regions offixedembryosatstage sides offixedembryosinhingepoint(hp)and surface areas ofcontrol andShroom3-MO-injected s.e.m.; ctl, Shroom3-MO-injected regions atstage18(mean± the nucleusforhingepointcellsincontrol and between thebasalcellsurfaceandlimitof ( ( reveals cellcorticesandDAPIreveals nuclei. side withShroom3-MO. Anti- anterior neuraltubeofanembryoinjectedonone closure. neural epithelialcellsduringtube constriction andapicobasalcellelongationin Fig. 2.Shroom3 isrequired forapical B a Ј Graph ofapicobasalcellheightandthedistance ) Schematic ofneuroepithelial cellshapeinA. ) E Ј oslpoeto fsae16 Dorsalprojection ofstage ) ). Cellshapechangeprogressednormally ( A n Cross-section through theclosing ) =12; Mo, Ј ). We quantified apicalconstriction n RESEARCH ARTICLE =25). Hingepointand n =11). ( D Dorsalprojection of ) Ј ␣ in thesupplementary 754-1108 C -tubulin staining Graphofapicalcell ) (DN-Shrm3) Xenopus 1433

DEVELOPMENT 3A,a extending throughapproximatelytheapicalthirdofcell(Fig. parallel arraysofMTsemanatingfromtheapicalcellsurface and in wedgingneuroepithelialcells(Fig.3A,a antibody against 1971). We examined MTsusingconfocalmicroscopy andan more densearrayofthesealignedMTs(Burnside,1973;Karfunkel, and increasingcellheightisassociatedwiththeassemblyofamuch contain parallelarraysofMTsalignedalongtheirapicobasalaxis, Like mostepithelia,neuralepithelialcells changes incelllength. We next pursuedthemechanismbywhichShroom3mediates cells MT arraysintheapicalregion ofneuralepithelial Shroom3 isrequired fortheassemblyofparallel 1434 epithelial celltypes,centrosome-like fociof tubulin intheneuralepitheliumbyimmunohistochemistry. Inmany the developing neuralplate,weexamined thedistribution of To begin tocharacterizethemolecularbasisofMTorganization in cells redistribution of are associatedwithShroom3-dependent Cell elongationandassemblyofapicalMTarrays 3D anddatanotshown). apicobasal MTarraysandthatfailed toconstrictandelongate(Fig. remained localizednormally, even incellsthatlacked robust and datanotshown). InShroom3morphants,ZO-1andPar3 at apicalcell-celljunctionsofelongatingneuralplatecells(Fig.3C example, incontrolembryos,ZO-1andPar3 weretightlylocalized markers weremaintainedintheabsenceofShroom3function.For Shroom3 morphants(Fig.3A,B).Second,wefoundthatapical are consistentwiththenormalappearanceofperinuclearMTsin was identicaltothatinnormalneuralcells(Fig.2B).Thesefindings (Fig. 2A)andthepositionofnucleirelative tothebasalcellsurface that inShroom3morphants,nuclearpositioningwas highlyuniform from generaldefectsinapicobasalcellpolarity. First,weobserved failure ofcellshapechangeinShroom3morphantsdidnotstem changes incellheightduringneuraltubeclosure. aligned MTsandareconsistentwiththenotionthatsuchdrive that Shroom3directstheassemblyofadiscretepopulation in cellslackingShroom3function(Fig.3A,B).Thesedatasuggest perinuclear MTnetworks appearedsimilarinbothcontrolcellsand disrupted incellslackingShroom3function.Corticaland contrast totheparallelMTarrays,overall MTassemblywas not fragment DN-Shrm3(Fig.S2C,c were observed following expression ofthedominant-negative aligned MTarraysintheapicalregion (Fig.3B,b was uniformlyassociatedwithafailure toassemblethethick, Xenopus 4A,B). Thisbroadaccumulation of distributed asathick cloudwithmany brighterfocipercell(Fig. Joshi, 1993). organized (MeadsandSchroer, 1995;Müsch,2004;Rizzoloand near theapicalcellsurface, fromwhichMTsarethought tobe 4A,a the robust apicobasally alignedMTarraysdescribedabove (Fig. tube closure,thuscorrelatingin time andspacewiththeassemblyof the apicalthirdofeachelongating neuralplatecellduring prior totheonset ofrobust apicalconstriction (stage16;Fig.S2A,B Finally, weobserved thatthefailure ofMTorganization andthe Strikingly, thefailure ofcellelongationinShroom3morphants By contrast,weobserved thatinneuralplatecells Ј Ј ). ), consistentwithprevious electronmicroscopy studiesin RESEARCH ARTICLE ␥ -tubulin accumulated apicallyattheearlyneurulastages, (Karfunkel, 1971). ␣ -tubulin. ApicalMTarrayswerereadilyapparent ␥ -tubulin inneuralepithelial Ј in thesupplementarymaterial).In ␥ -tubulin filled approximately Ј ␥ ). We observed thick, -tubulin areobserved Ј ). Identicalresults ␥ -tubulin was ␥ - control hingepointcells(green, ZO-1;red, anti- junctions. changes are eliminated,butZO-1remains apicallylocalizedatcell-cell magnification viewofhingepointcellslackingShroom3 function.Shape magnification viewofMTs incontrol hingepointcells(green, anti- parallel MTs inelongatingneuralepithelialcells. Fig. 3.Shroom3 isrequired forassemblyof discrete arraysof fragment. Aswas thecaseformorphants, cellsexpressing DN- 4E,e failure ofcellstoformaligned MTarraysandtoelongate(Fig. accumulation of that injectionoftheShroom3-MO completelyeliminatedthe function affected the apicalaccumulationof directly fromtheactionofShroom3. data