Foxi1e Activates Ectoderm Formation and Controls Cell Position in The

Home , Ectoderm

are abletocontribute toallthreegermlayersduring theearlyand cell transplantationexperiments have shown thatanimalcells,which isdepleted (HoustonandWylie, 2003).Further,when VegT single- ability ofanimalandvegetal cellstosortfrom oneanotherislost adhesion arenotyetknown. However, ithasbeenshown thatthe (Turner etal.,1989).Themolecularmechanismsofthisdifferential reaggregate separateaccordingtotheiroriginalregional identities different regions oftheblastulathatare mixed andallowed to specification begins duringtheblastulastage.Dissociatedcellsfrom 1998). endoderm andmesoderminsteadbecomeectoderm(Zhangetal., that have beendepletedofVegT program ofgeneexpression anddevelopment. Indeed,inembryos pathway adoptinganectodermal not undertheinfluenceofVegT considered adefault pathway ofembryonicdevelopment, withcells cells arespecified tobecomeectoderm.Ectodermhaslongbeen induce mesoderm.Bycontrast,littleisknown abouthow animal release nodal-relatedfactors, whichactuponequatorialcellsto (Zhangetal.,1998).Thesecells T-box transcriptionfactor VegT specified tobecomeendodermbythevegetally localizedmaternal best studiedinvegetal andequatorialcells.Vegetal cellsbecome respectively. Themolecularmechanismsoftheseevents have been vegetal cellsarespecified toformmesodermandendoderm, Animal cellsarespecified tobecomeectoderm,whileequatorialand embryo occursduringtheblastulastage(Heasmanetal.,1984). Specification ofthethreeprimarygermlayersin INTRODUCTION Accepted 30November 2006 1 KEY WORDS:FoxI1e,Xema differentiate accordingtotheirnewpositions.Because precursors ofectodermalstructures.Initsabsence,theylosecontactwiththeanimalcap,mixcellsothergermlayers ectoderm. Inaddition,FoxI1eisrequiredtomaintaintheregionalidentityofanimalcellsblastula,that the blastulastagefornormalformationofbothcentralnervoussystemandepidermis,twoearlyderivatives how theectodermbecomesspecified.Inthispaper, weshowthattheforkheadproteinFoxI1e(alsoknownasXema)isrequiredat releases nodalsignalsthatspecifytheadjacentmarginalzoneofblastulatobecomemesoderm.However, littleisknownabo Xenopus The segregationofthevertebrateembryointothreeprimarygermlayersisoneearliestdevelopmentaldecisions.In C.Wylie Christopher and Adnan Mir position inthe FoxI1e activatesectodermformationandcontrolscell (2007)doi:10.1242/dev.02768 Development 134,779-788 Foundation, 3333 Burnett Avenue,Foundation, 3333Burnett Cincinnati, OH45229,USA. *Author forcorrespondence (e-mail:[email protected]) Cincinnati, OH45267,USA. Training Program, UniversityofCincinnatiCollegeMedicine,POBox670555, adhesion, whichlimitscellmixingasprimarygermlayersbecomespecified. neural andepidermal.TheworkalsoshowsthatFoxI1eplaysaroleintheembryopoorlyunderstoodprocessofdifferenti downregulated intheneuralplate,itsroleandepidermalgeneexpressionmustprecededivisionofectodermi Division ofDevelopmentalBiology, CincinnatiChildren’s HospitalResearch Previous work hasshown that theprocessofectoderm , wheretheprocessisbestunderstood,endodermspecifiedbyavegetallylocalizedtranscriptionfactor, VegT, which 1,2 , MattKofron Xenopus , 1, * 1 , , Aaron M.Zorn Xenopus cells normallyfated tobecome , Ectoderm 2 Physician Scientist 1 , MatejBajzer blastula FoxI1e Xenopus is initiallyexpressedintheanimalregionofembryoandrapidly FoxI1 Xema (Surietal.,2005).Sequence similarityplaces was aforkhead-typetranscription factor previously identified as both epidermalandneuralstructures. mid-blastula stageonward, athighlevels inthecellsdestinedtoform embryos. Thisidentified several candidategenes,expressed fromthe marginal andvegetal regions formectoderm)relative towild-type dissected blastulae,andinVegT-depleted embryos (inwhichthe upregulated bothinanimalcapsrelative tovegetal massesof carried outAffymetrix chipanalysestoidentifymRNAs thatare specification. present attheblastulastage,actively initiateectodermal epidermal, norwhichsignalingmoleculesandtranscriptionfactors, about genesthatactupstreamofthedecisiontobecomeneural or (Luo etal.,2002;Tao etal.,2005).However, notmuchisknown Robertis andKuroda, 2004;Stern,2005)orepidermalstructures potential tocontribute toeitherneuralstructures(reviewed byDe population. Itisclearthatallofthecellsanimalcaphave the border oftheCNSandepidermiscontribute totheneuralcrest to Bmpantagonistsreleasedfromtheorganizer. Thecellsatthe side giving risetothecentralnervous system(CNS)uponexposure response tohighlevels ofBmpsignaling,andthoseonthedorsal cells ontheventral sideoftheembryogiving risetoepidermisin begin tosubdivide intodifferent ectodermalsubpopulations,with expression. Duringthegastrulastage,cellsofanimal region ectoderm domain,ratherthanactivators ofectodermalgene have beenreportedtoberepressorsofmesoderm formationinthe have bothbeenfoundtobeimportantinthisprocess. Bothmolecules zygotic forkhead-boxtranscriptionfactor Xema(Surietal.,2005), Smad4 ubiquitinligaseectodermin(Dupontetal.,2005)andthe during theblastulastage.Thematernallyandzygoticallyencoded specification ofanimalcellsbegins duringthe lateblastulastage. from thelateblastulatogastrulastages(Snapeetal.,1987).Thus, mid-blastula stages,becomegraduallyrestrictedtoectodermalfates 2 , MansoorHaque The genemosthighlyoverexpressed inVegT-depleted blastulae To identifycandidate genes thatmayaddresstheseproblems,we Little isknown abouttheinitialevents ofectodermspecification class ofgenes,and sowesuggestthemoresystematic name 1 , JanetHeasman RESEARCH ARTICLE 1 Xema are and in the al nto 779 ut

