ehsa D20892,USA. MD Bethesda, aaauOgawa Masaharu RIKEN BrainScienceInstitute,Wako, Saitama 351-0198,Japan. transition remaintobeelucidated. this Themolecularandcellularmechanisms thatregulate (CP). thecorticalplate to fromthepremigratoryzone just beforemigrating neuronschangetheirshapefrommultipolar to bipolar Then, zone. thepremigratory alsoknown as which is intermediate zone(IZ), neurites withinthesubventricular zone(SVZ) andlower immature neuronstransientlybecomemultipolar, withmultiple Afterthefinal division, 2003;Noctoretal.,2004). Nakajima, radial neuronalmigration(Nadarajahetal.,2001;Tabata and of migratingcerebralcortex neuronsidentified specific phasesin Recently, time-lapseimaging cortex, hippocampusandcerebellum. formation oflaminatedcorticalstructures,suchasthecerebral Proper positioningofneuronsisparticularlycrucialforthe neurons fromtheventricular zone(VZ)totheirfinal destination. radialmigrationof This uniquelayeringisachieved viacoordinated 1961). andSidman, occupying themoresuperficial layers(Angevine neurons positionedinthedeepestlayersandlater-generated neurons cerebral cortex inaninside-outmanner, withtheearliest-generated layerswithinthe In themammalianbrain,neuronssettleintosix INTRODUCTION Accepted 21March 2007 Development, Medicine, Shinjuku-ku,Tokyo 160-8582,Japan. 1 KEY WORDS:Neuronal migration,Cerebral cortex,Mouse,Cdk5,Map2(Mtap2) butnotin migrating neuronsatE14.5byinuteroelectroporation,weobservedwild-type WhenweintroducedGFPinto MicelackingCdk5exhibitabnormalcorticogenesisowingtoneuronalmigrationdefects. fibres. migratealongradialglial then bipolarshapewithinthesubventricularzone-intermediatezone(SVZ-IZ)and from amultipolarto thefinaldivision,neuronstrans After Recentstudiesidentifiedspecificphasesofradialmigrationcorticalneurons. period. neuronalmigrationduringtheembryonic layersthataregeneratedviacoordinated cortexconsistsofsix The mammaliancerebral Toshio Ohshima of pyramidalneuronsinthecerebralcortex radial neuronalmigrationandproperdendritedevelopment formultipolar-to-bipolar transitionduring Cdk5 isrequired (2007)doi:10.1242/dev.02854 Development 134,2273-2282 Medicine, Tokyo 105-8461,Japan. Molecular Neurobiology, InstituteofDNAMedicine,JikeiUniversitySchool Saitama 351-0198,Japan. axonal trajectoriesofcorticalpyramidalneurons. Inaddition,properradialneuronalmigrationgenerates an inside-outpatternofcerebralcortexformationandnormal neurons. ofpyramidal resultsinabnormalmorphology adeficiencyofCdk5 and required forpropermultipolar-to-bipolar transition, TheseresultsindicatethatC deficient mice,andtheaxonaltrajectoryofcorticalneuronswithincortexwasalsoabnormal. Cdk5- of TheamountofthedendriticproteinMap2wasdecreasedincerebralcortex dendriticmorphology. impaired severely callosalneurons,butnotlayerVIha knockout miceshowedinvertedlayeringofthecerebralcortexandlayerV conditional Cortex-specificCdk5 manner. withintheSVZ-IZinacell-autonomous impairment ofthemultipolar-to-bipolar transition thewild-typemigratingneuronsconfirmedspecific after 3-4days.Introductionofthedominant-negativeformCdk5into *Author forcorrespondence (e-mail:[email protected]) of MedicalScience,UniversityTokyo, Minato-ku, Tokyo, Japan. Keiko Saruta Laboratory forDevelopmentalNeurobiology, RIKENBrainScienceInstitute,Wako, 5 Laboratory forBehavioralGeneticsand 1 , Takuji Iwasato 1, 2 *, MotoyukiHirasawa