suggestthatthebroadapicalaccumulationof for maturemammalianneuroepithelialcells(Chennetal.,1998).These foci attheapicalcellsurface (Fig.4C,c closure was complete,our downregulated (Haigoetal.,2003).We foundthatafterneuraltube completion ofneuraltubeclosure,whenShroom3expression israpidly be mediatedbyShroom3,weexamined neuralepithelialcellsafterthe reported. To askwhether thisuniqueredistribution of such adistribution ininterphaseepithelialcellshasnotbeen previously region ofneuralplatecellswas unexpected, and,toourknowledge, with two different apical surface ofneuroepithelialcells.We obtainedidenticalresults concentration of in thesupplementarymaterial),demonstratingthatapical ( MTs indorsolateralhingepoint(DLHP)cellslackingShroom3 function. A. Arrow indicatesparallelMTarrays.( tubulin; red, propidium iodide,nuclei).( b Ј In lightofthesefindings, weasked whetherlossofShroom3 The broad,intensedistribution of Schematic ofacellshowninB.( ) Ј ). To confirm thisresult,weexpressed theDN-Shrm3 ␥ ␥ ␥ -tubulin intheneural plate,coincidentwiththe -tubulin was notafunctionoftheconstricting -tubulin antibodies(datanotshown). ␥ -tubulin antibodydetectedcentrosome-like C ) High-magnificationviewof B ␥ a ) High-magnificationviewof -tubulin throughouttheapical Ј Ј Schematic ofacellshownin ) ,D), similartothatdescribed ␣ ␥ -tubulin). ( -tubulin. We observed Development 134(7) ␥ -tubulin mayresult ( A ␥ ) High- -tubulin could D ) High- ␣ -

DEVELOPMENT naïve epithelialcellsinwhichtotestproteinfunction(seeDollaret cells (thesuperficial blastomeres)provide anexcellent sourceof that surroundthecleavage-stage previously describedgain-of-functionassayusingtheepithelialcells To furtherexplore theactivity ofShroom3,wetookadvantage ofour elongation innaïveepithelialcells Shroom3 issufficient toelicitapicobasalcell apical regions ofneuroepithelialcells. suggesting thatShroom3directsaredistribution of embryos werenotaffected (Fig.S3Ainthesupplementarymaterial), levels of supplementary material).Western blottingrevealed thatthetotal Shrm3 alsofailed toaccumulate Shroom proteinsmicrotubule govern architecture ␥ -tubulin inthedorsaltissuesofShroom3-MO-injected ␥ Xenopus -tubulin apically(Fig.S2Cinthe embryo (Fig.5A).These ␥ -tubulin in with neighboringcellslackingShroom3orwhencompared with expressing cellsdramaticallyincreasedtheirheightwhencompared and measuredtheapicobasallengthofthesecells.Indeed,Shroom3- the presenceoffunctionalShroom3protein(seeHaigoetal.,2003)] them withphalloidintodetectapicalactinaccumulation[indicating into apicobasal elongationinnaïve cells.We injected we first asked whetherectopicShroom3was sufficient toinduce protein encodedbythatmRNA. the effects ofinjectedmRNAs representthedirectactionof Roberts etal.,1992)andaretranscriptionallyquiescent,suchthat polarity characteristicofepithelialcells(Chalmersetal.,2005; al., 2005;Haigoet2003).Suchcellsdisplayarobust apicobasal Because neuralcellslackingShroom3functionfailed toelongate, Xenopus injected, leftsideiscontrol). ( unilaterally injectedwithShroom3-MO (rightsideis cells showninE. of theclosed tubulin stainingreveals celloutlinesintheventralregion o) ( box). neural tubeindicatinglocationofsectionshowninC(red surfaces ofmature neuroepithelial cells.( optical sectionsreveals centrosome-like fociattheapical ( in A(red box). closing neuraltubeindicatinglocationofsectionshown for sections showingelongatingneuralepithelialcellsstained Fig. 4.Shroom3 controls thedistributionof tubulin stainingforcellsshowninA.( ( in neuroepithelial cellsduringneuraltubeclosure. A embryos andrearedthemtoblastomerestages,stained High-magnificationprojection ofastackoptical ) regions (mean ±s.e.m.;ctl, superficial blastomeres incontrol and Shroom3-injected basal cellsurfaceandthelimit ofthenucleusfor apicobasal cellheightandthedistance betweenthe actin alsodisplayincreased cellheight.( expressing Shroom3. Cellswithaccumulatedapical superficial blastomeres ofan embryoectopically iodide reveals nuclei(red). ( staining reveals cellcortices(green) andpropidium superficial blastomeres ofacontrol embryo.Phalloidin Roberts etal.,1992).( et al.,2005;DollarHaigo2003; superficial blastomeres in ( cell elongationinnaïveepithelialcells. Fig. 5.Shroom3 issufficient todriveapicobasal A ␥ -tubulin (whitelinesindicatecelloutline).( Schematicdepictingtheepithelialcharacterof ) E Opticalsectionshowing ) Xenopus C ) ␥ -tubulin staininginaprojection of neural tube.( B RESEARCH ARTICLE Cross-section through ) Xenopus e C n Ј Cross-section through ) ) =26; Shroom3, ␥ ␣ -tubulin stainingfor -tubulin inanembryo D (see alsoChalmers B Schematicofthe ) Schematicofthe ) Shroom3 c Ј D Acetylated- ) Graphof ) a n ␥ Ј =19). ) -tubulin mRNA ␣ - 1435 ␣ -