DEVELOPMENT MATERIALS ANDMETHODS before separationintoepidermalandneuralpathways. initiation ofectodermalgenetranscription,andprobablyplaysarole differentiation. Thissuggeststhat for activation ofgenesinbothbranchesectodermal This shows that express genesnormallyexpressed inboththeCNSandepidermis. cells, they adoptcharacteristicsofectodermaldevelopment, and inhibition isoverridden byinjectionof downstream of to theanimalhalfofblastulaviainhibitionbynodalsignaling, in theepidermis.We thenshow that neurula stage.We show thatitremainsononlyinnon-ciliatedcells all ofthecells,andthatitisswitchedoff inthe CNS attheearly animal halfoftheembryoatblastulastage,but curiously, notin the formationofothergermlayers(Surietal.,2005). maintain theexpression domainofectodermalgenesbyinhibiting formation ofmesodermandendoderm.Itwas thereforeproposedto the blastulastage,andwhenoverexpressed, itinhibitedthe expressed onlyintheanimalhemisphereofembryo,startingat and Knochel,2005).Inaprevious study, lineages. Theseresultsshow that blastocoel, wherethey mixwith,anddifferentiate into,other contact withtheanimalregion oftheembryoandfall intothe adhesion atthemid-gastrulastage.Initsabsence,animalcellslose Lastly, weshow that and thedownregulation ofbothepidermalandneuralmRNAs. caused defectsinbothepidermisandnervous systemdevelopment, and henceexpression ofitsgeneproduct,intheearlyembryo. This oligodeoxynucleotide (SBMO)toblock To testthis,weusedasplice-blockingmorpholinoantisense of theseparationneuralandepidermalcellfates intheectoderm. role asaninhibitoroftheothergermlayers,andmayactupstream ectodermal differentiation, inadditiontoitspreviously identified mesoderm formation( Ornithine decarboxylase LightCycler systemasdescribedby Kofron etal.(Kofron etal.,2001). primers, andsemi-quantitative RT-PCR was carriedoutusingthe or five vegetal massespersample.cDNA was synthesizedusingoligodT Unless otherwiseindicated,inputwas two whole embryos,tenanimalcaps Total RNA was extracted aspreviously described (Zhang etal.,1998). RT-PCR RT-PCR. Each Affymetrix experiment was performedonce,andthenconfirmed by Trizol (Gibco)followed by purification throughRNeasycolumns(Qiagen). proteinase-K treatmentfollowed byphenol:chloroformextraction, orby at stage11.Total RNA forAffymetrix experiments was isolatedeitherby versus vegetal massmicroarrayexperiment, vegetal masseswereexplanted coated dishesin1 1967). Dissectionswereperformedateitherstage7or9onagarose- Staging was accordingtoNieuwkoop andFaber (Nieuwkoop andFaber, antisense oligobeforebeingfertilizedviathehosttransfertechnique. al., 1998)usingmanuallydefolliculatedoocytes injectedwith 7.5ng VegT Oocytes andembryos head specification ( The subclasses and20forkheadgeneshave beenidentified in in avariety ofdevelopmental processes.Currently, over ten transcription factor family includesalarge numberofgenesinvolved FoxI1e 780 In thiswork, wefirst confirm that FoxI1- - depleted embryosweregeneratedaspreviously described(Zhanget , whichwewillusethroughoutthispaper. Theforkhead RESEARCH ARTICLE class genesin FoxI1e ϫ VegT MMR andthenculturedinOCM.For theanimalcap , inthevegetal halfoftheembryo.Ifthis FoxI1a) FoxI1b is sufficient, whenoverexpressed ectopically, FoxI1e FoxI1e ( ODC Xenopus ) was usedasaloading control,andall andeye development ( ) is requiredforanimal:animalcell (Matsuo-Takasaki etal.,2005), are reportedlyinvolved inventral FoxI1e FoxI1e FoxI1e FoxI1e FoxI1e FoxI1e is involved intheactive FoxI1e may beanactivator of expression isconfined mRNA intovegetal is expressed inthe mRNA maturation, was foundtobe FoxI1c Xenopus. ) (Pohl VegT CGTTCATGTGGGCATAAGTG-3 GTTC-3 5 CACTCTTT-3 included restrictionsitesfor A coding-sequence-only mRNA, morpholinooligosandinjections in PBS+0.1%Tween-20. For co-stainingwith Blocking solutionwas 20%fetalcalfserumand4%bovine serumalbumin 488-conjugated (MolecularProbes)anti-mousesecondaryantibody. followed byHRP-(JacksonImmunoResearch),Cy5-(Jackson),orAlexa- ␣ Immunostaining andwhole-mountinsituhybridization 2003). New primersweregeneratedfor specific primersarelistedinHoustonandWylie (HoustonandWylie, experiments toverify reproducibilityofresults.Sequencesformarker- experiment was repeatedaminimumofthreetimesinindependent transcriptase-negative controlsfailed toproducespecific products.Each values werenormalizedto compared andfoldchangeswere calculated. to wild-typevegetal masses.Normalizedexpression levels were relative tocontrol vegetal massesandinwild-typeanimalcaps relative candidates thatwereupregulated in animal capsandvegetal massesatstage11.We thenlooked for embryos was compared.Second, expression was comparedbetween depleted earlygastrulastage(stage10)(Nieuwkoop andFaber, 1967) microarray experiments. First,expression in control andVegT- Gene expression changeswerecomparedintwo different Affymetrix embryos andinanimalcaps FoxI1e RESULTS xylenes, embeddedinparaplastandsectionedbeforeimaging. stage injections.For histology, embryoswerefixed, dehydrated,clearedin ng was usedforeight-cellstageinjections,and5ngwas usedfor32-cell 15 dextran (RLDX)orfluorescein-lysinedextran (FLDX) wereco-injected: at the32-cellstage.For tracinginjectedanimalcells,rhodamine-lysine (GFP)was injectedtotracethedescendentsofindividual D-tier cells protein greenfluorescent Four hundredpicogramsofmRNA encodingenhanced Lineage analysis mMachine kit(Ambion).Threemorpholinostargeting sites inpCS107.mRNA was synthesizedusingtheSP6mMessage CATGTTAAACCCCACAG-3 proteinase-K stepwas omittedtoensuresurvival ofthereactive epitope. modified fromHarland(Harland,1991)andthenstainedfor probe, embryoswerefirst processedbythewhole-mountinsituprotocol ATATGAGTGCATTTGATCCACA-3 approximately 40-fold inisolatedVegT GCCCCAGATAAAAG-3 GCTTGTGGATCAAATGCACTCATAG-3 synthesized andrejected:two weredesigned toinhibittranslation:5 injectate. tracers, wereinjectedsequentiallyratherthanmixed togetherinone For rescueexperiments, mRNA andSBMO,alongwithdifferent lineage mature andimmatureformsof designed totarget both.Intron-spanningprimersweredesignedtodetect sequenced. Two pseudoalleleswereidentified, andthemorpholinowas performed using designing primersagainst CTGTAAAGAAAA-3 found tobeeffective andnon-toxic:5 (only partiallyeffective). Aspliceacceptor-blocking oligo(SBMO)was against thesplicedonorsite:5 GGTCACAAATACACCTGTA-3 Ј -Tubulin stainingwas performedwith1:500DMEMantibody(Sigma), -GCAGCCACCAACTCTTCTCT-3 Ј ) and mRNA isupregulated in Ј , D:5 Sox X. laevis - 2 Ј -TGCTTTGAATTTGCAGTACATTG-3 (U: 5 Ј . Intronjunctionsequencewas determined by genomic DNA asatemplate,andtheproductwas FoxI1e Ј ODC , D:5 Ј X. tropicalis -TCTGCACATGAAGGAGCATC-3 Ј -TGAATGG TTATTACCTGGATCATCT-3 Ј ), andthenclonedintothecorresponding levels. Inallcases,water-only andreverse Cla Ј clone was generatedwithprimersthat Ј - TTGGGTCCAAGGTCC AATAA-3 Ј ). (ineffective), andonewas designed FoxI1e I and Ј Ј , D:5 , D:5 NRP-1 VegT genomic sequence.PCRwas Ј - Not Ј Ј mRNA (U:5 depleted vegetal masses, VegT- -depleted vegetal masses -TCGAGCG GC CGCACC- -CGTAGCTCCATTGCCT- txc n 5 (toxic) and (U: 5 FoxI1e Ј FoxI1e I (U:5 -ATAATTGCCCTTGC - Development 134(4) Ј -TGATCCGCTCTG- depleted FoxI1e was upregulated antisense-mRNA Ј -TCGAATCG - ␣ -tubulin. The Ј Ј mRNA were -CATGGA - Ј -CTAGCA - ), AP-2 Ј , D:5 (U: Ј Ј Ј ). - - Ј

