Biosciences, OsakaUniversity, Yamadaoka 1-3,Suita,Osaka565-0871,Japan. et al.,1998;ImondiandKaprielian,2001;Kadison (Oppenheim etal.,1988;Yaginuma andOppenheim,1991;Erskine distance away fromthefloorplatewithinventral spinalcord or bifurcaterostrocaudally, andsometurnlongitudinallyata crossing commissuralaxonshave alsobeenshown togrow caudally previously thought.Inchickandrodentspinalcord,somepost- post-crossing commissuralaxonsarelessstereotypical than contralateral surface ofthefloorplate.However, thetrajectoriesof commissural axons,whichturnrostrallyinclosecontactto the gradients mightaccountforthelongitudinalpolarityofmostspinal (Lyuksyutova et al.,2003;Bourikaset2005).Wnt4andShh rostrocaudally orientatedgradientsinandaroundtheventral midline commissural axonsinrodentandchick,respectively, byforming (Shh) controltherostrocaudalpolarityofpost-crossingspinal along thelongitudinalaxis. crossing, inparticular, thosethatregulate theirrostrocaudalpolarity that controlthepathfindingofcommissuralaxonsaftermidline Guthrie, 2004).Bycontrast,littleisknown aboutthemechanisms Shirasaki, 1997;StoeckliandLandmesser, 1998;Salinas,2003; neural tube(Tessier-Lavigne andGoodman,1996;Murakami adhesion molecules,distributed alongthedorsoventral axisofthe concerted actionsofchemoattractants,chemorepellentsandcell- the ventral midlinehave beenstudiedextensively; they relyonthe mechanisms thatguidethecommissuralaxonstowards andacross and turnintolongitudinalpathstowards theirtargets. The commissural axonsthatgrow circumferentiallyacrossthemidline half ofthenervous systemiscommunicatedtotheotherhalfby In bilaterallysymmetricorganisms, neuronalinformationfromone INTRODUCTION KEY WORDS:Commissuralaxons,EphrinA,Longitudinalpolarity, Hindbrain,Chick in regulatingthepolarityofpost-crossingcC-VCaxonsastheyturnintolongitudinalaxis. rostrocaudal pathfindingerrorsofpost-crossingcC-VCaxons.TheseresultssuggestthatephrinA/EphAinteractionsplayakeyr hindbrain andcC-VCaxonsexpressedEphAreceptors.Finally, perturbingephrinA/EphAsignallingbothinvitroandvivoled ephrin A.We furtherdemonstratedthatephrinA2wasdistributedinacaudal-high/rostral-lowgradientthecaudolateral for growingcC-VCaxons.Thisnon-permissiveness/repulsionwasinpartmediatedbyglycosyl-phosphatidylinositol(GPI)-anchored second-order vestibularneurons(cC-VC).We foundthatthecaudalhindbrainpossessedagradednon-permissive/repulsiveactivit the rostralturningofalaterallylocatedcommissuraltract,identifiedascaudalgroupcontralateralcerebellar-project that controltheirrostrocaudalturningpolarityarestillpoorlyunderstood.We usedthehindbrainasamodelsystemtoinvest Most post-crossingcommissuralaxonsturnintolongitudinalpathstomakesynapticconnectionswiththeirtargets.Mechanisms Yan Zhu lateral commissuraltractinchickhindbrain Ephrin A/EphAcontrolstherostralturningpolarityofa Development 133,3837-3846(2006)doi:10.1242/dev.02564 Accepted 3August 2006 1 [email protected]) *Authors forcorrespondence (e-mail:[email protected]; London SE11UL,UK. Centre forDevelopmentalNeurobiology, King’s College London,Guy’s Campus, SORST, JapanScienceandTechnology, Japan. Two recentstudieshave shown thatWnt4andsonichedgehog 1,2, *, SarahGuthrie 3 and FujioMurakami 2 Graduate SchoolofFrontier 1,2, 3 MRC * containing 30 with flamesharpened tungstenneedles.Thegrafts were soaked inGBSS were preparedinice-coldGey’s BalancedSaltSolutions(GBSS,Sigma) described (Chédotaletal.,1997;Zhu etal.,2003). Organotypic cultureofchickhindbrainswas carriedoutaspreviously Organotypic culture andgrafting Osaka University. out inaccordancewiththeguidelinesofAnimalStudiesCommittee of Hamilton (Hamburger andHamilton,1951).Animalstudieswerecarried at 38.5°Ctodesiredstages.Embryonicstagingwas basedonHamburger and Fertilised eggs (Takeuchi egg farm) wereincubatedinahumidifiedincubator Chick embryosandstaging MATERIALS ANDMETHODS crossing cC-VCaxons. a crucialroleincontrollingtherostrallydirectedturningofpost- lines ofevidence thatdemonstrateephrinA/EphAsignallingplays activity ismediatedatleastinpartbyephrinA.We presentmultiple permissive/repulsive activity forgrowing cC-VCaxons.This Here, weshow thatcaudalhindbrainpossessesagradednon- Puelles, 2003),turnrostrallyatadorsalpositioninthehindbrain. contralateral secondordervestibular neurons (cC-VC)(Diazand which weidentifiedascomingfromthecaudalgroupof turn longitudinallyatadistancefromtheventral midline. Theaxons, guidance ofpost-crossingcommissuralaxons. for uncovering novel molecularmechanismsthatregulate the Thus, thehindbrainrepresentsacomplex modelthatis wellsuited diversity ofpost-crossingcommissuralprojectionsinthehindbrain. components withdistinctspatialdistributions, accountforthe that multiplemolecularmechanisms,possiblyinvolving Shirasaki etal.,1995;andMurakami,2001).We envisage Petursdottir, 1991;Clarke andLumsden,1993;Glover, 1993; positions, intoeitherrostralorcaudaldirections(Glover and ventral aspectoftheneuraltube,but alsoatintermediate anddorsal spinal cord.Hindbraincommissuralaxonsnotonlyturnwithinthe hindbrain, awiderstructurealongthedorsoventral axisthanthe 2004). Thisdiversity iseven morepronouncedinthedeveloping In transplantationexperiments, graftsfromdonorandhosthindbrains In thepresentstudy, wehave focusedoncommissuralaxonsthat ␮ g/ml 3,3 Ј -dioctadecyloxacarbocyanine perchlorate(DiO, RESEARCH ARTICLE ing igate to 3837 ole y