DEVELOPMENT and alsoelongates. ( elongated andcontain abroad, diffuse accumulationof expressing Shroom3 stainedfor constrict andelongate.Parallelarrays ofMTs emanate from theapicalcellsurface.( throughout theapicalregion ofthecells(Fig.6E,e epidermis stainedfor Fig. 7.Expression ofShroom3 inepidermalcellsrecapitulates theevents underlyingneuroepithelial cellshapechange. a broader, morediffuse accumulationof cells have assumedawedgeshapeandhave increasedtheirheight, cell surface (Fig.6D,d ␥ At earlystages,priortoachangeincellshape,theaccumulated dramatic accumulationof embryo (seeFig.5A).Ectopicexpression ofShroom3 resulted ina naïve epithelialcellsthatsurroundthecleavage-stage To explore thishypothesisfurther, weagaintookadvantage ofthe on cellelongationstemsfromarelocalizationof Our dataintheneuralepitheliumsuggestthateffect ofShroom3 in naïveepithelialcells Shroom3 directs apicalaccumulationof both necessaryandsufficient toinduceapicobasalcellelongation. addition toitsroleingoverning apicalconstriction,Shroom3isalso cells insimilarregions ofuninjectedembryos(Fig.5B-D). Thus,in 1436 -tubulin inShroom3-expressing cellslocalized tightlytotheapical RESEARCH ARTICLE e ␣ Ј Anti- ) -tubulin. ( Ј ). Atlaterstages,whenShroom3-expressing ␥ ␣ -tubulin atapicalcellsurfaces (Fig.6). -tubulin staining of thecellshowninE. ␥ B -tubulin. Cellsexpressing Shroom3 inthispanel(markedbyanti-Myc,red; Epidermisexpressing Shroom3 stainedfor ) ␥ -tubulin was observed ␥ Ј -tubulin (Fig.4). ,e ␥ Љ ␥ ). -tubulin. ( -tubulin Xenopus E Asingleepidermiscell expressing highlevels ofShroom3 apicallyconstricts ) the spatialdistribution, ratherthanabsolutelevels, of material). Thesefindings thereforesuggestthatShroom3actstoalter the totalamountof naïve cellsexpressing Shroom3was notassociatedwithachangein western blottingdemonstratedthattheaccumulationof also sufficient todrive apicalredistribution of these resultsdemonstratethatShroom3isnotonlynecessarybut interaction betweenShroom3and immunoprecipitation experiments failed todetectaphysical ␥ occupies adomainimmediatelyapicaltotheregion ofaccumulated Shroom3 proteindidnotcolocalizewith epithelial cells.Moreover, wefoundthattheectopicallyexpressed cells. We suggestthatthiseffect on apicobasal cellelongation. the capacityofShroom3toorganize apicobasalMTarraysanddrive -tubulin (Fig.6E,e ␣ Consistent withourdatafromloss-of-functionmanipulations, Together withourloss-of-functiondata(Fig.4,andseeabove), -tubulin. Cellsexpressing Shroom3 (markedbyanti-Myc; ␥ Fig. 6.Shroom3 issufficient todriveapicalaccumulationof anti- surface viewofthesuperficialblastomere epitheliumstainedwith control embryoatearlystagesstainedwithanti- tubulin. ( view ofthesuperficialblastomere epitheliumstainedwithanti- tubulin. Mergedimageisshownin ectopically expressing Shroom3 atearlystagesstainedforanti- image isshownin ( and stages. EmbryohasbeenstainedtodetectMyc-Shroom3 (E;red) blastomeres ofanembryoectopicallyexpressing Shroom3 atlater propidium iodide).( D -tubulin innaïveepithelialcells. C Cross-section through superficialblastomeres ofanembryo ) Control epidermisstainedfor ) ␥ ␥ -tubulin ( -tubulin. ( C Cross-section through superficialblastomeres ofa ) e ␥ B Ј Ј -tubulin present(Fig.S3Binthesupplementary ; green). Mergedimageisshownin Shroom3-expressing embryoshowingsurface ) ,e c Љ E Ј ). Consistentwiththelocalizationdata,co- Cross-section through superficial ) (green, d Ј ␥ are onlyslightlyconstricted, butare ) -tubulin; red, propidium iodide). ␥ ␥ -tubulin (datanotshown). -tubulin localizationunderlies ␥ d -tubulin. ( Ј ( A (green, Control embryoshowing ) ␥ Development 134(7) ␥ -tubulin, but instead -tubulin inepithelial D ␥ -tubulin; red, Epidermis ) ␥ -tubulin. Merged ( A Control ) e ␥ ␥ Љ . -tubulin in b -tubulin in Ј ) ␥ ␥ - -

DEVELOPMENT accumulation of injection atthefour-cell stage,weobserved simultaneous at theapicalsurface. Whenweexpressed Shroom3bymRNA would bereflectedinthetimingofactinand that ifoneactivity was primaryandtheothersecondary, thensuch function assayusingnaïve blastomeres(seeFig.5A).We reasoned control actin-andMT-based cellshapechanges. (Fig. 1C)raisesthepossibilitythatShroom3mayindependently uncoupling ofcellelongationfromapicalconstrictioninneural cells alternatively, oneoftheeffects couldbesecondarytotheother. The Shroom3 coulddirectlyimpactbothMTandactinassembly, or Shroom3 Joint control of is cell-autonomousinneuralepithelialcells(Holtfreter, 1946). autonomous andisconsistentwithaprevious finding thatelongation demonstrates thatcellelongationmediatedbyShroom3is significantly elongatedintheapicobasalaxis(Fig.7E,e Shroom3-expressing cellswereapicallyconstricted and expressing epidermalcells.Inallcases,individual, isolated epidermal cellsexpressed Shroom3andweresurrounded bynon- Shroom3 intheepidermis.We foundmany casesinwhichsingle question, weagainexamined embryosmosaically expressing mediated changesinoverall tissuegeometry. To addressthis it isalsopossiblethatelongationaby-productofShroom3- It