DEVELOPMENT ae0.0485 0.0496 1.0026 Base VegT Uninjected wholeembryos VegT Uninjected base downstream of gene withexpression that isnormallysuppressedbynodalsignaling Therefore, relative tocontrols (Fig.1C). had increasedlevels of late neurula(stage18)stages.Embryos injectedwith (Agius etal.,2000),andcultured tothemid-gastrula(stage11)and stage with1.5ngofmRNA encodingCerS,asolublenodalinhibitor test thishypothesis,embryoswereinjectedvegetally atthetwo-cell signaling downstream ofVegT (inthemarginal zone),orboth.To embryo, eitherbyVegT directly(inthevegetal mass),or bynodal that itsexpression isnormallyinhibitedinthevegetal halfofthe VegT inboththeprospective mesodermandendoderm, itispossible in themarginal zone.As ligands invegetal cells,which,inturn,inducemesoderm expression from previous work that activation andlocalizedinhibitionin thevegetal cells.Itisknown be duetoeitherlocalizedactivation, ortoacombinationofglobal were notexpressed inthesamecells(Fig.2D). 2B,C). Analysisofmultiplesectionsshowed thatthesetwo markers respectively. We foundthat,infact, thereverse was thecase(Fig. FoxI1e co-stained tailbud embryos(stage30)for possibility that to develop ciliaontheirsurfaces (Deblandreetal.,1999).To testthe similar tothatof previously published. Biology (http://xenopus.nibb.ac.jp, cloneXL016g24),but not screen ofrandomcDNAs, bytheJapaneseNational InstituteofBasic salt andpepperexpression patternconfirms those shown inansitu developing nervous system,andmaintainedonlyintheepidermis.The early development. Atlaterstages,expression islostfromthe arrangement. Thispatternofexpression ismaintained throughout present inallcells(Fig.2A),but instead,ispresentinasaltandpepper that althoughitisexpressed intheanimalcapofblastula, itisnot (Suri etal.,2005).Additionally, insituhybridizationfor beginning afterthemid-blastulatransitioninprospective ectoderm previously reportedexpression pattern,whichshows expression with lower levels inthemesoderm(Fig.1B).Thisresultconfirms the expression revealed that mid-gastrula stage(stage11).Comparative analysisofgene mesoderm andendoderm,respectively, andculturedthemuntilthe equators andvegetal masses,representingprospective ectoderm, normal embryosatthelateblastulastage(stage9)intoanimalcaps, upregulated intheabsenceofVegT (Fig.1A).We thendissected regions ofcontrolembryosatstage10toshow that zones andvegetal masseswerecomparedwiththecorresponding semi-quantitative RT-PCR. VegT-depleted wholeembryos,marginal vegetal masses(Table 1).Thesemicroarrayresultswereconfirmed by FoxI1e compared withwild-typevegetal masses.Inwild-typeanimalcaps, FoxI1e specifies a 6.2330 Cap Table 1.Affymetrix microarray results for Expression of The saltandpepperpatternofexpression intheepidermis is bs 2.4000 12.2900 –whole embryos –base mRNA was overexpressed over 128-foldrelative towild-type mRNA bybothimmunochemistryandinsitu hybridization, FoxI1e VegT Xenopus ␣ FoxI1e -tubulin, whichisexpressed onlyincellsdestined . is expressed specifically inciliatedcells,we FoxI1e only intheanimalhalfofblastulacould VegT VegT FoxI1e ectoderm FoxI1e omlzdepeso Foldchange Normalized expression is expressed primarilyintheectoderm, activates expression ofnodal-class mRNA, asshown byRT-PCR, is upregulated intheabsenceof FoxI1e FoxI1e ␣ -tubulin proteinand ( Xema is anectodermal FoxI1e FoxI1e FoxI1e CerS ) 128.52 mRNA is 41.13 12.26 mRNA shows whole embryos. CerS are normalizedtoODC.( shows enrichmentof animal caps,equatorsandbasesdissectedfrom wild-typeembryos cadherin upregulated mRNAs encodingtheearlypan-ectodermalmarker gastrula stage(stage11).As determinedbyRT-PCR, stage (stage9),andvegetal explants werecultureduntilthemid- with 300or600pgof markers ofboththosetissues. Two-cell embryos wereinjected cap, incellsthatwillformboth epidermisandCNS,wetestedfor ectodermal markers. As at thelateblastulastageandassayedthemforexpression of and testedtheirfates intwo ways. First,weexcised vegetal masses normally express it,weinjectedvegetal cellswith explants compared withcontrol explants.( upregulated inVegT-depleted (VegT–) equatorial(eq)andvegetal(base) more extensively whatproperties upregulation oftheepidermalmarker epidermalkeratin. To test zone leadstoadownregulation ofseveral mesodermalgenesand differentiation (Surietal.,2005).Itsoverexpression inthemarginal FoxI1e FoxI1e absence ofVegT. Fig. 1. A C B mRNA atthetwo-cellstageincreases expression of FoxI1e Relative Expression has beenreportedtobeaninhibitorofmesoderm Relative Expression Relative Expression h epidermal markers the , 100 120 is anactivatorofectodermdifferentiation 100 150 200 250 300 100 150 200 250 300 20 40 60 80 50 50 0 0 0 mRNA isanimallylocalizedandupregulated inthe nn q eT qUijbs VegT-base Uninjbase VegT-eq Uninj eq RT-PCR analysisof FoxI1e asEutr Bases Equators Caps FoxI1e C Uninj 1ng CerS FoxI1e ) LossofNodalsignalingbyinjection1ng expression inprospective ectoderm.Data mRNA, dissected atthelateblastula is normallyexpressed intheanimal ODC FoxI1e epidermal cytokeratin FoxI1e RESEARCH ARTICLE FoxI1e FoxI1e B FoxI1e confers oncellsthatdonot ) Comparisonofisolated expression. ( FoxI1e FoxI1e A ) FoxI1e and in mRNA FoxI1e AP-2 is 781 E- ,