DEVELOPMENT for thewhole-mountAPinsitubinding. followed by5washes inPBS.Therestoftheprocedurefollows theprotocol were airdriedfor2hoursandfixed in99.5%ethanolfor 30seconds, (Calbiochem). after dyeinsertion,beforefixation.AllsamplesweremountedinMowiol Hindbrains wereorganotypically culturedfor1dayinvitro(div) to2div samples werestoredin4%PFA for4weeksatroomtemperature. Carmine mark,guidedbyascaledeyepiece graticule.For fixed hindbrains, (Wako), andDiIorDiDcrystalswereinsertedataspecificdistancefromthe rootofvestibuloacoustic ganglion(gVIII)was marked byCarmine the (PFA)/0.1 Mphosphate-buffered saline(PBS,pH7.4).The positionof cultured hindbrainsoropen-bookfixed in4%paraformaldehyde Invitrogen) wereinsertedbyaglassmicropipettewithbroken tip,on dicarbocyanine, 4-chlorobenzenesulfonatesalt(DiD)(MolecularProbes, perchlorate (DiI)or1,1 H sections, asdescribedabove, withafew modifications:(1)treatmentof0.3% solution. Sampleswereclearedin90%glycerolforimaging. diaminobenzidine tetrahydrochloride(DAB; 0.1%inTris-buffered saline) Immunohistochemistry andantibodies used atafinalconcentrationof20-30 ephrin A2(B3)(akindgiftfromDrH.Tanaka) (Yamada etal.,2001)was medium attheonsetofculture.Afunction-blockingantibodyagainstchick concentration of1500ng/mland2000ng/ml,respectively, to theculture mouse EphA3-FcChimera(R&DSystems)wereaddedatafinal div. HumanFcfragmentofIgG(JacksonImmunoResearch)orrecombinant medium attheonsetofculture,withafreshsupplementPI-PLCafter1 Invitrogen) atafinal concentrationof200mU/mlwas addedtotheculture Phosphatidylinositol-specific phospholipaseC(PI-PLC,MolecularProbes, treatment invitro PI-PLC, hFc,EphA3-FcandephrinA2functionalblockingantibody Small crystalsof1,1 Anterograde- andretrograde-labelling were discernedbyDiOsignalsandbright-fieldillumination. positioned usingafire-polishedpulledglasspipette.Thebordersofthegrafts transferred ontomillicellcultureinserts(CM,Millipore)andthegraftwas Molecular Probe,Invitrogen) for5minutes.Boththegraftandhostwere 3838 respectively. Partial cDNA fragments ofchick (Drescher etal.,1995)andDrN.Wada (University ofCalifornia,Irvine), were kindgiftsfromDrH.Tanaka (Iwamasa etal.,1999),DrU.Drescher riboprobes. Plasmidscontainingchick templates wereusedtogeneratedigoxigenin-labelled anti-senseorsense previously described(Henriqueetal.,1995).Thefollowing plasmid In situhybridisationonwhole-mount chickhindbrainswas performedas RNA insituhybridisationandprobes with 10%sheepserum.For APinsituonfrozensections, 30 was composedofHanks’BalancedSaltSolution(HBSS):PBSat1:1ratio, described (Chengetal.,1995)withsomemodifications:theblockingbuffer chick hindbrainsorfrozensectionswas carriedoutlargely aspreviously gifts fromDrH.Tanaka. Affinity probeinsitubindingonwhole-mount Chick ephrinA2-alkalinephosphatase(AP)andchickEphA3-APwere kind Affinity probe insitubinding described (Tashiro etal.,2000),except that0.04%NiSO Immunohistochemistry onflatwhole-mounthindbrainswas performedas (1:200, Vector Laboratories). Secondary antibodyusedwas biotinylated horseanti-mouseIgG(H+L) against chickephrinA2(kindgiftfromDrH.Tanaka) (Yamada etal.,2001). Hybridoma Bank,TheUniversity ofIowa) andamonoclonalantibody neurofilament associatedprotein(3A10)(fromtheDevelopmental Studies (DABCO, Sigma). mounted inMowiol (Calbiochem) with2.5%1,4-diazabicyclo-[2,2,2]octane 1:1000) was used;(3)Triton-X100 was usedat0.2%;and(4)slideswere 2 O Fluorescence immunohistochemistrywas performedon20 Primary antibodiesusedwereamonoclonalantibodyagainstchick 2 was omitted;(2)Alexa594-conjugated streptavidin (MolecularProbe, RESEARCH ARTICLE Ј -dioctadecyl-3,3,3 Ј -dioctadecyl-3,3,3 ␮ ephrin A2 Ј g/ml. ,3 Ј -tetramethylindodicarbocyanine EphA3 , ephrin A5 Ј ,3 , EphA4 Ј -tetramethylindo- 4 and was addedto ␮ , EphA5 ␮ m sections ephrin A6 m frozen and Plasmid solution(1 and extra-embryonic membraneswereremoved fromthehindbrainarea. (Nakamura andFunahashi,2001).Briefly, stage20-21eggs werewindowed In ovo electroporationwas carriedoutessentiallyasdescribedpreviously In ovoelectroporation cDNA: 831-1620ofEphA3;410-1219 hindbrains. PrimersforRT-PCR correspondtonucleotidepositionson EphA7 inserted intothedorsalhindbrain 700-1200 book configurationsatvarious stages.SmallcrystalsofDiIwere revealed by DiIanterograde-labellingonfixed hindbrainsinopen The developmental courseofthelateralcommissuraltractwas first lateral commissuraltractinchickhindbrain Developmental courseofacerebellar-projecting commissural axons. controlling thelongitudinalpolarityoflaterallyturning modelsystemforinvestigating theguidancemechanisms our pathway (Fig.1J,K).Thus,wedecidedtousethisaxon tractas to beoneoftheearliesttractsthatgrow alongthislongitudinal longitudinal pathwithinthelateralhindbrain(Fig.1),anditappears drew ourattentionbecauseofitstwo features:itturnsintoarostral the commissuraltractsindeveloping chick hindbrains.Onetract of hindbraincommissuralaxons,wefirstsystematicallysurveyed To investigate themechanismsthatcontrolrostrocaudalturning RESULTS was usedforstatisticalanalysis. turning caudally. Thedatasetsfitbinomialdistribution; thus,Fisher’s test categories: sampleswithallaxonsturningrostrallyand below theturningpoint.These sampleswerearbitrarilydivided intotwo measures asdescribedabove becauseoftheretrogradelylabelledcellsright PLC andEphA3-Fcinvitro,itwas unfeasible toperformquantitative U-test was subsequentlyperformedon the processeddata. MetaMorph (Version 6.1,Universal ImagingCorporation).Mann-Whitney Quantitative measurement onfluorescenceimageswas performedusing Data quantificationandstatistics laser-scanning confocalmicroscope(MRC1024ES,BIORAD). (BX-60, Olympus).Insomecases,fluorescenceimageswereobtainedbya digital (CCD)camera(Axiocam,Zeiss)linked toanuprightmicroscope Fluorescence andbrightfieldimageswerecapturedwithacharge-coupled Image recording Vector (Promega) forsubsequentriboprobepreparation. 