ispossiblethatShroom3drives elongationcell-autonomously, but autonomously Shroom3-mediated elongationoccurscell- epithelium. subcellular events observed inhingepoint cellsintheneural remarkable accuracy thecellshapechangesand underlying expression ofShroom3inepidermalcellsrecapitulates with Fig. 7D,d in cellsthathave yettofullyconstricttheirapical surface (compare expresssing epidermalcellswas associatedwithcell elongationeven for normalneuralplatecells,accumulationof with MTassemblyandcellelongation(Fig.7C,D,d develop abroad,diffuse accumulation of in neuralepithelialcells,Shroom3-expressing epidermalcells MT arraysemanatingfromtheapicalsurface (Fig.7B).Finally, as Importantly, theelongationis associatedwithassemblyofrobust cells apicallyconstrictandelongatedramatically(Fig.7A,B,b the matureepidermis(seeVize etal.,1991). into earlyembryostomosaicallyexpress Myc-taggedShroom3in function ontheMTcytoskeleton, weusedplasmid DNA injections hinders imagingofMTs.To analyzetheresultofShroom3gain-of- However, thevery denseyolkof apical constriction,cellelongationand of neuroepithelialcellshapechange,includingactinaccumulation, Expression ofShroom3inblastomeresrecapitulatesmany features cells assembly ofapicobasallyalignedMTs inepithelial Expression ofShroom3 issufficient todrive Shroom proteinsmicrotubule govern architecture accumulation ofonecomponent priortothatoftheother. less than1hour(Fig.8A,B). In nocasedidweobserve apical accumulated large amountsof accumulation was notstrictly interdependent.Somecellsthat apical surfaces ofShroom3-expressing cellsalsoargued thattheir To begin toprobethispossibility, weturnedagaintoourgain-of- Compared withcontrolepidermalcells,Shroom3-expressing Moreover, thepatternsofaccumulated Ј with Fig.S2A,a ␥ -tubulin andactinatthe apicalcellsurface within ␥ -tubulin andactindistributionby Ј in thesupplementarymaterial).Thus, ␥ Xenopus -tubulin didnotaccumulateactin ␥ ␥ blastomeres significantly ␥ -tubulin thatisassociated -tubulin andactinatthe -tubulin accumulation. ␥ ␥ -tubulin accumulation -tubulin inShroom3- Ј ). Asisthecase Ј ). Thisresult Ј ). anti- actin; arrow indicatesaccumulatedactinwithlittle of thesuperficialblastomere epitheliumstainedwithphalloidin( ( blastomere epitheliumstainedwithphalloidin( ( epithelium stainedwithphalloidin( at stage6showingsurfaceviewofthesuperficialblastomere The in non-neuralepithelialcells based elongationandaccumulationof Shroom familyproteins are associatedwithMT- phalloidin ( surface viewofthesuperficialblastomere epitheliumstainedwith adhesive organ ofamphibian embryos(PerryandWaddington, 1966). expressed inepithelial cells ofthecementgland(Fig.9A,B),an related proteinShroom1(Hagens etal.,2006;Staub1992). activity isaconserved featureofthisproteinfamily, westudiedthe induce diverse cellshapechanges.To askwhetherMT-regulating can coordinatetheresponseof both MTandactincytoskeletons to the actincytoskeleton, but ourdataabove demonstratethatShroom3 al., 2006).Previous reportshave associated eachoftheseproteinswith proteins thatarecloselyrelatedinsequencetoShroom3(Hagens et tubulin (Fig.8D,d high levels ofactinfailed toaccumulate significant amountsof (Fig. 8D,d and Fig. 8.Shroom3 coordinates thepolarizedassemblyofbothactin c C Љ Control embryoatstage7.5showingsurfaceviewofthesuperficial ) ). ( In situhybridizationrevealed thatbothShroom3andShroom1were ␥ ␥ Xenopus D -tubulin ( -tubulin cytoskeletons. Shroom3-expressing embryoatstage7.5showingsurfaceview ) Ј a ,d Ј ) andanti- Љ , mouseandhumangenomeseachcontainadditional , bracket), andconversely somecellsthataccumulated d Љ ). Bracketindicatesaccumulated Ј ,d Љ , arrow). ␥ -tubulin ( ( A a Control embryoatstage6showing ) b Љ .( ). Ј ) andanti- RESEARCH ARTICLE B Shroom3-expressing embryo ) c ␥ Ј ) andanti- -tubulin ( ␥ ␥ -tubulin withlittle -tubulin. ␥ -tubulin b ␥ Љ -tubulin ). d Ј 1437 ) and ␥ -

DEVELOPMENT increased theirheightbyover 30 elongation occursindependentlyofapicalconstriction;indeed,cells As inneuralcells,cementglandcellsundergo periodsinwhichcell radical apicobasalelongation,but constrictonlyslightly(Fig.9C-E). This strongexpression was intriguingbecausethesecellsundergo a 1438 include apical constrictionandapicobasal cellelongation. morphogenetic tissuemovements. Two suchcellshapechanges Cell shapechangesmake animportantcontribution to actin cytoskeletons Shroom familyproteins influencebothMTand DISCUSSION at cell-celljunctions. cell surface, Shroom1drove accumulation of whereas Shroom3drove accumulationof role forShroom1ingoverning MTarchitecture. Interestingly, tubulin innaïve epithelialcells(Fig.10A,B,b ectopic expression ofShroom1didelicitapicalaccumulation and didnotinduceapicalconstriction(datashown). Nonetheless, Shroom1 failed toelicitapicalaccumulationofactinmicrofilaments assay system(seeFig.5A).Unlike