DEVELOPMENT overlapping expression. cells and83ciliatedwere counted.There were fiveinstancesof analyzed forexpression ofthetwomarkers.Sixty-one high-power picture isshowninC.( mount (B),andstainedembryoswere thenembeddedandsectioned.A are expressed indifferent cellpopulations.Stainingwasdoneinwhole for immunostaining for and Additionally, mRNAs encodingtheendodermalmarkers the neuralmarker FoxI1e not co-localizewithepidermalcilia. Fig. 2. 782 where they arealreadyexpressed, weinjected could furtherincreaseexpression ofthesemarkers intheectoderm, resembling epiboly (80%, hemispheres partiallyenveloped thevegetal masses inamanner Fig. 3D).However, pigmentedcellsin n cells formedaggregates onthesurface ofthevegetal masses(100%, cultured tosiblingearlytailbud stages,thepigmentedmesodermal ectoderm (illustratedinFig.3C). Incontrolvegetal hemispheres but excluded theanimalcellsthatwould normallygive riseto mesoderm, asindicatedbythepresenceofarow ofpigmentedcells, included alloftheprospective endodermand partoftheprospective cell stagewith600pgof blastula stage(stage9)inembryosthathadbeeninjectedatthe two- hemispheres wereseparatedfromanimalatthe late between thegermlayersasthey becomespecified. Vegetal cells displaydifferential adhesive propertiesthatlimitcellmixing including ingressionandconvergent extension. Additionally, all while mesodermalcellsundergo acomplex seriesofmovements, development. Ectodermalcellsundergo epibolyduringgastrulation, layers normallyexhibit specific behaviors duringearlystagesof characteristics ofdeveloping ectoderm.Cellsofthedifferent germ behave asnormalendodermandmesoderm orifthey adopt mesoderm, wewanted toknow whetherthe cells misexpressing it doses hadnoeffect onectodermalgeneexpression (datanotshown). FoxI1e expression inanimalcapsatstage11.Interestingly, highdosesof the animalcytoplasm atthetwo-cell stageandthenexamined gene =18) andinsomecasesextended away fromthemass(44%, As FoxI1e endodermin at stage10showingsaltandpepperexpression. ( FoxI1e FoxI1e RESEARCH ARTICLE mRNA (600pg)causedanimalcapstodissociate, andlower (purple, arrowheads) demonstratesthatthesetwomarkers is notexpressed inallectodermalcellsanddoes is notnormallyexpressed intheendodermand were decreased(Fig.3B).To determineif Sox-2 ␣ -tubulin (brown, arrows) andinsituhybridization n theneuralcrestmarker and FoxI1e n =20, Fig.3E).Further, immunostaining D mRNA. Thevegetal hemispheres ) Twenty high-powerfieldswere ( A ) Insituhybridizationfor FoxI1e FoxI1e -injected vegetal FoxI1e Slug B , C mRNA into ) Co- -positive (Fig. 3A) Xsox17 FoxI1e n =18, ␣ . positive cellslocatedintheepidermis. shown inwholemount(B).ThesectionDshowsGFP/ 10-30 pg tier cells.GFPsignalisrestricted totheendoderm.( mount andtransversesectionofembryosinjectedwithGFPalonein D- hemispheres ( embryos ( causes theirdescendentstoenterothergermlayers. Immunostaining for and cultured tostage11.( PCR analysisofvegetalexplantsinjectedwith300-600pg Fig. 3. marker cytokeratin increased, including Fig. 4.Injectionof explants, whichwereabsentfromcontrols (Fig.3F,G). We conclude against for cilia,aspecializationof pigmented mesodermalcellsformedalayeraround theexplant( that extendedfrom bases,whereas in vegetal hemispheres, pigmentedmesodermalcellsformedaggregates of vegetalhemispheres ectopicallyexpressing reduced. ( FoxI1e slug ␣ -tubulin revealed thepresenceofciliaonsurfaces ofthe F FoxI1e C ), butdemonstratedsurfaceciliationon ) Schematicofexperimentaldesigntodeterminebehavior and . ( B G expression issufficient forectodermformation. ) Endodermalmarkers ). AP-2 mRNA causescellstoenterothergermlayersas FoxI1e ␣ E-cadherin , theneuralmarker -tubulin tomarkciliawasnegativeincontrol A mRNA inD-tiercellsatthe32-cellstage ) Expression ofectoderm-specificmarkersis Xenopus , epidermalmarkers Sox17 FoxI1e- Sox-2 epidermis, withanantibody ␣ positive hemispheres, FoxI1e and and theneuralcrest Development 134(4) endodermin FoxI1e B epidermal . ( , D D ) Co-injectionof ) Incontrol -positive FoxI1e ( A , C ) Whole- were FoxI1e E mRNA ). RT- -

DEVELOPMENT morpholino oligo (SBMO)thatspansthesplice-acceptor siteofthe out aloss-of-functionanalysis. We designedasplice-blocking is requiredforbothneuralandepidermal differentiation, wecarried differentiation isa normalfunctionof formation. To testthehypothesisthatactivation ofectoderm already known function ofinhibitingmesodermandendoderm FoxI1e alone ( and GFP( tissues intailbud embryosin89%ofinjected with positive cellswereobserved inbothmesodermalandectodermal from moreanimallylocalizedblastomeres(Fig.4B,D).GFP- vegetally derived region oftheembryoandmove intotissuesderived many ofthedescendantsthesesinglevegetal cellstoleave the map (Fig.4A,C).Co-injectionof only wererestrictedtothedeveloping gut,as expected fromthefate The descendantsofvegetal blastomeresinjected withGFPmRNA to endoderm),togetherwithmRNA encodingthelineagetracerGFP. into onevegetal cellatthe32-cellstage(normallyfated tocontribute localization intheembryo.We injected10-30pgof gene expression characteristicofectoderm. that FoxI1e specifies As shown above, Next, wesoughttodeterminewhether FoxI1e n include moleculesthatmediateadhesionand/ormigration. =92) (Fig.4).Thissuggeststhatdownstream targets of n =109), but onlyin5%ofembryosinjectedwithGFP overexpression invegetal cellsleadstobehavior and Xenopus FoxI1e ectoderm can activate ectoderm,inadditiontoits FoxI1e FoxI1e mRNA, however, caused FoxI1e , andifso,whetherit also controlscell FoxI1e mRNA FoxI1e FoxI1e 20, 40or60ngoftheSBMO, into theanimalregion ofeachcell. development, byinjectingwholeembryosat thetwo-cell stagewith first examined theeffects ofglobal lossof inherently toxictoembryoniccells. isnot control animalcaps(Fig.5C).Therefore,lossofFoxI1e depleted ofFoxI1e showed thesamelevel ofmesoderminductionas blastula stage(stage9)weretreatedwithactivin. Animalcaps cell stagewith40ngSBMOandanimalcapsexcised atthelate mesoderm