Functional GenomicsSection,CDBRB,NIDCR,NIH, 3 4 Department ofAnatomy, KeioUniversitySchoolof , AshokB.Kulkarni 8 Department ofMolecularNeurobiology, Institute 5 , ShigeyoshiItohara 4 Laboratory forCellCulture 6 Hashimoto Research Unit, 2 and KatsuhikoMikoshiba 2 , HidenoriTabata 7 Department of 5 , Mistuhiro Hashimoto transition duringtheirradialmigration. multipolar-to-bipolar the neurons have severe impairmentof We foundthat neurons inthedeveloping cerebralcortex. elucidated indetail. tobe abnormalitiesneed neurons (Gilmoreetal.,1998),but these priori forproper corticogenesisinthedeveloping mousebrain. dependent multipolar-bipolar transitionofmigratingneuronsisa thatCdk5- ofmutantmiceindicated analysis Further cerebral cortex. ofpyramidal neuronsinthepostnatal defective axonal trajectories of radialmigrationresultsinabnormal dendriticdevelopment and ofcorticalneuronstotransition fromthemultipolarphase inability Usingthismutantmouseline,wedemonstratethatthe 2004). mice (Iwasato etal.,2000)and generated cortex-specific Cdk5-deficient micebymating lethality, we To overcome thisearly (Ohshima etal.,1996). perinatal periodbeforecerebralcortex layerformationiscompleted the cerebralcortex, neocortical In throughout thebrain(Ohshimaetal.,1996;Gilmore1998). 2001). al., 1996;Gilmoreet1998;Chae1997;Ohshima about mainlybytheregulation ofneuronalmigration(Ohshima et andthisisbrought thedeveloping mousebrain, cortical structuresof and itsactivating subunit p35have crucialrolesintheformationof Previous studiesbyusandothergroupsrevealed thatCdk5 activity. forkinase Genome Informatics)oritsisoformp39,iscrucial eitherp35(CdK5r1–Mouse neuron-specific regulatory subunit, AssociationofCdk5 witha 2001). neurons (Dhavan andTsai, kinaseactivity ismainlydetectedinpostmitotic its other Cdksbut aeln td niae htteeaemigrationdefectsin thereare studyindicated that labelling Abirth-date II toVstallbelow thesubplate(Gilmoreetal.,1998). 3 , Tetsuji Mutoh eew eotoraayi ftemgaoybhvorof Here wereportouranalysisofthemigratorybehaviour Cdk5 isauniqueserine/threoninekinasewithclosehomologyto Cdk5 1,8 –/– mice exhibit substantialmigratorydeficits 4 6 , Tomoko Adachi , KazunoriNakajima Cdk5 fCdk5/fCdk5 RESEARCH ARTICLE –/– neurons destinedforlayers 1 Cdk5 , Hiromi Suzuki mice (Hirasawa etal., Cdk5 3,7 –/– , –/– mice dieinthe embryos Emx1Cre Cdk5 Cdk5 Cdk5 form dk5 is 2273 1 , d –/– –/– –/–

DEVELOPMENT to the generated bytheknock-instrategy; Crerecombinaseactivity was restricted bandfo ako aoaoy A The obtained fromJacksonLaboratory, MA. (Hirasawa etal.,2004).TheYFP-Hmouseline(Feng2000)was (Ohshima etal.,1996). microscope (FV300,Olympus).Ten totwentyoptical ta. 97.Dt r shown asmeans±s.d. Dataare et al.,1997). major dendritethat graduallytaperedoff from thecellbody(Threadgill neurons weredefined ascellsthathadapyramidal cellbodywithone Pyramidal Tuj1-positive cells was calculatedinrandomlyselectedfields. Thepercentageofpyramidal neuronsamong fluorescent microscope. Cellimageswereacquiredwith a (Molecular Probes,Eugene,OR). followedDenver, byAlexa-Fluor 488 PA) atroomtemperaturefor1 hour then incubatedwithanti- Thecellswere X-100 inPBS)atroomtemperaturefor30minutes. Triton were incubatedwithblockingbuffer (1%bovine serumalbumin and0.2% Afterwashing withPBSthreetimes,cells atroomtemperature. minutes in0.1Mphosphatebuffer for10 with 