Shroom3,expression of throughout theapicalregions ofthesecells(Fig.9F,G,g time andspacewithabroad,intenseaccumulationof the thickarraysofMTsincementglandwereagaincorrelated (Fig. 9F,f apicobasally alignedMTsthatemanatefromtheapicalcellsurface cell elongation,cementglandcellscontainrobust arraysof constriction was negligible (seestages22-25inFig.9D). We next testedtheactivity ofShroom1usingournaïve blastomere Consistent witharoleforShroom1andShroom3inMT-mediated RESEARCH ARTICLE Ј ) (PerryandWaddington, 1966;Picard,1976).Moreover, ␮ m duringatimewhenapical ␥ -tubulin acrosstheapical ␥ Ј -tubulin specifically ), consistentwitha Ј ). ␥ -tubulin ␥ - and thesetwo cellshapechangescanoccur independentlyinboth Finally, Shroom3drives bothapicalconstriction andcellelongation, assembly orapicalconstriction (Fig.10A)(Fairbank etal.,2006). accumulation of another family member, Shroom2,caneachdrive apical can accumulateindependently(Fig. 8).Moreover, Shroom1 andalso example, inShroom3-expressing blastomeres,actinand these effects areatleastpartiallyindependentofoneanother. For accumulation isnotyetclear, but thereisevidence tosuggestthat 2005). How thisactin-myosinsystemislinked with Shroom3-mediated apicalconstrictioninnaïve cells(Hildebrand, mouse neuralplate,andthatmyosinIIfunctionisessential for apical localizationofmyosinIIinbothculturedcellsand the remain obscure,but itwas shown recentlythatShroom3drives the ␥ associated withcellelongationandcandrive apicalredistribution of show thatanothermemberofthisfamily, Shroom1,isalso Prat etal.,1996;Yoder andHildebrand,2006).Inthisstudy, we al., 2006;Hagenset2005;Haigo2003;Hildebrand, some way withtheactincytoskeleton (Dietz et al.,2006;Hagens of asmallfamily ofproteins,allwhichhave beenassociatedin apicobasal cellelongationinepithelialcells.Shroom3isamember tubulin, theassemblyofrobust apicobasalMTarrays,and necessary andsufficient toinduceanapicalredistribution of shape changesduringneuraltubeclosure.We show thatShroom3is additional functionforShroom3inthecontrolofMT-mediated cell accumulation (Haigoetal.,2003).We now demonstrateanovel, sufficient toinduceapicalconstrictionandcoincidentactin Previously, wedemonstratedthatShroom3isnecessaryand -tubulin innaïve epithelialcells. The mechanismsbywhichShroomfamily proteinsfunction show nuclei. shown inGmergedwithpropidium iodidestainingto nuclei. ( merged withpropidium iodidestainingtoshow accumulation of gland are associatedwithabroad apical fcmn ln el vrtm sae 19-25).( of cementglandcellsovertime(stages constriction (blue)andapicobasalcellelongation(red) epithelial cells.( sections of gland epithelialcellshape.( schematic diagramillustratingthechangeincement tissues (grey). ( gland epithelialcells(red) andothersurrounding diagram ofthesectioninC,illustratingcement eetgad ( cement gland. hybridization reveals Shroom1 expression inthe phalloidin toreveal cellshapes.( section through thecementgland,stainedwith ( the invaginatingnasalandoticplacodes. Shroom3 expression inthecementgland,andalso elongation innon-neuralcells. Fig. 9.Shroom familyproteins andcell ␥ -tubulin, but neithercanelicitsignificant actin G RobustapicalMTarraysinthecement ) ␣ -tubulin stainingofcementgland D B f ) Graphofchangesinapical Ј ␥ Insituhybridizationreveals ) ) -tubulin. ( ␣ -tubulin stainingshowninF F g Projection ofoptical ) Ј ) Development 134(7) ␥ c -tubulin staining Ј Aschematic ) ( A Insitu ) C Cross- ) ␥ ␥ -tubulin -tubulin E )A ␥ -

DEVELOPMENT ␥ cell elongationremainstobediscovered. Thebroaddistribution of (Fig. 10C). affecting bothapical constrictionandapicobasalcellheightening directs accumulationofboth actomyosinand propose apreliminarymodelwhereby apicallylocalizedShroom3 Combining ourdatawiththatofothers(e.g.Hildebrand,2005), we neural andcementglandepithelialcells(Fig.1C;Fig. 9D). Shroom proteinsmicrotubule govern architecture [indicated byanti-Mycstaining(red)] accumulateexcess of blastomeres stainedfor accumulation of Fig. 10.Shroom1 expression issufficient to elicitapical view ofblastomeres stainedfor accumulate highlevelsof and Haigoetal.(Haigoal.,2003). current workandalsoontheofHildebrand(Hildebrand,2005) changes inneuralepithelialcells.Modelisbasedondatafrom the cell-cell boundaries.( -tubulin inelongatingneuralepithelial cellshasnottoour Finally, theroleplayedby ␥ -tubulin innaïveepithelialcells. C ModelforShroom3-mediated cellshape ) ␥ ␥ -tubulin atcell-celljunctions.