inductionbyactivin. Embryoswereinjectedatthetwo- cap assaytoseeifembryosdepletedofFoxI1e respondnormallyto weperformedananimal using asplice-blockingmorpholinoistoxic, stage (Surietal.,2005).To determineif translational startsiteresultsinembryonicdeathbythegastrula previously reportedthattheuseofamorpholinotargets the that itinhibits stage-14 embryospreviously injectedwiththeSBMOconfirmed gene. RT-PCR analysisofrandomhexamer primedcDNA from During earlytailbud stages,FoxI1e-deficient embryos failed to neural-fold formationwas also delayeddose-dependently(Fig.5D). controls reachedmid-neurulastage (stage16).Cementglandand during gastrulation,but gastrulatedcompletelybythetimesibling beginning atthegastrula stage.Allinjectedembryosweredelayed To determinetherole of - deficient embryosdisplayed dose-dependentabnormalities FoxI1e mRNA maturation(Fig.5A).Ithasbeen by SBMOinjectionatstage11.5. ( 2 reduced atstage23. PCR analysisof efficiently inhibitmRNAmaturation.( targeting thesplicedonorsite,doesnot product. ( used asapositivecontrol forunspliced are affected. GenomicDNAtemplatewas maturation bytheSBMO.Bothpseudoalleles demonstrating inhibitionofmRNA showing oligobindingsiteandRT-PCR sox-2, FoxG1 (stage 27,E).( (stage 19,D)andintailbudstageembryos development, shownattheneurulastage were dose-dependentabnormalitiesin cell stagewithadoserangeofSBMO.There abnormalities inembryos. maturation andcausesdevelopmental Fig. 5.AnSBMOprevents mRNA ( induction similartouninjectedcaps. animal capsshowedlevelsofmesoderm with 1 animal capsdepletedofFoxI1eandtreated G D are partiallyrescued by50pg ) , E Xbra, Xwnt-8 FoxI1e Embryosinjectedanimallyatthetwo- ) ␮ g/ml ActivinA.SBMO-injected B ) An alternative morpholino, ) Analternative in embryonicdevelopment, we RESEARCH ARTICLE ( F BF-1 ) XBra Epidermal cytokeratin,slug, and ) FoxI1e FoxI1e and Xlhbox6 and E. keratin,slug chordin FGF8 mRNA depletion function onearly ( A are unaffected expression in ) Schematic FoxI1e are all and C ) RT- mRNA. sox- 783

DEVELOPMENT restoring normalciliaformation. of FoxI1e infive repeatsof theexperiment, while (dorsal mesoderm). mesodermal), stage (stage11.5)wereassayedforexpression of To testtheeffect onmesodermalpatterning,embryosatthegastrula reduced, showing thattheeffect was pan-neural,ratherthanregional. ( levels ofanteriorneural( general roleinectodermaldifferentiation, weassayedtheexpression slug encoding pan-neuralgenessuchas of expression of resistent no consistentchange(Fig.5G).Reintroductionofmorpholino- ( resulted inlossofciliacellsthatreceived themorpholino. cell attheeight-cellstageandstainedfor animal, ventral cellattheeight-cellstage. Inanormalembryo,this 10 ngSBMO,togetherwithRLDX asalineagetracer, intoone cadherin genes. mRNAs encodingthepan-epidermal genessuchas FoxI1e of geneexpression atstage23byRT-PCR inembryosinjectedwith acceptor site-targeting oligofortherestofourexperiments. Analysis is notefficiently inhibited(Fig.5B).We thereforeusedthesplice injected withthismorpholinoshows that severe effects (datanotshown). RT-PCR analysisofembryos the splicedonorsiteof early tailbud stage.Amorpholinooligonucleotidedesignedagainst the SBMOresultedindeathofembryosbyosmoticlysis The epidermallesionsinembryosinjectedwiththehighestdoesof structures andbegan todevelop gaps intheirepidermis(Fig.5E). elongate properlyalongtheanteroposterioraxis,hadreducedhead ininjectedcells.( normal ciliapattern Control embryos( Fig. 6.LossofFoxI1ecausesarescuable lossofepidermalcilia. 784 of determine ifthiswas duetolackofcilia,wedissectedouttherole deficient embryos,unlike controlembryos,failed todothis.To larva over thefloorofculturedish.We noticedthatFoxI1e contains cilia,thebeatingofwhich causesglidingmovements ofthe 5F) E Xlhbox6/HoxB9 , F To testwhether From thetailbud stage(stage22)onward, the FoxI1e . SBMO-resistant ) (Fig. 5F). mRNA shows adownregulation ofbothneuralandepidermal RESEARCH ARTICLE FoxI1e and in theepidermisusing epidermal cytokeratin Xwnt8 mRNA producesamodestbut reproduciblerescue A epidermal cytokeratin ) genes.Bothwereconsistentlyandequally , B FoxI1e FoxI1e ) injectedwithRLDX(red) inananimal,ventral Xbra (lateral andventral mesoderm)and and mRNA wasinjectedaftertheSBMO, plays aroleinpatterning,ratherthan FoxI1e FoxI1e FoxG1/BF-1 Xwnt8 C Sox-2 mRNA causedsimilarbut less , Xenopus D were unaffected bydepletion were reduced,asthose ) Co-injectionofSBMO ␣ , E-Cadherin -tubulin (green) havea FoxI1e , andtheneuralcrestgene ) andposteriorneural fate map.We injected Xenopus mRNA maturation chordin and Xbra epidermis slug chordin showed (pan- (Fig. E- - neural markers epidermal cytokeratin absence ofFoxI1e. Inuninjectedanimalcaps, to analyzetheirabilityexpress neuralmarkers inthepresenceand presence andabsenceofFoxI1e, andinjectedwithBmp inhibitors, cultured alone,toanalyzetheirabilityformepidermisinthe specification. Inthisexperiment, wetherefore comparedcaps to Bmpinhibitorsfromtheorganizer, whichcauseneural be expected toformonlyepidermalmarkers, asthey arenotexposed ectoderm formation.Animalcapsculturedalonefromstage7would actively promotes in thosemarkers, wewould concludethatFoxI1e expression would beunchanged.If,however, therewereareduction mesoderm intheanimalcap,levels ofearlyectodermalmarker works onlybyrepressingendodermand factors. IfFoxI1e cells oftheanimalcaparenever exposed tomesoderm-inducing of theanimalcapbeforemid-blastulatransitionensuresthat caps atstage7,beforetheonsetofzygotictranscription.Removal 60 ngSBMOintotwo-cell stageembryosanddissectedtheanimal activation ofectodermintheearlyembryo.To dothis,weinjected n morpholino-resistant mRNA rescuesthelossofcilia(6%abnormal, mRNA withGFPmRNA asalineagelabel.Reintroductionof the injectionofSBMOandRLDXwithanother abnormal, injected cells(non-fluorescentclones)inthesameembryo(67% decreased numbersofcilia,comparedwithcellsderived fromnon- labeled cellsintheepidermis.However, thesehaddramatically injected withRLDX,togethertheSBMO,alsohadclonesof immunostaining