4%paraformaldehyde(PFA) Afterwashing withPBS,culturedneuronswerefixed medium for3days. E14.5 corticalneuronsof Dissociated neuronal culture andimaginganalysis Female andtime-lapseimaging In utero electroporation Cdk5 Mice MATERIALS ANDMETHODS 2274 and provided withcareasperRIKENBSIguidelines. YFP-H micetoobtain controls. astrocyte feederwas culturedoutside,at3 dissociated cellswereculturedonfibronectin-coated dishes,onwhichan ng/ N-2 supplement(Invitrogen), 5%fetalbovine serum,5%calf4 of DMEM/Ham’s F-12mixture(Sigma,StLouis,MO)supplementedwith andwashed inculturemediumwhichconsisted dissociated, at37°C, 10minutes was excised, incubatedwith0.25%trypsininPBSfor Briefly, thecerebralcortex (Hayashi etal.,2002)withslightmodification. mice ( (Cappelloetal.,2006). E10 asdescribed merged. intervals werecapturedevery 30minutesandallfocalplaneswere San Diego, CA)andwas incubated at37°Cin5%CO glass-bottom dishwithNeurobasalmediumcontainingB27(Invitrogen, (Millicell-CM, 0.4 of thecerebralcortex wereembeddedincollagengelonapermeablefilter 2003). Briefly, coronalbrainslices(200 slices was conductedasdescribedpreviously (Tabata andNakajima, Time-lapse imagingofbrain CRC-Cdk5-DN, respectively. Cdk5-DN and CAG-RFP-CAG togenerateCGC- inserted intoCAG-EGFP-CAG and Cdk5-DN(N144)was then generated inthesameway usingCAG-RFP. CAG-RFP-CAG was downstream oftheEGFP inCAG-EGFP. amplified withpCAGGS byPCR,andinsertedintothe 2.3 kbfragmentcontainingtheCAG promoterandthe a To generateCAG-EGFP-CAG (CGC), was generatedasfollows. DN) (CGC-Cdk5- same cells,bi-expression vector CAG-GFP-CAG-Cdk5-DN andEGFPinthe Inordertoexpress bothCdk5-DN 2001). Nakajima, asreportedpreviously (Tabata and progenitors (i.e.radialglialcells), migratingneuronsaswelltheir neurons, andCAG-EGFP labelled T etal.,1991). Miyazaki (Niwa J. was kindlyprovided byDr inserted, respectively, intopCAGGS, which from pEGFP-N1andpDsRed2-N1(Clontech,Palo Alto,CA)were andCAG-RFP, thecDNA ofEGFPandDsRed2 generate CAG-EGFP T 2003). Tabata andNakajima, 2001; (Tabata andNakajima, previously subjectedtoinuteroelectroporationasdescribed pregnant, were well asICRmales(JapanSLC,Shizuoka,Japan)and,afterbecoming ␣ -GPwsgnrtda ecie Tbt n aaia 01.To 2001). 1-EGFP was generatedasdescribed(Tabata andNakajima, ␮ l EGE,8ng/ –/– Emx1 fCdk5 Cdk5 RESEARCH ARTICLE mice weregeneratedandmaintainedasdescribedpreviously Reeler xrsinrgo Iaaoe l,20)anddetectedasearly expression region (Iwasato etal.,2000) /–; eeoyoe werematedwith heterozygotes Emx1 mice (B6C3Fe-a/a-rl,JacksonLaboratory)werematedwith ␮ ␮ Cre/+ m poresize,Millipore).Thefilter was placedona reeler ai G,pncli n tetmcn The l basicFGF, penicillinandstreptomycin. d5loxP Cdk5 , CxCdk5KO) wereanalysedalongwithlittermate Cdk5 ␤ YPHmtn ie Micewerefedadlibitum ;YFP-H mutantmice. III-tubulin antibody(1:500Tuj1, Covance, +/+ -flanked miceweregeneratedasdescribed ␣ -GPefcetylble migrating 1-EGFP efficiently labelled and Cdk5 ␮ Emx1Cre m) fromtheanteriorone-third –/– Cdk5 ϫ were culturedasdescribed Emx1Cre 10 4 eeoyoemlsas heterozygote males conditional cells/cm z -sections at5 mouse linewas Eco 2 Hin 2 RI sitewas on alaser in culture Cdk5 dIII site ␮ KO m t3°.Thebrainswereembeddedin2%agarose,cutinto100 at 37°C. inthesamefixative for3-4weeksinthedark Thebrainswerekept at P10. Consortium, 2005)andIMAGE clones,respectively. Mouse cDNAs for were insertedintothedorsalthalamusorcerebralcortex of Several smallcrystalsofDiI (pH 7.4),brainswereremoved fromtheskulls. in0.1Mphosphatebuffer Briefly, afterintracardiacperfusionwith4%PFA DiI tracingwas performedasdescribedpreviously (Ohshimaetal.,2002). 