( -tubulin. Shroom1-expressing cells(right) ␥ ␥ -tubulin. ( -tubulin duringShroom3-mediated b Ј Shroom1-expressing cells ) ␥ ( A ␥ -tubulin, thus B -tubulin at ) Surfaceview Section ) constriction in of actinandmyosinappeartobeuniversal, driving apical evolutionarily diverse celltypes?Apicallocalizationandcontraction How conserved isthemechanismofcellshapechangeacross vertebrates andinvertebrates Common mechanismsofapicalconstrictionin similar proteinsmediateShroom-dependent mitosis (Ludersetal.,2006),soitistemptingtospeculatethat as GCP-WD,areknown toregulate be importantinthefuturetodeterminehow MTs (Gunawardane etal.,2000;Lajoie-Mazenc al.,1994).Itwill minus-ends, but inthemitoticspindleitassociateswithsidesof 1994). Ininterphasecells, most epithelia, we find thatinneural platecellsengagedinapical nature ofcellpolaritywithinepithelia duringmorphogenesis.Unlike One interestingimplicationofthe resultsinthisstudyconcernsthe Cell polarityindeveloping epithelia into focus. comprehensive pictureofthesecellshapechangeswillbebrought understanding ofsuchdiscrepancieswillberequiredbefore a disrupts apicalconstrictioninvertebrates (Brounsetal.,2000).An to betrue,asitislossofaRhoGAP, ratherthanaRhoGEF, that (Haigo etal.,2003;Hildebrand,2005).Infact, theopposite appears constructs clearlycannotblockShroom-mediatedapicalconstriction Drosophila (Rogers etal.,2004).Finally, thecrucialroleforaRhoGEFin directs theapicallocalizationofEB1,aplus-endbindingprotein a MTminus-end-associatedprotein(Figs4,6),whereasDRhoGEF2 Moreover, Shroom3controls theapicalaccumulationof heightening (LeeandGoldstein,2003;NancePriess,2002). roundworm isnotapparentlyassociatedwithapicobasal cell discrepancies mustbenoted.For example, apicalconstrictioninthe ubiquitous coincidenceofcellheighteningandapicalconstriction. some casesbyasinglemolecule,thusappearstounderliethealmost et al.,2004).Avery closelinkageofMTandactinorganization, in plus-end-associated proteinEB1(Dawes-Hoang etal.,2005; Rogers also inMTorganization, inthiscasethroughlocalizationoftheMT this moleculeisimplicatedinnotonlymyosinIIlocalization,but Nikolaidou andBarrett,2004;Rogersetal.,2004).Like Shroom3, cell elongation(Barrettetal.,1997;Hacker andPerrimon,1998; be bothnecessaryandsufficient toinduceapicalconstrictionand exchange factor forthesmallGTPase Rho,DRhoGEF2,appearsto governance ofthesetwo cellshapeevents in Recent studieslikewise implicateasingle moleculeinthe elongation andapicalconstrictionbyorganizing bothMTsandactin. 1977). 1984; Schroeder, 1970;Sweetonetal.,1991;Viamontes andKirk, Takata, 2000;NishiiandOgihara,1999;Schoenwolf andFranks, Hertzler andClark,Jr, 1992;Kametal.,1991;Kominami and shrimps, sanddollars,mice,chicksandfrogs(Burnside,1973; constricting cellsacrosstaxa,including Likewise, anincreaseincelllengthisobserved inapically Priess, 2002;NishiiandOgihara,1999;Young etal.,1993). vertebrates (Hildebrand,2005;LeeandGoldstein,2003;Nance proteins regulate thedistribution of the MTsthatelongateneuralepithelialcellsandhow Shroom of knowledge beenreportedinnon-mitoticcells,but itisreminiscent Although suchunifyingfeaturesareimportant,several We have shown herethatShroom3governs bothapicobasal ␥ -tubulin distribution inmitoticspindles(Lajoie-Mazencetal., is perhapssurprisinggiven thatdominant-negative Rho Volvox , Drosophila ␥ -tubulin isgenerallyassociatedwithMT ␥ RESEARCH ARTICLE -tubulin. Specific proteins,such , Caenorhabditis elegans ␥ -tubulin targeting during ␥ ␥ -tubulin redistribution. -tubulin interactswith Volvox Drosophila , Drosophila ␥ -tubulin, 1439 . An and ,

DEVELOPMENT Burnside, B. Burnside, Bacallao, R.,Antony, C.,Dotti,Karsenti,E.,Stelzer, E.H.andSimons,K. Aaku-Saraste, E.,Oback,B.,Hellwig,A.andHuttner, W. B. Davidson, L.A.andWallingford, J.B. Chenn, A.,Zhang,Y. A.,Chang,B.T. andMcConnell,S.K. Chalmers, A.D.,Pambos,M.,Mason, J.,Lang,S.,Wylie,C.andPapalopulu, Brun, R.B.andGarson,J.A. http://dev.biologists.org/cgi/content/full/134/7/1431/DC1 Supplementary materialforthisarticleisavailableat Supplementary material to J.B.W. andbytheNIH/NIGMS(1RO1GM74104). a Career Award intheBiomedicalSciencesfrom theBurroughs Wellcome Fund by aUTAustin/UniversityCo-opUndergraduateResearch FellowshiptoH.M.S., Brouns, M.R.,Matheson, S.F., Hu, K.Q.,Delalle,I.,Caviness,V. S.,Silver, J., Barrett, K.,Leptin,M.and Settleman,J. Aaku-Saraste, E.,Hellwig,A.andHuttner, W. B. References laboratory forthegiftof for helpfuldiscussionsandreading ofthemanuscript.We thanktheKirschner Sisson forcriticalinsights,andA.Ewald,J.Gross, J.HildebrandandS.Haigo We thankP. Fairbank,E.KiesermanandA.Ellisfortechnicalassistance,J. constriction, 1440 Bre, M.H.,Pepperkok,R., Hill,A.M.,Levilliers,N.,Ansorge,W., Stelzer, E. morphogenetic events involving epithelialsheets. depolarization maybeacommonmechanismunderlying developing epithelia.Moreover, wesuggestthatpartial discrete cytoskeletal elementsthatdrive cellshapechangesin family proteinsplayacentralroleincontrollingtheassemblyof apically localized(Fig.3).Insum,ourdatasuggestthatShroom in embryoslackingShroom3function,ZO-1andPar3 remain whereas bothactin-andMT-based cellshapechangesaredisrupted change. Thisinterpretationisconsistentwithourfindings that polarity canbeuncoupledfromthoseelementsdriving cellshape findings suggestthatcytoskeletal