865-1507 of [EphA3 (Nishida etal.,2002;Feldheim2004)andRCASB(P)mockvector EphA3 E6. with afew dropsofcoldPBS.Eggsweresealedforfurtherincubationuntil Square Porator(ECM830,BTX).Theembryoswerecooledimmediately five squarepulsesof15V, 50msdurationwereappliedwithanElectro anode andcathodewereplacedinparalleloneithersideofthehindbrain into theIVthventricle. Two silver wire(0.3mmdiameter)electrodesas (Hatanaka andMurakami,2002)at2:1ratio. co-electroporated withanEGFPexpression vector pCAGGS-EGFP (Niwa etal.,1991).Eachoftheabove threeconstructs(1mg/mlinPBS)was were amplifiedbyRT-PCR andclonedintoanexpression vector pCAGGS and thevector re-ligated].Full-lengthchickephrinA2-codingsequences within thelateral aspectofthecontralateralhindbrain (Fig.1B).At 24, DiIlabelledatractthatcrossed themidlineandgrew rostrally vestibuloacoustic ganglion(gVIII)(schematicsinFig.1A).Atstage In thecasesofcaudalturningerrorcC-VCaxonsafteradditionPI- Two avian retroviral constructsusedforelectroporationareRCASB(P)- ⌬ were generatedbyRT-PCR fromtotalRNA preparedfromE6chick ⌬ C (akindgiftfromDrJ.G.FlanaganandM.Nakamoto) C insertwas deletedfromRCASB(P)-EphA3 EphA7 . ThecDNA fragmentswereclonedintopGEM-TEasy ␮ l) colouredwithFast Green(w/v0.05%)was injected EphA4 ␮ ; 826-1641of Development 133(19) m caudaltotherootof ⌬ C by Cla EphA5 I digestion ; and

DEVELOPMENT lateralmost tractappearstoconvergefrompoint~900-1000 aturning (K) Ahighermagnificationviewoftheboxedarea inJ.Bluearrowheads inJ,Kindicatethepositionoflateralmostlongitud (3A10) onastage24flat-mountedhindbrain. against aneurofilament-associated protein ( to thecentralprojections ofgVIII( The lateralcommissuraltractwaslocated right gVIIIlabelledbyDiD(whitearrowhead). (white arrow) andthecentralprojection ofthe with thelateralcommissuraltractlabelledbyDiI organotypically cultured stage26hindbrain, contralateral totheDiIinjectionsites.( indicate retrogradely labelledneurons cerebellar plate.Whitearrowheads inC,D,G,H the longitudinaltractandinvadedeveloping stage 27+2div(H),axonscouldbeseentoleave in itsappearanceanddevelopmentalcourse.At closely resemble thatinthefixedsamplesboth anterograde labellingintheorganotypiccultures lateral commissuraltractlabelledbyDiI injection sitesofDiI.Scalebar:400 red asterisk),andthelateralcommissuraltract(bluearrowhead). WhiteverticallineinBindicatesthemidline.Theasterisk located inthelateralextreme ofthehindbraincanbeseeninK:centralprojection ofgV(purplearrow) andofgVIII(roo 27 ( the caudalhindbrain,700-1200 crystals were insertedintothelateralmarginof preparation, withDiIanterograde labelling.DiI ( organotypically cultured hindbrainsatstage21 inset inD).( invade thecerebellar plate(whitearrow inthe shows thatsomeaxonsfromto thetractturn High magnificationoftheblue-boxedarea inD tract grew tothebaseofcerebellar plate. commissural tract.(D)Thelabelled the progression ofalaterallylocated Ephrin A in longitudinal turning polarityofcommissuralaxons Ephrin Ainlongitudinalturning ran sideby withinthelateralaspectof the hindbrain(Fig. protein. Atstage24,threeneurofilament-positive longitudinaltracts stages with3A10,anantibody againstneurofilamentassociated We thenimmunostainedflat-mountchickhindbrains ofvarious positioned furtherlaterallytothe gVIIIcentralprojection(Fig.1I). hindbrains. We foundthatthelateralcommissural tractwas and DiD,respectively, onorganotypically culturedstage26 tract withthatofthecentralprojectiongVIII,labelled DiI longitudinal fascicle (LLF).First,wecomparedtheposition ofthis tract inrelationtootherlongitudinaltractswithinthelateral hindbrains (seelatersections). influence thepathfindingofthistractinorganotypically cultured in vitroallowed ustoinvestigate thetissuesandmoleculesthat than thatinvivo. Thepossibilityoffollowing thegrowth ofthistract although thelabelledtractinculturesappearedtobelessfasciculated labelling onorganotypically culturedhindbrains(Fig.1E-H), commissural tractcouldberecapitulatedbyDiIanterograde- cerebellar primordium(Fig.1D).Thedevelopment ofthislateral defasciculated fromthelongitudinaltractandturnedinto thecerebellarplate(Fig.1C). Bystage27,someaxons of stage 26,thetractextended furtherrostrallyandreached thebase ( fixed hindbrainsatstage24( the gVIIIroot. ( ( commissural tractinchickhindbrains. Fig. 1.Developmentofalateral J n n A , =3), stage23( =3) andstage27( K Schematicshowinganopen-bookhindbrain ) We next investigated therelative positionofthiscommissural Immunohistochemistry usinganantibody ) n =3), cultured for2daysinvitro (div).The E-H B-D ) DiIanterograde labellingof n =8), stage25( ) DiIanterograde labellingon n =4), respectively, showing n =3), stage26 n ␮ =10) andstage n m caudalto =6). ␮ m inB-J;100 I ) An ␮ m intheinsetDandK. ␮ m caudaltogVIII(indicatedbyared arrow inJ).Three longitudinaltracts material). (Diaz andPuelles,2003)(see Fig.S1inthesupplementary cerebellar-projecting secondordervestibular neurons(cC-VC) tract asthatformedbyacaudal groupofthecontralateral Further anatomicalcharacterisation ledtotheidentificationofthis pioneering tractexploring thislateralmostterritoryofthehindbrain. longitudinal tracts(Fig.1J,K), implyingthatitcouldbea nearest neighbour–thegVIIIcentralprojectionsandother positive lateralmosttractappearedtogrow separatelyfromits revealed byDiIanterogradelabelling.At stage24,the3A10- lateralmost 3A10-positive longitudinaltract was thesametractas that ofDiIanterogradelabelling.We thereforeconcludedthatthe cerebellum fromstage27(datanotshown), againconsistentwith cerebellar primordiumatstage26,andbegan toinnervate the later stagesshowed thatthelateralmosttractreachedbaseof stage (compareFig.1Jwith1B).Neurofilamentstaining at with thatoftheDiI-labelledlateralcommissuraltractatasimilar root ofgVIII,andtheappearancetractcorresponded well commissures ataturningpointabout900-1000 The lateralmostofthethreeappearedtoconverge from projections ofthetrigeminalganglion(gV)andgVIII,respectively. 