1,1’-dioctadecyl-3-3-3 with BrdU(100 For bromodeoxyuridine(BrdU)experiments, pregnant damswereinjected in situhybridisation and injection,immunohistochemicalstaining Bromodeoxyuridine Our previous study of Morphological features ofmigrating stage Cdk5-deficient corticalneurons attheembryonic RESULTS pentobarbitone at50 Pregnant miceweredeeplyanesthetisedwithsodium M.O., unpublished). of Adex-CAG-Lyn-Venus willbedescribedelsewhere (T.M., M.H.and Lyn (amino acids246-307)totheN-terminusofVenus. Thedetailedfeature generated byfusingthesequenceofpalmitoylation siteoftyrosinekinase was Lyn-Venus promoter. (Nagai etal.,2002)undercontroloftheCAG calledVenus improved YFP CAG-Lyn-Venus membrane-targeted expresses Adex- Theadenoviral vector 2004). described (HashimotoandMikoshiba, The adenoviral vector constructionwas carriedoutbasicallyaspreviously Adenovirus preparation, infectionandtime-lapseimaging and actin( antibody forCdk5(C-8),Map2(Chemicon),NF-M(NF-150,Chemicon) withthefollowing primaryantibodies:polyclonal (Ohshima etal.,2005) extraction andwesternblottingwerecarriedoutasdescribed Western blotanalysis Cdk5 Cdk5 deconvolution Deblursoftware (Leica)was usedfordeblurringthe (Leica).The software ASMDW by multipoint time-lapsestagecontrolled and ofaninverted microscope(DMIRE2,Leica) microscope setupconsisted multidimensional live tissueimaging(Leica,Heidelberg, Germany). The ALeicaASMDW workstation was usedfor carried outasdescribedabove. brainsliceswere (E14.5).Preparation andcultureof removed later were embryosatE12.5.The total) wereinjectedintotheventricle ofthe morphology of migrating later-born We first analysedthe mutant cortex (Gilmore etal.,1998). positionedabnormallyunder thesubplatein layers IItoVare marginal zoneandsubplate,but later-born neuronsthatconstitute VI neuronsmigratenormally andsplitthepreplateinto ihavbaoeadphotographedwitharhodaminefilter. and with avibratome perchlorate (DiI)tracing 1.Prfi etos(8 P10. Paraffin sections Student’s SD n Premieresoftware (Adobe,SanJose,CA). ASMDW and images. Then,thedeconvoluted imageswere processedtoAVI movies by spoe.Fri iuhbiiaininthecoronalsectionsfrom For insituhybridisation as probes. (Ferland etal.,2003), coronal sectionsfromCxCdk5KO andcontrolmiceatP10using was performedasdescribed(Ohshimaetal.,2002)inthe hybridisation Cruz, CA)andmonoclonalantibodyforMap2(Chemicon).Insitu polyclonal antibodiesforGFP(MBL,Tokyo, Japan)andCdk5(C-8,Santa usingthefollowing primaryantibodies: 1998; Ohshimaetal.,2001) frozen sections(15 For theimmunohistochemicalanalysis, (Gilmoreetal.,1998). described werecutandstainedwithmonoclonalanti-BrdUantibodyas buffer) –/– –/– embryosatE16.5werestainedusing , embryos atE18.5orCxCdk5KO miceandtheirlittermatecontrols t -test. ␣ atn im) aaaesona ensd andanalysedby mean±s.d. -actin, Sigma).Dataareshown as P ␮ <0.05 was consideredstatisticallysignificant. /,itaeioel attheindicatedageandanalysed g/g, intraperitoneal) Neurod1 ␮ )werecutandstainedasdescribed(Gilmoreetal., m) ␮ Er81 / oywih.Aeoia etr (2.0 bodyweight.Adenoviral vectors g/g Ј -3 Cdk5 ␮ Ј Abre l,20)and (Arber etal.,2000) )o ie ri 4 F n01Mphosphate M 0.1 PFA in (4% offixed brain m) -tetramethylindiocarbocyanine and –/– Dcx mice indicatedthatearly-bornlayer eetheFANTOM3 (TheFANTOM were Cdk5 Neurod1 Cdk5 –/– Development 134(12) Cux2 ern.To visualise neurons. –/– and (Nieto etal.,2004) neurons Dcx Cdk5 ␮ Cdk5 m sections as probes. ϫ z -stacked 10 +/+ +/+ Foxp2 6 and and pfu