elementsmaintainingoverall cell do not(Aaku-Sarasteetal.,1996;Aaku-Saraste1997).These markers remainapicallylocalizedduringneuraltubeclosure,others epithelial cellsduringneuraltubeclosure:whereassomepolarity downregulation ofapicobasalpolaritypreviously reportedforneural Xenopus distribution was observed inShroom1-expressing cellsinthe coincident withexpression ofShroom3,andasimilar epithelial cells(Fig.4).Thisodddistribution of distribution throughouttheapicalregion ofelongatingneural surface. Instead,wefind that membrane, norisitlocalizedincentrosome-like fociattheapical neural tubeclosure andneurogenesis. Neuroepithelial cellsdownregulate theirplasmamembrane polaritypriorto polarity ofmammalianneuroepithelial cells. Am. Zool gastrulation. RhoGEF mediateasignalingpathwayforthecellshapechangesinDrosophila the formationofapolarizedepithelium. (1989). ThesubcellularorganizationofMadin-Darbycaninekidneycellsduring 679. remodeling oftheneuroepithelium priortoneurogenesis. functional tightjunctions,butnotZO-1,duringneuraltubeclosure – Press. Konnerth), pp.125-136.ColdSpringHarbor, NY: ColdSpringHarborLaboratory embryos. In vertebrate development. N. and theneuralplate. (Ambystoma mexicanum):adrugstudyandcellshapeanalysisoftheepidermis Development p190 RhoGAPisrequired formorphogeneticprocesses inneuraldevelopment. Bronson, R.T. andSettleman,J. during polarizationinMDCKIIcells. H. andKarsenti,E. This distribution of (2005). aPKC,Crumbs3andLgl2control apicobasalpolarityinearly RESEARCH ARTICLE cement gland(Fig.9). . 13 (1973). Microtubules andmicrofilaments inamphibianneurulation. Imaging inNeuroscienceandDevelopment , 989-1006. Cell ␥ 127 -tubulin isnottightlyassociatedwiththeapicalcell 91 , 4891-4903. , 905-915. (1990). Regulationofmicrotubule dynamicsandnucleation J. Embryol. Exp.Morphol J. Embryol. Xenopus Development (1983). NeurulationintheMexicansalamander ␥ ␥ -tubulin mayreflectthepartial -tubulin antibody. Thisworkwassupported (2000). Theadhesionsignalingmolecule J. CellBiol ␥ Mech. Dev (2005). Visualizing morphogenesis infrog(2005). Visualizing -tubulin accumulatesinabroad 132 J. CellBiol (1997). TheRhoGTPaseandaputative , 977-986. Mol. Cell.Neurosci . . 74 111 . , 275-295. 69 . (1996). Lossofoccludinand , 3013-3021. 109 , 71-81. (ed. R.Yuste andA. , 2817-2832. Dev. Biol (1998). Intrinsic (1997). . 11 ␥ , 183-193. -tubulin is . 180 ␥ -tubulin , 664- Holtfreter, J. Hildebrand, J.D.andSoriano,P. Gunawardane, R.N.,Lizarraga,S.B.,Wiese,C.,Wilde,A.andZheng,Y. Hildebrand, J.D. Job, D.,Valiron, O.andOakley, B. Haigo, S.L.,Hildebrand,J.D.,Harland,R.M.andWallingford, J.B. Fristrom, D. Hertzler, P. L.andClark,W. H.,Jr Hagens, O.,Ballabio,A.,Kalscheuer, V., Kraehenbuhl,J.P., Schiaffino, M.V., Hagens, O.,Dubos,A.,Abidi,F., Barbi,G.,Van Zutven,L.,Hoeltzenbein,M., Hacker, U.andPerrimon,N. Fairbank, P. D.,Lee,C.J.,Ellis,A.,Hildebrand,J.Gross, J.M.and Dawes-Hoang, R.E.,Parmar, K.M.,Christiansen,A.E.,Phelps,C.B.,Brand, Kominami, T. andTakata, H. Karfunkel, P. Kam, Z.,Minden,J.S.,Agard, D.A.,Sedat,J.W. andLeptin,M. Dollar, G.L.,Weber, U.,Mlodzik,M.andSokol,S.Y. T.Dietz, M.L.,Bernaciak, M.,Vendetti, F. andHildebrand,J.D. Lajoie-Mazenc, I.,Tollon, Y., Detraves,C.,Julian,M.,Moisand,A.,Gueth- Lee, J.Y. andGoldstein,B. Nikolaidou, K.andBarrett, K. T.Luders, J.,Patel,U.K.andStearns, Nieuwkoop, P. D.andFaber, J. Picard, J. Perry, M.andWaddington, C.H. Nance, J.andPriess,R. Meads, T. andSchroer, T. A. Nishii, I.andOgihara,S. Müsch, A. Messier, P. E. 99 actin-binding protein, isrequired forneuraltubemorphogenesisinmice. positioning ofanactomyosinnetwork. amphibian cells. Development shrimp Sicyoniaingentis:invaginationisaccompaniedbyorientedcelldivision. during neuraltubeclosure. Shroom inducesapicalconstrictionandisrequired forhingepointformation Top. Dev. Biol (2000). gamma-Tubulin complexesandtheirrole inmicrotubule nucleation. 645-690. in Xenopus. 7 Drosophila. family ofoncogenesandcontrols cellshapechangesduringgastrulationin 4118. activity ofshroom-family proteins. Differential actin-dependentlocalizationmodulatestheevolutionarilyconserved standard nomenclature forproteins related toApxandShroom. Smith, P. R.,Staub,O.,Hildebrand,J.andWallingford, J.B. Hum. Genet of thenovelKIAA1202geneare associatedwithX-linkedmentalretardation. Tommerup, N.,Moraine,C.,Fryns,J.P., Chelly, J.etal. Cell Biol three-dimensional fluorescence microscopy. Drosophila gastrulation:analysisofcellshapechangesinlivingembryosby and localizationintheretinal pigmentepithelium. Wallingford, J.B. Lethal giantlarvaebyDishevelled. the control ofmyosinlocalization. A. H.andWieschaus,E.F. dollar Scaphechinusmirabilis. cells: presence ofgamma-tubulininthemitoticspindle. al. Hallonet, C.,Debec,A.,Salles-Passador, I.,Puget,A.,Mazarguil,H.et elegans gastrulation. 