for normal patternsofciliaontheirsurfaces, asrevealed by large clonesoffluorescentcellsintheepidermis,whichshowed (Dale andSlack,1987).EmbryosinjectedwithRLDXalonehad cell contributes tomostoftheepidermisononesideembryo If FoxI1e wererequiredformaintaining animalcellsinthe ectodermal specification thattakes placeduringtheblastulastage. molecular mechanismsofthis process, orwhetheritispartofthe again (Turner etal.,1989).However, very littleisknown about the and cellsfromdifferent regions thataremixed willrapidlysortout is limitedcellmixing,asindicated bytheexistence ofafate map, regional celllocalization isstrictlycontrolledintheblastula.There control positionsofcellsexpressing itintheembryo.Itisknown that mightbeto raises thepossibilitythatoneoffunctionsFoxI1e expressing itmoved totheectodermratherthanendoderm.This One ofthegain-of-functioneffects ofFoxI1e was thatvegetal cells cells withintheembryo FoxI1e ectodermal fates, bothneuralandepidermal,inanimalcap cells. 7F). Fromthesedata,weconcludethatFoxI1e actively promotes stage 7,mesodermalgenetranscriptswerealmostundetectable(Fig. cmBmp7 reduced levels ofneuralgenesrelative tothoseinjectedwith injected with epidermal markers relative touninjectedanimalcaps,andthatthose that animalcapsdepletedofFoxI1e expressed reducedlevels of We foundbyRT-PCRexcised atstage7andcultureduntil14. with with theSBMOattwo-cell stage,andsomewerefurtherinjected (Hawley etal.,1995).Inthisexperiment, embryoswereinjected reduction inepidermalmarkers andanincreaseinneuralmarkers encoding cmBmp7,whichinhibitsallBmpsignaling,causesa =68) (Fig.6A-F). We next wanted todetermineifFoxI1e isnecessaryforthe cmBmp7 controls thelocalization ofectodermal mRNA alone(Fig.7A-E) n =66). To show thatthiseffect isspecific, wefollowed cmBmp7 mRNA atthefour-cell stage.Animal capswere Sox-2 ␣ , mRNAs areexpressed, but notthoseofthe -tubulin (0%abnormal, mRNA anddepletedofFoxI1e expressed NRP-1 or . As expected incapsexcised at NCAM. Development 134(4) Injection ofmRNA n E-cadherin =52). Embryos FoxI1e and

DEVELOPMENT mesodermal marker induction requires thepresence of markers are reduced. ( uninjected (epidermal)animalcaps.LossofFoxI1edownregulates expression ofthesemarkers.cmBMP7neuralizescaps,so of RLDX-injected embryoshadsignalrestricted toneuralstructures give risetodescendents inthenervous system.Eighty-seven percent embryos tothetailbud stage(stage35-37).Thiscellwould normally 32-cell stage(cellA1)(Dale and Slack,1987)culturedthe alone orRLDX+15ngSBMO into asingledorsalanimalcellatthe that normallycontribute tothenervous system.We injectedRLDX embryo (72%, there was acollectionofRLDX-positive cellsintheabdomenof reached theirnormalpositionwithintheembryo(Fig.8B).Instead, of theembryos,indicatingfewer descendentsoftheinjectedcellhad was adramaticdecreaseintheamountofRLDXsignalat surface cells elsewhere. Inembryosco-injectedwith15ngSBMO,there the vast majorityofsignalisintheepidermis,withonlyoccasional extra-epidermal cellswas theCNS.However, even intheseembryos, n was thecaseinembryosthatwereinjectedwithRLDXalone (92%, are thereforelocatedatthesurface ofthetailbud stageembryo.This descendents ofthiscellarenormallyrestrictedtotheepidermis and (stage 40)andstainedforciliawith the 32-cellstage(cellA4).Embryoswerefixed atthetailbud stage alone orRLDX+15ngSBMOintoasingleventral animal cellat regions oftheembryo.To testthishypothesis,weinjectedRLDX hemisphere, thenremoving itwould causethemtorelocateother FoxI1e specifies with cmBMP7.Animalcapswere excisedatstage7andcultured tostage14.( Fig. 7.FoxI1eactivelypromotes ectodermformation. =60, Fig.8A).Oftheremaining8%,mostfrequentlocation of C B A

Relative Expression Relative Expression Relative Expression 1200 1600 2000 100 120 100 120 400 800 20 40 60 80 20 40 60 80 0 0 n 0 Xenopus =60). We performedsimilarexperiments forcells Xbra nn opoiocBP SBMO+ cmBMP7 Morpholino Uninj SBMO+ cmBMP7 Morpholino Uninj C-E nn opoiocBP SBMO+ cmBMP7 Morpholino Uninj A ) Theneuralmarkers at stage11incapsexcised7.Wholeembryoexpression isshownasareference. ectoderm FoxI1e. Cytokeratin E-Cadherin Therefore, Sox-2 ␣ -tubulin antibody. The Sox-2, NCAM FoxI1e Embryos were injectedatthetwo-cellstagewithSBMOandthenfour-cell stage is necessaryforexpression ofbothepidermalandneuralgenes.( cmBMP7 cmBMP7 cmBMP7 and NRP-1 are inducedrelative tocontrols incmBMP7-injected (neural)caps.This F E D rise predominantly totheCNS),ratherthanA4, gave corresponding Experiments inwhichweinjected theA1blastomere(whichgives brush borderalongwithRLDX-negative cells(Fig.8D). visible alongloopsoftheintestinal epitheliumandcontributed tothe morpholino, large groupsofRLDX-positive cellswereclearly shown). Inembryos co-injectedwiththesplice-blocking some cellswerefoundscattered inothergermlayers(datanot agreement withquantitative fate mapstudies (DaleandSlack,1987), The structureofthecellswas clearlyvisiblebyDIC(Fig. 8C).In only, cellswerelocatedintheskinorandnervous system. the identitiesofectopiccells.InembryosinjectedinA4withRLDX epithelium haddifferentiated (stage47)sothatwecoulddetermine develop tothestageatwhichfeedinghad started,andthegut possibilities was thecase,weallowed embryostreatedasabove to appropriate totheirnew locations.To determinewhichofthese CNS orepidermis,they maychangetheirfate completelytoone may migratetoinappropriatelocationsbut retaintheir identitiesas a locationaffected bylossof nervous systemandinthegut(25%, In embryosco-injectedwithSBMO,signalwas foundbothinthe embryos thathadcellsinotherstructures,primarilytheepidermis. ( n =45). Aswiththeventral injection,therewereasmall number of A There arethreepossibilitiesregarding thestatusofcellsthathave ,

B Relative Expression Relative Expression Relative Expression 100 120 100 150 200 250 300 350 400 100 200 300 400 500 600 ) B 20 40 60 80 50 Epidermal cytokeratin 0 0 0 Uninj WEst nn opoiocBP SBMO+ cmBMP7 Morpholino Uninj nn opoiocBP SBMO+ cmBMP7 Morpholino Uninj 11.5 nn C3n C60ngAC 30ngAC Uninj AC and FoxI1e. NRP-1 E-cadherin NCAM Xbra RESEARCH ARTICLE n They mayeventually die,they =32). are expressed in F ) Expression ofthe cmBMP7 cmBMP7 the epidermal 785