1J,K). Themedial-mostandthemiddletractswerecentral RESEARCH ARTICLE s inB-Iindicatethe t ofgVIIIindicatedby inal tract.This ␮ m caudaltothe 3839

DEVELOPMENT inhibition ishighcaudallyandlow rostrally. repulsive) tocC-VCaxons.Furthermore,thedegree ofthis turning pointofcC-VCaxonsisinhibitory(nonpermissive/ n most labelledcC-VCaxonsgrew throughthegraft (Fig.2C,D, from therostralpathofcC-VCandputbacktoitsoriginalposition, (Fig. 2B,D, cC-VC axonsstalledorturnedaway withoutentering thegraft 1.8 mmcaudaltogVIII)causedamoremarked effect; almostall (Fig. 2A,D, percentage ofaxonscontinuedtogrow intoandthrough thegraft axons stalledorturnedaway beforeenteringthe graft, yetasmall close tobut caudaltotheturningpointofcC-VCaxons,many graft was taken fromabout1.2mmcaudaltogVIII,aposition anterograde-labelling ofthetractasdescribedinFig.1.When Behaviour ofcC-VCaxonswas thenobserved following VC tract,inorganotypically culturedstage25-26hindbrains. in thislocationweregraftedtointersecttherostralpathofcC- tissue posteriortotheturningpointofcC-VCaxons.Thetissues cC-VC axons,wefocusedontheguidancepotentialofhindbrain To investigate themechanismsthatcontrolrostralturningof growing cC-VCaxons nonpermissive/repulsive activityforrostrally Caudal hindbrainpossessesagraded 3840 =10). Theseresultssuggestthatcaudalhindbrainposteriortothe RESEARCH ARTICLE n n =12). Agraftfromafurthercaudalposition(about =12). Bycontrast,whenapieceoftissuewas cut ephrin Abindingtoitsreceptors(Yates etal.,2001;Dufour used thesolublerecombinantmouseEphA3-Fctoblockspecifically the potentialinvolvement ofephrinAsinthecaudalinhibition,we Vanderhaeghen, 1998;Wilkinson, 2000;Wilkinson, 2001).To test CNS,oftenwiththeirgradeddistribution (Flanaganand the members areinvolved inadiverse rangeofguidanceevents in mediating thecaudalinhibition. n indicated byinvasion ofmany cC-VCaxonsintothegraft(Fig.3A2, PI-PLC totheculturemediumalleviated thecaudal inhibition,as to enterthegraftasshown inFig.2B(Fig.3A1, depicted inFig.2B.Without addingPI-PLC,almostallaxonsfailed inhibitory activity inthecaudalhindbrain,using graftingscheme ligands. We firsttestedwhetherPI-PLCtreatmentcanalleviate the PLC, whichinturnresulttheremoval ofthesemembrane-attached molecules. TheGPIanchorcanbeenzymaticallycleaved byPI- both theephrinAandSemaphorinVIIfamilies areGPI-anchored families (Yu andBargmann, 2001;Dickson,2002).Amongthese, Neuropillin/Plexin-Semaphorin, Robo-SlitandUnc5H-Netrin limited numberofreceptor-ligand families, namelyEph-ephrin, Inhibitory axonguidancecuesintheCNShave beenattributed toa caudal inhibition PI-PLC andEphA3-Fctreatment alleviatethe =20). ThissuggestsGPI-anchoredmolecule(s)isinvolved in Among theGPI-anchoredinhibitorymolecules,ephrinAfamily indicates s.d.).Scalebar:400 the graft(* C. ( other images.Whitedotsoutlinetheborder ofgraftsinA- removed forbetterillustrationofaxonsinthegrafts illumination. DiOsignalwasdisplayedonlyinC,but byDiOsignalcombinedwithbright-field discerned into thehosts,thusborders ofthegraftswere ( Majority ofcC-VCaxonscouldgrow through thegraft along thecC-VCpathwascutandputbackascontrol. through thegraftinA.( whereas asmallproportion ofthecC-VCaxonsstillgrew awaybeforeor turned enteringthegraftinB( caudal togVIII,respectively. AlmostallcC-VCaxonsstalled the graftswere approximately 1.2mm(A)and1.8(B) ( nonpermissive/repulsive activityforthecC-VCaxons. Fig. 2.Caudalhindbrainpossessesagraded n A =10). Allgraftswere soakedinDiObefore transplanting , B D ) Two typesofgraftingexperiments.Thepositions ) Quantificationofthepercentage ofaxonsentering P <0.0001, Mann-Whitney C ) Apieceofrostral hindbrain ␮ m. Development 133(19) U n test) (error bar =12). Additionof n =12),

DEVELOPMENT reached ~700 – low inrostralandhighcaudal.Theextent ofthegradient half oftheneuroepitheliumanditsexpression appeared tobegraded 2002). Inthecaudalhindbrain,ephrinAwas enrichedinthelateral consistent withprevious reports(Karametal.,2000;Nishida n found astrongephrinAactivity inthecaudalhindbrain(Fig.4A, 25 hindbrains,usingEphA3-APaffinity probeinsitubinding,and To testthis,wefirstinvestigated thedistribution ofephrinAonstage predicted theexpression ofthesemoleculesinthecaudalhindbrain. the caudalhindbrain,presumablythroughEphAreceptor, The involvement ofephrinAinmediatingtheinhibitoryactivity of in stage25chickhindbrain Distribution ofephrinAligandandEphAreceptor hindbrain. ephrin Aisinvolved inmediatingtheinhibitoryeffect ofthecaudal supplementary material).Taken together, theseresultssuggestthat weaker thanPI-PLCandEphA3-Fc(seeFig.S2inthe is et al.,2001)was addedtoculturemedium,althoughtheeffect function-blocking antibodyagainstchickephrinA2(B3)(Yamada shown). Thecaudalinhibitioncouldalsobereduced