2825-2837. activation. used reiteratively inepithelialfoldingandpotentiallyselectstheoutcome ofRho New York: Garland. Development nucleation. targeting factorrequired forcentrosomal andchromatin-mediated microtubule Morphol Experientia cement gland. 2117-2127. hemisphere isrequired forinversionoftheVolvox embryo. 5 polarized epithelialcells. , 18. , 1-9. , 485-497. (1994). Recruitmentofantigenicgamma-tubulinduringmitosisinanimal . . (2004). Microtubule organization andfunctioninepithelialcells. 15 (1976). Ultrastructure ofthecementglandXenopuslaevis. 148 (1988). Thecellularbasisofepithelialmorphogenesis. Curr. Biol 34 (1946). Structure, motilityandlocomotioninisolatedembryonic Nat. CellBiol (1971). Therole ofmicrotubules andmicrofilaments inneurulation , 111-117. (1978). Microtubules, interkinetic nuclearmigrationandneurulation. Genes Dev . Dev. Biol . 118 , 193-208. 116 129 , 289-296. 49 J. Cell.Sci. (2005). Shroom regulates epithelialcellshapeviatheapical J. Morphol , 55-73. , 578-590. , 127-140. , 387-397. (2006). Shroom2 (APXL)regulates melanosomebiogenesis . . 14 Development 25 . , 1822-1826. 12 (1999). Actomyosincontractionofthe posterior . Cell Motil.Cytoskeleton , 30-56. (2002). CellpolarityandgastrulationinC.elegans. 8 1 , 274-284. (2003). MechanismsofcellpositioningduringC. , 137-147. , 193-200. . Curr. Biol (1995). Polarityandnucleationofmicrotubules in (1998). DRhoGEF2encodesamemberoftheDbl (2005). foldedgastrulation,cellshapechangeand 79 (2000). Cellularbasisofgastrulationinthesand Biol. Bull. (1994). , 27-62. (1999). Shroom, aPDZdomain-containing (2004). ARhoGTPasesignalingpathwayis (1992). Cleavageandgastrulationinthe Nature Development J. Biol.Chem (2003). Microtubule nucleation. 130 . (1966). Theultrastructure oftheXenopus 13 (2006). GCP-WDisagamma-tubulin Normal Table ofXenopuslaevis(Daudin). J. CellSci. , 307-320. 199 , 2125-2137. 437 , 287-297. Development , 1376-1380. . 132 281 32 118 Development , 273-288. , 4165-4178. , 20542-20554. , 5191-5203. Development 134(7) (2005). Regulationof 112 J. CellSci. Development (2005). Disruptions , 365-370. BMC CellBiol 133 (2006). Anew Tissue Cell (2006). (1991). Curr. Opin. , 4109- 107 (2003). J. 126 , Cell Traffic Curr. 20 , . ,

DEVELOPMENT Prat, A.G.,Holtzman,E.J.,Brown, D.,Cunningham,C.C.,Reisin,I.L., Shroom proteinsmicrotubule govern architecture Rizzolo, L.J.andJoshi,H.C. Rogers, S.L.,Wiedemann,U.,Hacker, U.,Turck, C.andVale, R.D. Rodriguez, O.C.,Schaefer, A.W., Mandato,C.A.,Forscher, P., Bement,W. Roberts, S.J.,Leaf,D.S.,Moore, H.P. andGerhart,J.C. Schoenwolf, G.C.andFranks,M.V. Schroeder, T. E. Sive, H.L.,Grainger, R.M.andHarland, channel. H. F. Kleyman, T. R.,McLaughlin,M.,Jackson,G.Jr, Lydon, J.andCantiello, retinal pigmentepitheliuminvivo. organizing centerandassociatedgamma-tubulinduringthepolarizationof manner. Drosophila RhoGEF2associateswithmicrotubule plusendsinanEB1-dependent interactions incellmovementandmorphogenesis. M. andWaterman-Storer, C.M. Biol establishment ofpolarizedmembranetraffic inXenopuslaevisembryos. cell shapesduringbendingoftheavianneuralplate. based uponlightandelectron microscopy. 462. . 118 (1996). Renalepithelialprotein (Apx)isanactincytoskeleton-regulated Na+ Curr. Biol , 1359-1369. J. Biol.Chem (1970). NeurulationinXenopuslaevis.Ananalysisandmodel . 14 , 1827-1833. . 271 , 18045-18053. (1993). Apicalorientationofthemicrotubule Dev. Biol (2003). Conservedmicrotubule-actin (1984). Quantitativeanalysesofchangesin J. Embryol. Exp.Morphol J. Embryol. . 157 (2000). , 147-156. Nat. CellBiol Dev. Biol Early Developmentof (1992). The . 105 . 5 , 599-609. , 257-272. . (2004). 23 , 427- J. Cell Viamontes, G.I.andKirk,D.L. Viamontes, Sweeton, D.,Parks,S.,Costa,M.andWieschaus,E. T.,Stearns, Evans,L.andKirschner, M. Staub, O.,Verrey, F., Kleyman,T. R.,Benos,D.J.,Rossier, B.C.and Young, P. E.,Richman,A.M.,Ketchum,S.andKiehart,D.P. Yoder, M.andHildebrand,J.D. Vize, P.Vize, D.,Melton,D.A.,Hemmati-Brivanlou,A.andHarland,R.M. of inversioninVolvox. conserved componentofthecentrosome. Genes Dev Morphogenesis inDrosophila requires nonmusclemyosinheavychainfunction. invaginations. Drosophila: theformationofventralfurrow andposteriormidgut 119 laevis thatparticipatesinamiloride-sensitivesodiumchannelactivity. Kraehenbuhl, J.P. Harbor Press. Manual Xenopus laevis:ALaboratory Cytoskeleton associated protein implicatedincytoskeletalorganization. 367-387. Assays forgenefunctionindevelopingXenopusembryos. , 1497-1506. . 7 , 29-41. 64 Development , 49-63. (1992). Primarystructure ofanapicalprotein from Xenopus J. CellBiol 112 (1977). Cellshapechangesandthemechanism (2006). Shroom4 (Kiaa1202)isanactin- , 775-789. . 75 , 719-730. . ColdSpringHarbor, NY: ColdSpring (1991). Gamma-tubulinisahighly RESEARCH ARTICLE Cell 65 , 825-836. (1991). Gastrulationin Cell Motil. Methods CellBiol (1993). J. CellBiol (1991). 1441 . 36 . ,

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