DEVELOPMENT and CPin at thepremigratoryzoneorabipolarmorphologyupperIZ exhibited eitheramultipolarmorphology neurons GFP-labelled Whenweexamined E17.5embryos, supplementary material). Cdk5 and theirfibres extended toward thepiaincerebralcortex of radialglialcellsexhibited abipolarmorphology, GFP-labelled most GFP-positive cellswerelocalisedinthepremigratoryzone. AtE16.5, examined byimmunostainingwithanti-GFPantibody. neurons electroporation, andthemorphologiesofGFP-labelled days(E18.5)afterinutero (E16.5),3(E17.5)or4 fixed 2 Embryoswere 2003)atembryonicday14.5(E14.5). Nakajima, 2001;Tabata and electroporation (Tabata andNakajima, anenhancedGFP(EGFP) byinutero cortex, weintroduced the morphologyofmigratingcorticalneuronsincerebral Cdk5-dependent acquisitionofpyramidalmorphology n aaia 03 otre l,20) Inthe 2003;Noctoretal.,2004). and Nakajima, reported(Tabata zone (SVZandlower IZ)totheCPaspreviously bipolar transitionduringradialmigrationfromthepremigratory F-aeldneuronsinthe GFP-labelled Bycontrast, themidline(Fig.1A). then turnedsharplytowards theIZand towards neurons extended theiraxons GFP-labelled were detectedinthecerebralcortex of after labelling(E18.5),noGFP-positive cellsinabipolarshape Even 4days observed inthepremigratoryzoneorCP(Fig.1B). neuronswerenot bipolar keeping theirmultipolarmorphology; hw) Axonal trajectoriesofGFP-labelled shown). +/+ as well Cdk5 +/+ embryos (Fig.1A),indicatingmultipolar-to- Cdk5 –/– Cdk5 mro seFg 1inthe S1 Fig. embryos (see –/– Cdk5 brain migratedupward, –/– Cdk5 embryos (datanot –/– Cdk5 neurons ran +/+ brain, notebplrsaewti h pe Z However, careful into thebipolarshapewithinupperIZ. in theCP, but themajorityofRFP-positive neuronstransformed a delayedmigrationofneuronsexpressing Cdk5-DNwas observed In thebrainswithaCAG-RFP dilutionof20%(1:4)or50%(1:1), observed (Fig.2). RFP, different degrees ofmigrationdefectswere CRC-Cdk5-DN indifferent concentrationsbydilutingwithCAG- Whenweapplied expression. lower level ofCdk5-DN indicating a low (Fig.2), intensity ofRFPintheseneuronswas relatively Few neuronswerebipolarandthe had themultipolarshape(Fig.2). positionedwithinthepremigratoryzoneand positive neuronswere RFP- the CRC-Cdk5-DNplasmid(100%Cdk5-DN),most Whenweintroducedonly CAG promoterwithinasingleplasmid. DN), inwhichRFPandCdk5-DNweretranscribedviatheirown CAG promotervector, CAG-RFP-CAG-Cdk5-DN (CRC-Cdk5- adual oftheneuronsexpressing Cdk5-DN, weutilised behaviour To examine themorphologyandmigratory (Hatanaka etal.,2004). impairment ofthetransformationfromamultipolartobipolarshape but not radialmigration, into migratingneuronscausesadelayin Introduction ofthedominant-negative formofCdk5(Cdk5-DN) dominant-negative formofCdk5 Morphological features of neurons expressing the Cdk5 during themigrationfrompremigratoryzonetoCPin indicate aspecific deficit ofmultipolar-to-bipolar transition Theseresults obliquely withinthecortex (arrowhead inFig.1B). 