whena graft asinFig.2Acouldalsobealleviated byEphA3-Fc (datanot slightly weaker thanPI-PLC.Inhibitionimposedbythemorerostral medium (Fig.3B2, when EphA3-Fc(withoutclustering)was addedtotheculture 3B1, the medium,almostallcC-VCaxonsfailed toenterthegraft(Fig. 2003). WhenpurifiedhumanFc(hFc)region ofIgGwas addedto polarityofcommissuralaxons Ephrin Ainlongitudinalturning extending rostrallyintothecaudalcerebellum,andcaudally intor8 activity (Fig.4B, appeared toberoughlycomplementarythatoftheephrinA binding. Inthelateralhalfofhindbrain,EphAdistribution by thegraftingexperiment (Fig.2). hindbrain correlatewiththecharacteristicsofinhibitionrevealed of ephrinAactive domainanditsgradednatureinthecaudal =3). EphrinAactivity was alsoobserved intherostralcerebellum, We next examined EphAreceptoractivity byephrinA2-APinsitu n =7). Bycontrast,many cC-VCaxonsgrew throughthegraft ␮ m caudaltothegVIIIroot.Thus,bothposition n =9). Itwas distributed inalateralcolumn n =7), althoughtheeffect ofEphA3-Fcwas cDNAs: mount hindbrains,withantisenseprobesofthefollowing chick respectively. Insituhybridisationwas performedonstage25flat- expressed inthecC-VCneuronsandlateralcaudal hindbrain, We next examined whichEphAreceptorsandephrinAligandswere developing cC-VCsystem components oftheephrinA/EphAactivityin Ephrin A2,EphA5andEphA3are themain hindbrain. a mannerthatitcaninteractwiththeephrinAincaudal and blackarrows). Thus,EphAisexpressed inthecC-VCaxons cC-VC commissurewas positive forEphAsignal(Fig.4C,white around thelateralmantlezone(Fig.4C,arrowhead). Furthermore, positive cC-VCneuronswerelocatedintheEphA-positive area with APsignalonthesamesection(Fig.4C,bottompanel).DiI- images werethenoverlaid tocomparethelocalisationofDiIsignal binding, toreveal EphAactivity (Fig.4C,middlepanel).Thesetwo immediately (Fig.4C,toppanel),followed byephrin A2-APinsitu those sectionscontainingretrogradelylabelledneuronswereimaged supplementary material).Suchhindbrainswerecryosectionedand organotypically culturedstage25hindbrains(asinFig.S1Athe neurons, wefirstretrogradelylabelledcC-VCneuronson 4B, comparewithFig.S1A,Binthesupplementarymaterial). retrogradely labelledcC-VCneuronsattheequivalent stage(Fig. r7 andpossiblyrostralr8was wellcorrelatedwiththepositionof with ataperingend.ThecaudolateralEphA-positive domaininr6, ephrin A6 antibody againstchickephrinA2.Onalateralparasagittalsectionof We furtherstudiedtheexpression ofephrinA2proteinwithan hindbrain andaroundthefloorplate,respectively (datanotshown). ephrin A2 more medial.Ofthethreeknown membersof shown) with the presumptive locationofcC-VCneurons(Fig.5A,B;datanot ephrin A5 To confirmthatEphAreceptorswereindeedexpressed incC-VC EphA3 . Amongthese,only was expressed inthelateralregion ofthehindbrains,while and EphA5 ephrin A6 , EphA4 stronger inthelateralregion, while , EphA5 were expressed intheventral halfofthe grafts. Scalebar:400 in thegraft.Whitedotsoutline entering thegraft,reducing theinhibition culture mediumledtomanyaxons (A2) orunclustered EphA3-Fc(B2)tothe caudal graft.However, addingPI-PLC axons were prevented from enteringthe culture medium(B1),almostallcC-VC medium (A1)orwithonlyhFcaddedto normal culture scheme asinFig.2B.With illustrated usingthesamegrafting experimental procedures followed caudal hindbrain. alleviate theinhibitoryactivityof Fig. 3.PI-PLCandEphA3-Fccould EphA5 , EphA7 RESEARCH ARTICLE and , ephrin A2 EphA3 ephrin A ( A ␮ , were expressed in B m. ) The , ephrin A5 in chick,only EphA3 3841 was and

DEVELOPMENT ␮ VC neurons andtheiraxonsappeartobeEphApositive.Scalebar:400 signals were tracedontotheAPsignalsbyred dots(bottompanel).cC- commissure. Thetopandmiddlepanelswere superimposedandDiI in themiddlepanelC;blackarrow showstheEphA-positive The samesectionwassubjectedtoephrinA2-APinsitubinding,shown white arrowhead, andcC-VCcommissures indicatedbywhitearrow). retrogradely labelled(retrogradely labelledcC-VCneurons indicatedby panel showsatransversesectionofhindbrainwithitscC-VCneurons the floorplateisalsoEphApositive(seelowerpanelsinC).( of presumptive cC-VCneurons. Aventralcolumnofcellsadjacentto bracket indicatesEphA3-positivedomaincorrelating withtheposition binding onastage25hindbrainrevealed EphAactivity. Theblue ephrin Agradientinthecaudalhindbrain.( activity. Thebluebracketindicatestherostrocaudal extent ofthelateral 3842 together withEGFP was electroporated inovo into the rostral affect thegrowth ofcC-VCaxons.AnephrinA2expression vector We next explored whetherasourceof ephrinA2coulddirectly ofcC-VCaxons and turning Ectopic expression ofephrinA2causesstalling detected bytheAP-fusionproteins inthedeveloping cC-VCsystem. main componentsthataccountfortheephrinA/EphAactivities these data,weconcludethatephrinA2,EphA5andEphA3are the hindbrain withitssignalstrongerincaudalsections(Fig.5D).From ephrin A2immunoreactivity was detectedinthelateralhalfof a rostral-low/caudal-high gradient(Fig.5C).Ontransverse sections, hindbrain, ephrinA2was distributed posteriorto theoticvesicle, in Fig. 5C)(Chengetal.,1995;Drescher1995).Inthecaudal of ephrinA2inthetectum,consistentwithprevious studies(insetof a stage26brainstem,wedetectedcaudal-high/rostral-low gradient ( Fig. 4.DistributionofEphAandephrinAinstage25hindbrains. A m inA,B;100 ) EphA3-APinsitubindingonastage25hindbrainrevealed ephrinA RESEARCH ARTICLE ␮ m inC. B ) EphrinA2-APinsitu C ) Top ␮ CB, cerebellum; gV, trigeminalganglion;ov, oticvesicle.Scalebar:400 stage 26hindbrain.a-ccorrespond approximately topositionsa-cinC. ( ephrin A2protein intectumandcerebellum from thesamebrain. the caudalhindbrainisindicatedbyarrows a,bandc.Insetshows section ofastage26brainstem.ThegradeddistributionephrinA2in neurons. ( indicate expression domainscorrelating withthelocationofcC-VC stage 25hindbrains,respectively. Thebluebracketsinbothimages Fig. 5. 