DEVELOPMENT into theblastocoel. ( RLDX +SBMO(B)andthenstainedfor blastomere, aprecursor ofepidermis,withRLDX(red) alone(A)orwith control stage11embryoinjected withFLDXaloneintoA4atthe32-cellstage.( sectioned andembedded.( located intheinteriorofembryo(B).Atstage47,embryoswere SBMO,thecellsare alone, injectedcellsco-localizewithcilia(A).With described above intheA4cell,andfixed andbisectedthemat Fluorescein-dextran (FLDX),withorwithoutthe SBMO as took embryosthathadbeeninjectedwithalineagemarker, FoxI1e normal gutepithelium(datanotshown). the CNS,but whenco-injectedwithSBMO,they alsocontributed to results. InRLDX-onlyembryos,themajorityofcellscontributed to ( Fig. 8.FoxI1econtrols regional positionofectodermalcells. 786 Fig. 9.FoxI1eisrequired fornormalectodermalcelladhesioninthegastrula. the intestinalepithelium. cells giverisetomorphologicallynormalendodermalstructures, including ( embryo injectedwithRLDXinA4.islocalizedtotheepidermis. A F-H , To testwhenthedescendentsofanimalblastomereslacking B ) Co-injectionwithSBMOcausescellstolocalizethegut.These Stage25embryosinjectedatthe32-cellstageinA4 ) RESEARCH ARTICLE first leave theectodermandenterother germlayers,we E-G ) Thiseffect isrescued bymorpholino-resistant mRNAinjected at thetwo-cellstage.* C-E ) DICandfluorescent imagesofacontrol ␣ -tubulin (green). With RLDX -tubulin (green). With express it,andthatitsabsencefromcells do normally activates ectodermgeneexpression incellsthatdonotnormally differentiation. Gain-of-functionexperiments show thatit These dataidentifyFoxI1e asplayingamajorrolein ectoderm DISCUSSION another andendupinotherlocationstheembryo. the gastrula.Initsabsence,affected cellslosecontactwithone suggest that morpholino atthe32-cellstageasbefore(Fig.9E-G).Thesedata cytoplasm atthetwo-cell stage,followed byinjectionofthe injection of5-10pgthe Fig. 10.Modelofgerm-layerformation. animal cap(94%, on thefloorofblastocoel,whichhadlostadhesionto bisected, agroupofFLDX-positive cellswerefounddissociated the surface ofthegastrula.Instead,whenembryoswere However, atstage11,therewas reducedsignalintheectodermon patch offluorescenceintheventral ectoderm(datanot shown). embryos wereidenticaltoFLDX-onlyembryos,withasmall stages 9,10,11and18.At9theSBMO-co-injected C , D ) Co-injectionofSBMOcausesthe cellstodisaggregate andfall ( A , B ) Brightfieldandfluorescent imagesofahemisected FoxI1e n =65, Fig.9A-D).Thiseffect was rescuedby regulates theadhesionofanimalcells P FoxI1e =8.8 ϫ 10 mRNA intotheanimal –5 , ** Development 134(4) P =0.03.

DEVELOPMENT good fate mapintheearly early embryo.Ithasbeenknown formany yearsthatthereisa progenitors. before thedivision oftheectodermintoepidermal andneural cells formingtheneuralplate,whichsuggeststhatitplaysarole differentiation. Aftergastrulation,FoxI1e israpidlylostfrom suggests thatitplaysaroleinbothepidermalandneural neural markers aredownregulated afterdepletionofFoxI1e ectodermal genes.Inaddition,thefact thatbothepidermaland express itcausesdownregulation oftheexpression of FoxI1e specifies regional identityoftheanimalcells. essential rolesintheactivation ofectodermalgenesaswell functional analysisofFoxI1e, whichrevealed thefact thatit plays general toxicityoftheembryo, andthusallowed amoredetailed binding toaspliceacceptorsite. Thisoligodidnotcauseany morpholino oligothatblocks maturationofthemRNA, by we coulduse.Afterscreeninga numberofsequences,wechosea However, itstoxicitymadeuslookforalternative sequences that analysis usingasequenceinthesameregion ofthemRNA. publication oftheSurietal.paper, wehad startedafunctional analysis pastthispointcouldnotbecarriedout.Before the oligo thatcausedtheembryostodieatgastrulastage,so that mesoderm specification. Thiswork usedanantisense morpholino suggested thatitsprimaryrolewas toactasaninhibitor of analysis. this expression pattern,aswellitsfunction,requirefurther surrounded bynon-expressing cells.Themechanismsunderlying expression, bymutualinhibitionmechanisms,to one cell asynchronous. Alternatively, intercellularsignalingcouldrestrict cells, but notatthesametime,oncecelldivisions have become control ofexpression, inwhichcaseitwould beexpressed inall This variegated patternofexpression couldbeduetocellcycle (http://xenopus.nibb.ac.jp), but hasnotbeenpreviously published. website thatdocumentstheresultsofanexpression screen in whichitisexpressed. Infact, thisinformationispresentona hybridizations donotshow expression inevery cellofthetissues remain pluripotent. state toamorespecified state,andinitsabsence,animalcells function ofFoxI1e istoswitchanimalcellsfromapluripotent according totheirnew positions.Thissuggeststhatthenormal differentiate asectopicectoderm,but insteadbecomespecified contact withtherestofanimalcellpopulationdonotdieor position intheembryo. shown thataforkhead-classtranscriptionfactor controlscell represents thefirst developmental systeminwhichithasbeen regional identityofanimalcellsarecontrolledbyFoxI1e. This known. Thedatapresentedhereshow thatmoleculescontrol of thesenascentgermlayersintheblastula,itsmechanismisnot differential adhesionintheembryo,andmaintain theseparation specification occursgenesareexpressed thatsomehow control 1983). Althoughitisgenerallyunderstoodthatasgerm-layer recombination experiments (Nieuwkoop, 1973;Smith andSlack, by blastomeresortingexperiments (Turner etal.,1989)andtissue mixing duringearlydevelopment, afact thathasalsobeenshown vertebrate development. However, afate maprequireslimitedcell the attractive featuresofusing The dataalsoshow thatFoxI1e controlscellpositioninthe A previous functionalanalysisof The expression patternof It isinterestingtonotethatanimalcellslackingFoxI1e thatlose Xenopus ectoderm Xenopus FoxI1e Xenopus embryo. Infact, thisisoneof is interestingbecauseinsitu FoxI1e as amodelforearly (Suri etal.,2005) nodal expression, downstream