100 subventricular zone-intermediatezone; VZ,ventricularzone.Scalebar: A neurons arrested withintheSVZ-IZinmultipolarshape (arrows in autonomous. WhenonlyCdk5-DNwasused(100%),RFP-positive disturbance ofneuronal migrationbyCRC-Cdk5-DNwasalsocell- ratios forinutero electroporation ofE14.5mouseembryos. ( DN. neurons expressing Cdk5- transformation withintheSVZ-IZin Fig. 2.Cell-autonomousimpairmentofthemultipolar-to-bipolar and thepercentage ofcellsineachbincalculated. Cdk5-DN. Thecorticalmantlewas divided intotenequallyspacedbins processes (arrows in A in CPbutsomeoftheRFP-positive neurons havebranchedleading migration delaywasobserved.RFP-positive neurons havebipolarshape arrest inacell-autonomous manner. WhenCdk5-DNwasusedat50%, that defectiveCdk5activityinmigratingneurons causedmigration of 100%(a)and50%Cdk5-DN(b)are showninA coronal sectionsatE18.5.Highermagnificationsoftheindicatedareas distribution andmorphologyofRFP-positivecells(red) were analysedin Ј ). Thisresult matchedthephenotypeof Cdk5 at thisstage.(B)Bycontrast,in SVZ-IZ, GFP-positiveneurons ofmultipolarshape(b)are observed toward thepiaandtheiraxons(arrowhead) toward theVZ.In Migrating neurons intheCPextendtheirleadingprocesses Fig. 1. Visualisation ofmigratingneuronsFig. 1.Visualisation in Cdk5 electroporation. cells byinutero into theCPwithtypicalbipolarshape(a)in magnification. (A)Overtheperiodof3days,neurons migrated Insets showareas indicatedbyarrows (a,b)athigher fixed atE17.5.Sectionswere stainedwithanti-GFPantibody. Scale bar:25 subventricular zone;IZ,intermediateVZ,ventricularzone. run obliquewithintheSVZ-IZ.CP, corticalplate;SVZ, the SVZ-IZinamultipolarshape(a),andtheiraxons(arrowhead) –/– ␮ Mixtures ofCRC-Cdk5-DNandCAG-RFPwere usedin different m. ( neurons. +/+ –/– B ) BindistributionofRFP-positiveneurons ateachratioof and ( mice byintroduction ofT B ␮ ) m. Cdk5 Ј b). MZ,marginalzone;CP, corticalplate;SVZ-IZ, –/– mouse embryosatE14.5,andbrainswere Cdk5 RESEARCH ARTICLE ␣ EGFP wasintroduced into( –/– Cdk5 1-EGFP plasmidintoVZ mice neurons arrested in –/– neurons, indicating Ј . Dose-dependent Cdk5 Cdk5 +/+ +/+ A mice. , A and Ј ) The 2275 A )

DEVELOPMENT A-GPadtoewt G-d5D.Thesedifferences were CAG-EGFP andthosewith CGC-Cdk5-DN. betweenbrainsliceswith hours neurons werealreadydifferent at4 ofGFP-positive Themorphologyandbehaviour Noctor etal.,2004). 2003; asdescribed(Tabata andNakajima, and tailingprocesses), (alsoidentified asleading which aneuronhasoneortwo neurites has extended multiple(more thanthree)neurites,or‘bipolar’,in each neuronwas classified aseither‘multipolar’,inwhichaneuron afteronsetofobservation, GFP-positive neuronsat4and24hours of Basedonthemorphologyandbehaviour 2003). and Nakajima, described (Tabata daysafterelectroporation,aspreviously 2 from intervals for24hours prepared atE16.5andimaged30-minute andsliceswere EGFP-CAG-Cdk5-DN (CGC-Cdk5-DN) plasmid, plasmid, inwhichEGFPwas usedforthevisualisation,or theCAG- inuteroatE14.5witheithertheCAG-EGFP were electroporated Brains we conductedtime-lapseobservation ofbraintissueslices. ofneuronsexpressing Cdk5-DN, behaviour To studythemigratory expressing Cdk5-DN ofneurons Time-lapse imagingofmigratorybehaviour 2A branchedleadingprocesses(Fig. positive neuronsintheCPhad highermagnification revealed thatsomeRFP- observation at 40.15±14.25%; Cdk5-DN,7.75±3.77%, transformation wasimpaired inneurons expressing Cdk5-DN(GFP, slice andsubjectedtostatisticalcomparison.Multipolar-to-bipolar multipolar tobipolarduringa20-hourperoid wascalculatedforeach ( slices. percentage ofeachisshownforCAG-EGFPandCGC-Cdk5-DNbrain at 4hours(4h)and24(24post-electroporation, andthe observed bipolar shape(bipolar)ormultipolar(multipolar)when behaviour, GFP-positiveneurons were dividedintotwo categories: Cdk5-DN (Cdk5-DN)atE14.5.