6B,C, turn away fromtheborderofectopicephrinA2domain(Fig. expression ofephrinA2causedmostlabelledaxonstostallator on thegrowth ofthecC-VCaxons(Fig.6A, (data notshown). Ectopicexpression ofEGFP alonehadnoeffect showed goodco-localisationwiththeco-electroporatedEGFP expression ofephrinA2asvisualisedbyEphA3-APinsitubinding 27-28, andcC-VCaxonswereanterogradelylabelled.Ectopic hindbrain atstage18-20.Hindbrainsweretaken forculture atstage A/EphA signalling bothinvitroandovo. cC-VC axons.We testedoutthispossibilitybyperturbing ephrin possibility thatephrinAisinvolved intherostralturning polarityof inhibitory activity for cC-VCaxonsinthecaudalhindbrainraise Our findingsthatephrinAcontributes tothelaterallylocated inappropriate ofsomecC-VCaxons caudalturning Perturbing ephrinA/EphA signallingleadto ovo. of ephrinA2couldelicitstallingandturningcC-VCaxons in caudal hindbrain. neurons, andephrinA2protein isdistributedinagradient D m inA,B;200 Ephrin A2immunostainingonthree transversesectionsfrom a ) n EphA5 =15/17). Thesedataprovide directevidence thatasource C ) EphrinA2immunohistochemistryonalateralparasagittal and ␮ m inC,insetCandD. ( EphA3 A , B ) EphA5 are expressed inpresumptive cC-VC and EphA3 in situhybridisationon Development 133(19) n =15/16), whereas

DEVELOPMENT majority ofsamples ( was electroporated,caudal-turning axonsweredetectedinthe samples ( electroporated, cC-VCaxons turned rostrallyinthemajorityof selected forfurtheranalysis.When anRCASBmockvector was electroporated domainsspanning thecC-VCturningpointwere hindbrains atstage27-28byDiI anterogradelabelling.Sampleswith We thenanalysedcC-VC axontrajectoriesoftheelectroporated electroporation ofRCASBmockvector ( ectopic expression ofEphA3 binding thatamarked reductionofephrinAactivity was causedby the electroporateddomain.We firstconfirmedbyEphA3-APinsitu stage 20-21chickembryos.EGFPwas co-electroporated to discern by inovo electroporationunilaterallyintothecaudalhindbrainsof (Nishida etal.,2002).We thereforeintroducedRCASB-EphA3 the maskingofendogenousephrinAbyectopicEphA3 endogenous ephrinAligandactivity inthistissue,possiblyowing to the developing chickcerebellumcausesa dramaticreductionof Feldheim etal.,2004).Overexpression ofexogenous EphA3 cytoplasmic domain(RCASB-EphA3 retroviral vector encodingamutantEphA3withtruncated ( added, cC-VCaxonsturnedrostrallyinthemajorityofsamples due totheFcpartoffusionprotein,becausewhenhFcalonewas more thanhalfofthesamples( inappropriate caudalturningofsomecC-VCaxonstookplacein specificantagonistforephrinAsignallingwas applied, more population (datanotshown). WhenrecombinantEphA3-Fc,a samples displayingtherostralturningofentirecC-VCaxon majority ofsamplesexamined ( treatment resultedinthecaudalturningofsomecC-VCaxons caudally forashortdistance(datanotshown). Bycontrast,PI-PLC (Fig. 7A),althoughsomeofthem( within thelateralaspectofhindbraininmostcases( control preparations,DiIlabelledcC-VCaxonsturnedrostrally cultured stage25-26hindbrainswithPI-PLCorEphA3-Fc.In polarityofcommissuralaxons Ephrin Ainlongitudinalturning n =21/29) (Fig.7C). To perturbephrinA/EphAsignallinginovo, weemployed a In vitroperturbationwas achieved bytreatmentoforganotypically n =41/55) (Fig.8A-D).However, whenRCASB-EphA3 n =33/49) (Fig.8E-I). DiI-labellingatstage27- n ⌬ =17/27) (Fig.7D).Thiseffect isnot n C ( =25/32) (Fig.7B),withonly7/32 n n =8/8) (Fig.8J),but notby =5/25) occasionallyturned ⌬ C) (Nishidaetal.,2002; n =4/4) (datanotshown). n =20/25) ⌬ C in ⌬ ⌬ ⌬ C C C caudally (whitearrow) ( ( test). ( caudally(white arrow).samples, someaxonsturned ( rostrally.in 20/25samples,allaxonsturned ( represented bytheratioofcaudalversus rostralturning axons andis Metamorph. Thedegree ofinappropriatecaudalturning was fluorescence intensityofcaudal-androstral-turningaxonsusing allowing ustoperformquantitative analysisbymeasuringthe 28 appearedtoresultinfewer ornoretrogradelylabelledcells, on organotypicallycultured stage25-26hindbrains.( caudally.VC axonsturning Fig. 7.BlockingephrinA/EphA signallinginvitro ledtosomecC- D ) With EphA3-Fc, in17/27samples,somecC-VC axonsturned ) With C ) With hFc, in 21/29 samples, all cC-VC axons turned rostrally. hFc,in21/29samples,allcC-VCaxonsturned ) With alone hasnoeffect oncC-VCaxons( VC axonswere anterogradely labelledwithDiI.( hindbrains were takenforculture atstage27-28,andcC- electroporated inovoataround stage20.Electroporated together withfull-lengthchickephrinA2were ofcC-VCaxons. stalling andturning Fig. 6.Ectopicallyexpressed ephrinA2induces caudally (C1)( awaytoextend axons eitherstalled(B1,C1)orturned the interfaceofanectopicephrinA2domain,mostcC-VC 200 with respect totheirresponsiveness toephrinA.Scalebar: cC-VC axonsmightrepresent aheterogeneous population domain inbothB2andC2,suggestingthepossibilitythat amount ofaxonsgrew intotheephrinA2expression C1, respectively, withEGFPsignalsremoved. Asmall ␮ m. P <0.015, Fisher’s test).Scalebar: 400 n =15/17). ( cC-VC axonswere anterogradely labelled A2-C2 RESEARCH ARTICLE B ) SameimagesastheA1- ) With PI-PLC,in25/32 ) With n =15/16). ( EGFP aloneor A P <0.0001, Fisher’s ) Without PI-PLC, ) Without A1 B1 ␮ m. ) EGFP , C1 3843 ) At