ofVegT, reduction inectoderm-inhibitoryfactors. Inareasofhighlevels of determining moleculescouldreachfurthervegetally duetoa Alternatively, intheabsenceofVegT vegetal cells(Xanthosetal.,2001;Zhang1998). of bothneuralandepidermallineagesappearinmarginal and early embryo,because,intheabsenceofVegT, molecularmarkers activators of of theblastulaintothreecellpopulations(Fig.10).Thematernal model tobeproposedofthecellinteractionsthatleaddivision P2(u ta. 02 n riyedlk1(Tao etal.,2005) AP-2 (Luoetal.,2002)andgrainyhead-like1 (HoustonandWylie, 2003),aswelltheepidermalproteins Xlim5 (Dupontetal.,2005)Xoom(Hasegawa etal.,2001)and ectodermin factors known tobeimportantinectodermdifferentiation suchas including thefunctionalrelationshipbetweenFoxI1e andother adhesion. Many elementsofthismodelrequirefurthertesting, of surface proteinsthatcontrolcellpositionbydifferential layers. Cellmixingacrossthisboundaryisprevented byexpression precise boundarybetweentheectodermandothertwo germ mutual inhibitionissetupthatgeneratesaprogressively more Dupont, S.,Zacchigna,L.,Cordenonsi,M.,Rugge,M. M.,Soligo,S.,Adorno, Deblandre, G.A.,Wettstein, D.A.,Koyano-Nakagawa, N.andKintner, C. De Robertis,E.M.andKuroda, H. Dale, L.andSlack,J.M. Agius, E.,Oelgeschlager, M.,Wessely, O.,Kemp,C.andDeRobertis,E.M. References HD046387). (RO1 HD45737,T32 Children’s HospitalResearch Foundation,andtheNationalInstitutesofHealth The authorsgratefullyacknowledgefinancialsupportfrom theCincinnati Hasegawa, K.,Sakurai,N.andKinoshita,T. Harland, R.M. lowest, inhibited. Inthemostanimalregion, wherenodalexpression is emerge. of aglobalmechanismgerm-layerformationisbeginning to little detailconcerningitsdownstream targets. However, theoutline control thespatialandtemporalexpression ofFoxI1e, andweknow likely thattherewillbesignalingpathways inadditiontonodalsthat the many earlyCNSfactors includingSox2andSox3.Itisalso Heasman, J.,Wylie,C.C.,Hausen,P. andSmith,J.C. Hawley, S.H.,Wunnenberg-Stapleton, K.,Hashimoto,C.,Laurent, M.N., Kofron, M.,Klein,P., Zhang,F., Houston,D.W., Schaible,K.,Wylie,C.and Houston, D.W. andWylie,C. Luo, T., Matsuo-Takasaki, M.,Thomas,M.L.,Weeks, D.L.andSargent,T. D. Matsuo-Takasaki, M.,Matsumura,M.andSasai,Y. induction inXenopusembryos. for Xenopusembryos. Ectodermin, aSmad4ubiquitinligase. and Piccolo,S. in theskinofXenopusembryos. oftheciliatedcells (1999). Atwo-stepmechanismgeneratesthespacingpattern Development Development (2000). EndodermalNodal-related signalsandmesoderminductioninXenopus. Xenopus embryos. and functionsasatransmembraneprotein forgastrulationmovementin determination ofsinglevegetalpoleblastomeres ofX.laevis. Dev. signals inembryonicXenopusectodermleadstodirect neuralinduction. Watabe, T., Blumberg,B.W. and Cho,K.W. Development Xlim5 regulates thedifferential adhesionproperties ofearlyectodermcells. embryo. Heasman, J. development inXenopus. (2002). Transcription factor AP-2isanessentialanddirect regulator ofepidermal gastrulation. Xenopus Foxi1aforventralspecification ofthecephalicectodermduring The identification oftherolesFoxI1e allows amorecomplete 9 , 2923-2935. FoxI1e Dev. Biol. Development (2001). The role of maternal axin in patterning theXenopus (2001). Theroleaxininpatterning ofmaternal (1991). Insituhybridization:animproved whole-mountmethod 99 127 130 FoxI1e (2005). Germ-layerspecificationandcontrol ofcellgrowth by expression isactivated. Once , 527-551. , 1173-1183. , 2695-2704. 237 Dev. GrowthDiffer. , 183-201. Methods CellBiol. (1987). Fatemapforthe32-cellstageofXenopuslaevis. expression mustbepresentthroughoutthe Dev. Biol. 132 (2003). TheXenopusLIM-homeodomainprotein , 3885-3894. Annu. Rev. CellDev. Biol. Development (2004). Dorsal-ventral patterning andneural (2004). Dorsal-ventralpatterning 245 Cell 43 , 136-144. 36 , 25-31. RESEARCH ARTICLE 121 , 685-695. , (2001). Xoom is maternally stored(2001). Xoomismaternally , 87-99. the influenceofectoderm- 126 (1995). DisruptionofBMP , 4715-4728. FoxI1e (2005). Anessentialrole of FoxI1e (1984). Fatesandstatesof 20 , 285-308. Cell is expressed, a expression is 37 , 185-194. Genes , 787 and

DEVELOPMENT Stern, C.D. Stern, Snape, A.,Wylie,C.C.,Smith,J.andHeasman, Smith, J.C.andSlack,M. Pohl, B.S.andKnochel,W. Nieuwkoop, P. D.andFaber, J. Nieuwkoop, P. D. 788 questions. 119 of commitmentsingleanimalpoleblastomeres ofXenopuslaevis. 317. properties oftheorganizerinXenopuslaevis. helix) transcriptionfactorsinXenopusdevelopment. Egg tilltheEndofMetamorphosis oftheDevelopmentfromFertilized A SystematicalandChronologicalSurvey 39. origin, spatialorganization,andmorphogeneticaction. , 503-510. RESEARCH ARTICLE (2005). Neuralinduction:oldproblem, newfindings,yetmore Development (1973). Theorganizationcenteroftheamphibianembryo:its 132 (2005). OfFoxandfrogs: Fox(forkhead/winged (1983). Dorsalizationandneuralinduction: , 2007-2021. (1967). . Amsterdam: North-Holland. Normal Table ofXenopuslaevis(Daudin): J. Embryol. Exp.Morphol. J. Embryol. Gene (1987). Changesinstates Adv. Morphog. 344 , 21-32. Dev. Biol. 78 10 , 299- , 1- Xanthos, J.B.,Kofron, M.,Wylie,C.andHeasman,J. Zhang, J.,Houston,D.W., King,M. L.,Payne,C.,Wylie,C.andHeasman,J. Turner, A.,Snape,A.M.,Wylie,C.andHeasman,J. Tao, J.,Kuliyev, E.,Wang, X.,Li,Wilanowski,T., Jane,S.M.,Mead,P. E. Suri, C.,Haremaki, T. andWeinstein, D.C. Development the initiatorofamolecularnetworkspecifyingendoderminXenopuslaevis. 252. is establishedbefore gastrulationintheXenopusembryo. Xenopus embryos. (1998). TheroleVegT ofmaternal inestablishingtheprimarygermlayers 1021-1034. Grainyhead-like 1isessentialforepidermaldifferentiation. and Cunningham,J.M. suppression ofmesendoderm. expressed inthegastrulastageXenopusectoderm,isrequired forthe 128 , 167-180. Cell 94 (2005). BMP4-dependentexpression ofXenopus , 515-524. Development (2005). Xema,afoxi-classgene 132 , 2733-2742. Development 134(4) (2001). Maternal VegT(2001). Maternal is (1989). Regionalidentity J. Exp.Zool. Development 251 132 , 245- ,

DEVELOPMENT