( mouse brainsliceselectroporated witheitherCAG-EGFP(GFP)orCGC- CAG-EGFP orCGC-Cdk5-DN. Fig. 3.Time-lapseimagingofmigratingneurons expressing either 2276 Ј b, arrows). C RESEARCH ARTICLE ) Thepercentage ofGFP-positiveneurons thattransformedfrom ( B A ) Basedontheirmorphologyand ) Representative results are shownfor n =4); *, P <0.001. oma hssae However, in form atthisstage. to shapes inthepremigratoryzones andintheCP, which juststarts Inthecontrolslices,weobserved bothmultipolarand bipolar 2004). layer Vneuronsasdescribedpreviously (HashimotoandMikoshiba, mostly atthedorsal telencephalonandlabelled infection occurred Onthisschedule,adenoviral daysafteradenoviral infection. from 2 for48hours infection systemandrecordedmigratorybehaviour We introducedCAG-Lyn-Venusstage. atE12.5usingtheadenoviral neurons. Fig. 4.Time-lapseanalysisofmigratorybehaviour analysis ofmigratorybehaviours of Therefore,wefocusedonthe ofcorticogenesis. stages advanced brain sliceswereparticularlysensitive to mechanical stressinthe Cdk5 in We carriedouttime-lapse recordingsofmigratorybehaviour neurons of Time-lapse imagingofmigratorybehaviour bipolar transitioninthepremigratoryzone. indicate thatneuronsspecifically requireCdk5formultipolar-to- Theseresultsclearly bipolar inthosewithCGC-Cdk5-DN(Fig.3C). multipolarneuronsbecame only7.75%(s.d.3.77)of whereas positive neuronsbecamebipolarinthebrainsliceswithCAG-EGFP, hours,40.15%(s.d.14.25)ofmultipolarGFP- Within 20 material). inthesupplementary (Fig.3A,B;seeMovie 1 24hours at enhanced shown foreachgenotype.*, morphology andbehaviourat2448hours,thepercentage is positive neurons were classifiedasmultipolarorbipolarbytheir culture. Scalebar10 hours (24h)and48(48of E14.5. Representative imagesare shownofmigratingneurons after24 Adex-CAG-Lyn-Venus atE12.5,andbrainslicecultures were startedat ;seMve2i h upeetr aeil.Time-lapse imaging inthesupplementarymaterial). 4; seeMovie 2 hours (Fig. GFP-positive cellsremainedinmultipolarshapesat48 –/– brain slicesfromdifferent stagesofcorticogenesis. ( A ) Cdk5 +/+ and Cdk5 P <0.01. –/– Cdk5 mouse embryoswere infectedwith –/– Cdk5 lcs ahigherpercentageof slices, –/– Development 134(12) neurons atanearly ␮ Cdk5 Cdk5 m. ( B –/– Cdk5 ) GFP- –/– –/–

DEVELOPMENT Emx1 neuronal differentiation inmigrating To evaluate consequences ofdefective neuronaldifferentiation. polarities ofcerebralcorticalneuronsdescribedherearesecondary 2001), itispossiblethatthedefectsinradialmigrationandcellular Cdk5 isinvolved inneuronal differentiation (Dahvan andTsai, (CxCdk5KO) miceweregenerated as Cortex-specific (Cappello etal.,2006). expressed inthecerebralandhippocampalcorticesasearlyE10 in we cannotexamine thepostnatalmorphologyofpyramidal neurons However, cortical pyramidal neuronsinthepostnatalcerebralcortex. upeetr aeil.Teerslsindicatethatdefective observed inmigrating morphology isauniquephenomenon results These supplementary material). uigrda ernlmgainin during radialneuronalmigration neuronal migration. stageof neurons