DEVELOPMENT 3844 in thesupplementary material).Therostralextent ofanephrinA execute rostralturning intolongitudinalaxis(Fig.4C;seeFig.S1B lateral hindbrain,cC-VCaxons coursetowards thepialsurface and of theventricular andmantle zone.Uponreachingthecontralateral The developing cC-VCaxonsextend circumferentiallyattheborder hindbrain The functionofephrinA gradientinthecaudal regulates thepolarityofpost-crossingcommissuralaxons. represents thefirstdemonstrationthatephrinA/EphAsignalling located hindbraincommissuraltract.To ourknowledge, this plays aroleinthecontrolofrostralturningpolaritylaterally In thisstudy, wehave demonstratedthatephrinA/EphAsignalling DISCUSSION of therostralturningpolarityEphA-expressing cC-VCaxons. signalling inthecaudalhindbrainiscruciallyinvolved inthecontrol employing PI-PLCandEphA3-Fc,suggestthatephrin A/EphA that turnedrostrally. axons, withthecaudalturningaxonsbeingonaverage 27%ofthose higher incidenceandpercentageofinappropriateturning summarised inFig.8K.ThesampleswithEphA3 The inovo perturbationdata,togetherwiththeinvitrodata RESEARCH ARTICLE ⌬ C showed both and (2)inovo introductionofRCASB-EphA3 turning pointwas transplantedtointersecttheaxonalpath (Fig.2A); a pieceofcaudalhindbrainlocatedposteriorandclosetotheir this possibility:(1)cC-VCaxonswereonlypartiallyinhibitedwhen than formingacaudalinhibitorybarrier. Two observations support the rostralturningpolarityofcC-VCaxonsbyitsgradient,rather axons turnrostrally. ThisraisesthepossibilitythatephrinAcontrols immunohistochemistry, reachestheregion withinwhichthecC-VC EphA3-APinsitubindingandephrinA2 gradient, demonstratedby rely onitsability tocausegrowth conecollapseandretractionof role ofephrinAintopographic projectionisgenerallythoughtto 2002; McLaughlinetal.,2003; GarelandRubenstein,2004).The thalamocortical projection(Vanderhaeghen etal.,2000;Yue etal., subcortical targets, thehippocamposeptalsystemand the projection ofretinalganglionic axonstovarious corticaland topographic projectionofseveral CNScircuits,includingthe an ephrinAgradienthasbeendemonstratedintheestablishment of translated intoapreferenceforturningrostrally. Theimportanceof the rostrocaudalspanoftheirgrowth cones,whichisinturn zone, encounterarostral-low/caudal-high ephrinAgradientacross envisage thatcC-VCaxons,uponentering their presumptive turning the rostralturningofcC-VCaxons(datanotshown). Therefore,we to but notoverlapping thecC-VCaxon turningpointdidnotdisrupt electroporated withRCASB-EphA3 C,D,G,H. inappropriate ( caudalturning significantly higherincidenceandamountof caudally.turning SampleswithRCASB-EphA3 in E-HandI,respectively. Arrows inF,H,I indicateaxons orange arrows indicatethedatapointsofsamplesshown sample valuessortedinanascendingorder. Thered and axons. Dataare represented inascatterchart, withthe represented astheratioofcaudal-overrostral-turning mock vector(Control). Thedegreeis ofcaudalturning ( sample indicatedbyEGFPisoutlinedwithwhitedots. EphA3 binding onahindbrainco-electroporated withRCASB- EphA3 higher degreeerror ofcaudal-turning whenRCASB- U VC axons. ovo ledtoinappropriateofsomecC- caudalturning Fig. 8.InterferingwithephrinA/EphAsignallingin of A,B,E,F, respectively. ( regions(C,D,G,H) Highermagnificationsofaxonturning to betterrevealpoint. theaxonsattheirturning images asinA,E,respectively, withEGFPsignalsremoved indicate thedomainofelectroporation. (B,F)Thesame EphA3 ( and cC-VCaxonswere anterogradely labelled. 27-28, electroporated hindbrainswere takenforculture reveal thedomainofsuccessfulelectroporation. Atstage 21. AnEGFPexpression vectorwasco-electroporated to electroporated inovointocaudalhindbrainatstage20- K A-D -test). Scalebar:200 ) Quantification of caudal turning error Quantificationofcaudalturning inhindbrains ) RCASBvectorwaselectroporated. ( ) ⌬ ⌬ ⌬ C andEGFP. Theelectroporated domainofthis C waselectroporated. ( C waselectroporated. EGFPsignalsinA,E RCASB vectororRCASB-EphA3 ␮ I m inA,B,E,F,I,J; 100 ) Adifferent samplewitha P <0.0001, Mann-Whitney J ) EphA3-APinsitu Development 133(19) ⌬ ⌬ C versusRCASB C toaregion caudal E-H ⌬ C was ␮ ) RCASB- ⌬ m in C showed

DEVELOPMENT all cC-VCaxons turnrostrally, implyingmechanisms otherthan of cC-VCaxonsenteringtheectopic ephrinA2domain(Fig.6).Yet in theirsensitivity to ephrinA,asevidenced bythesmallpercentage (Fig. 3).Second,cC-VCaxonsmight beaheterogeneouspopulation weaker thanPI-PLCtreatment inalleviating thecaudalinhibition rostral turningofcC-VC.First,the effect ofEphA3-Fcappearstobe might functioninconcertwithephrin A/EphAinthecontrolof of alateralhindbraincommissuraltract. A/EphA signallingisimportanttoensuretherostralturningpolarity gradient ofephrinAinthecaudolateralhindbrainandthat tracts. Ourresultshave demonstratedarostral-low/caudal-high exist tocontrolthelongitudinalturningpolarityoftheselateralaxon We postulatedthatmolecular cuesdistributed inthelateralhindbrain further fromthefloorplate,withinlateralaspectofhindbrain. commissural tracts,many ofwhichturnlongitudinallyatadistance and thehigherbrainstructures,hostsadiversity of the developing hindbrainasarelaystation betweenthespinalcord turn rostrallyimmediatelyaftercrossingthefloorplate.However, Bourikas etal.,2005).Inbothcases,thespinalcommissuralaxons axons inmiceandchick,respectively (Lyuksyutova etal.,2003; plate werefoundtodirecttherostralturningofspinalcommissural until recentlywhenWnt4andShhgradientsadjacenttothefloor turning ofpost-crossingcommissuralaxonshave remainedelusive However, themolecularcuesthatdirectlyinstructrostrocaudal Matise etal.,1999;Zou2000;ShirasakiandMurakami,2001). surgically orpharmacologically(StoeckliandLandmesser, 1995; when theirpre-contactwiththefloorplatewas perturbedgenetically, Post-crossing commissuralaxonsmake rostrocaudalturningerrors commissural axons Rostrocaudal polarityofpost-crossing turning neurons, aswelldefiningthecaudallimitofitsgrowth. the topographyofgVIIIafferents tothesecondaryvestibular hindbrain, raisesthepossibilitythatephrinAgradientmightcontrol within theephrinAgradientandstopsatcaudalextreme ofthe Finally, thetrajectoryofgVIIIdescendingafferent, whichgrows in thechickandmousespinalcord(ImondiKaprielian,2001). has beenproposedforephrin-B,inthecontrolofcommissuralaxons intermediate position(Y.Z. andF.M., unpublished).Asimilarrole emanating fromthecaudalendofhindbraintoturnatan as aninhibitorybarrierthatobligesthecommissuralaxons level ofephrinAinthecaudalextreme hindbraincouldalsofunction cerebellum throughthelateralhindbrain(Zhuetal.,2003).Thehigh olivary axonsthatextend from thecaudalhindbraintowards the structures, suchasthecerebellum.Onecandidateisinferior through thelateralhindbrain,especiallythosedestinedfordorsal the rostrocaudalpolarityofotheraxontractsthatgrow longitudinally upon growing intoagradientofephrinA. expressing axonscouldbeinducedtochangetheirgrowth polarity Nevertheless, thesedatarendersupporttoourmodelthatEphA- preferentially inducesturningversus collapseinoursystem. 2005).Atpresent,itremainsunknown whichcondition al., Xenopus to beadsorintheformofagradientcouldinduceturning showed that,inadditiontogrowth conecollapse,ephrinA5bound VC axons.Ourfindinggainssupportfromrecentinvitrostudiesthat expressed ephrinA2couldinduceturningaswellstallingofcC- has remainedunclear. Here,weshowed invivo thatectopically whether ephrinAcanguideaxonsbyinducinggrowth coneturning EphA-expressing axonsinaconcentration-dependentmanner. Yet polarityofcommissuralaxons Ephrin Ainlongitudinalturning Several observations pointtothepossibilitythatadditionalfactors It isconceivable thatthecaudalephrinAgradientalsocontrols and chickretinalaxons(Weinl etal.,2003;Weinl et Diaz, C.andPuelles,L. Clarke, J.D.andLumsden,A. Cheng, H.J.,Nakamoto,M.,Bergemann,A.D.andFlanagan,J.G. Chédotal, A.,Bloch-Gallego,E.andSotelo,C. different responsiveness tothesameguidancecue(s). diversity couldbeachieved througheachaxontractdisplaying hindbrain tosettheroutesforthesetracts.Inaddition,high combination ofdifferent guidancecuesthatintricatelypave the trajectories ofhindbraincommissuralaxonsmayrequirea the controlofrostrocaudalturningpolaritycC-VC.Thediverse ephrin Agradientinthecaudallateralhindbrainthatplaysarole hindbrain commissuraltract,cC-VC.Thisledustoidentifyan hindbrain atagrosslevel, withrespecttoadeveloping lateral hindbrains todissectouttheguidancepropertiesofcaudallateral hindbrain, weusedtransplantationonorganotypically cultured Taking advantage oftherelatively large sizeofthedeveloping Conclusion investigation. rostrocaudal turningofcommissuralaxonsawaits further Guthrie,2003).Theroleof thesemoleculesincontrolling and to bedistributed highinthecaudalhindbrainatst27(Chilton Frizzled receptors,respectively. Furthermore,Sema3Calsoappears or attractants,dependingonwhetherthey actthroughRykor (Liu etal.,2005).Thus,Wntproteinsmightacteitherasrepellents repulsive gradientsofWnt1andWnt5a,actingviatheRykreceptors caudally directedgrowth ofcorticospinalaxonsis controlledbythe spinal commissuralaxons.Indeed,arecentstudyhasshown thatthe chemoattractive roledemonstratedforWnt4intherostralturningof would have tofunctionaschemorepellents,ratherthanhaving the to controltherostralturningoflateralcommissuralaxons,they hindbrains ofdeveloping chick(Hollydayetal.,1995).Ifthey were shown toexpress inacaudal-high/rostral-low mannerinthelateral Briscoe andEricson,2001).Wnt4Wnt3ahave beenpreviously ventral region ofthedeveloping neuraltube(Martietal.,1995; located commissuralaxons,asitsactivity islargely confinedtothe likely candidatetocontroltherostrocaudalturningoflaterally only around27%oftherostral-turningaxons.Shhisnotavery turning caudallyupondisruptionofephrinA/EphAsignallingwas insensitive cC-VCaxons.Third,theaverage percentageofaxons ephrin Amightcontroltherostralturningofthese Briscoe, J.andEricson, Bourikas, D.,Pekarik,V., Baeriswyl,T., Grunditz,A.,Sadhu,R.,Nardo, M. References http://dev.biologists.org/cgi/content/full/133/19/3837/DC1 Supplementary materialforthisarticleisavailableat Supplementary material in-Aid from MEXTandDevelopmenttravelfellowship. SORST, JapanScienceandTechnology Corporation,InoueFoundation,aGrant- Nishida forcriticalreading ofthemanuscript.Thisworkwassupportedby for RCASB-EphA3 Dr N.Wada for fusion proteins, and We thankDrH.Tanaka forephrinA2antibody, ephrinA2-APandEphA3-AP Chilton, J.K.andGuthrie,S. dextranamine experiments invitro. other hindbraincerebellar afferents inmidtermchickembryos:biotinylated phenotype setsinthechickembryohindbrain. semaphorins andtheirneuropilin receptors. development ofthetopographicretinotectal projection map. Complementary gradientsinexpression andbindingofELF-1Mek4 in axons. cerebellum containstopographic cuesthatguidedevelopinginferiorolivary neural tube. longitudinal axisofthespinalcord. and Stoeckli,E.T. Development Curr. 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