frommultipolartobipolarshapeseven attheearly differentiation ofmigrating oprbet thosein comparable to Cdk5 We foundthatmultipolar-to-bipolar transitionofcorticalneuronsin specific cortex- of ofCdk5inthedevelopingcerebral cortex Loss and bHLH genesthatupregulate theexpression ofdoublecortin( neurogenesis andneuronaldifferentiation isinducedbyproneural A recentstudysuggestedthecouplingofneuronalmigrationwith Neuronal differentiation of neurons invivo. Cdk5 generatedfrom difference inthepercentageofpyramidal neurons andobserved no isolated fromthecerebralcortex ofE14.5brains ofcorticalcells weconducteddissociatedcultures definition, morphology ofpyramidal neuronsat3DIV. Usingthesame of culturedcorticalneuronsfromratcerebralcortex exhibit the thisdefinition, they foundthatabout45% from thecellbody;with graduallytapersoff singledendritethat a asaneuronwith neuron pyramidal a (Threadgilletal.,1997)defined Threadgilletal. brains. of early-stage Cdk5-dependent acquisitionofpyramidalmorphology Cdk5 ots hs eclue corticalneuronsfrom wecultured To testthis, that Cdk5isrequiredfornormaldevelopment ofpyramidal neurons. euiie the utilised we To deletethe knockout system. cortex ofthepostnatalmousebrain,weusedCre/loxPconditional In ordertoinvestigate theCdk5loss-of-functionphenotypein Cdk5-deficient cortexinthepostnatalstage by defective neuronaldifferentiation. migration defectsinCdk5-deficient corticalneuronsarenotcaused RA in mRNAs fortheexpression of hybridisation in situ RAepeso eesin levels expression mRNA pyramidal neuronswiththosefrom andcomparedthe brains d5poenwsosre inthecerebral cortex fromCxCdk5KO Cdk5 proteinwas observed subjected towesternblotanalysis (Fig.6).Asubstantialdecreasein andhomogenisedbrainsampleswere dissected outfromthebrain, andP10.Thecerebralcortex was E15.5, postnatalday2(P2) CxCdk5KO mice, wedeterminedthelevels ofCdk5protein at Cdk5 eas hr r opooia defectsinpyramidal neurons Because therearemorphological To confirm thelossofCdk5expression inthecortex of p35 –/– +/+ –/– Cre –/– iewas impairedandthismightaffect themorphologyof mice /+ mro 4.% ..52 t3DV(e i.S inthe Fig.S2 (see embryos (46.2%,s.d.5.2)at3DIV embryos (45.8%,s.d.5.7)comparedwiththosefrom RA nmgaignuos(ee l,20) Because mRNAs inmigratingneurons(Geetal.,2006). Cdk5 ; Cdk5 mice becausethey dieperinatally(Ohshimaetal.,1996). Cdk5 conditional knockoutmice +/– Cdk5 +/+ Emx1Cre with and –/– fCdk5 Cdk5 Cdk5 embryos revealed impairedtransitionof Cdk5 mouse, inwhichCrerecombinaseis Cdk5 –/– +/+ / fCdk5 Cdk5 embryos atE16.5. Cdk5 ern Fg ) hrfr,initial Therefore, neurons (Fig.5). –/– –/– neurons intheSVZandIZwere gene inacortex-specific manner, Cdk5 Cdk5 mice. ern cusnral,and neurons occursnormally, –/– Emx1 neurons invivo –/– –/– Neurod1 Cre/+ Cdk5 neurons, weperformed embryos, itispossible Cdk5 ; fCdk5/– Neurod1 conditional KO , –/– Dcx embryonic by mating and and Cdk5 Cdk5 Dcx Dcx p35 +/+ –/– ) Cdk5 aeil,adde betweentwo andthreeweeksofage. material), anddied inthesupplementary the earlypostnataldays(seeFig.S3A in beingsignificantly lower theirbodyweight retarded growth, with 1.38±0.04, CxCdk5KO, control,1.32±0.84; atday ofbirth; and littermatecontrolsatP0(i.e. difference inbodyweightwas evident betweenCxCdk5KO mice MostCxCdk5KO micesurvived theirperinatalperiodandno inset). from whichCdk5was notremoved inCxCdk5KO mice(Fig.6C, positive interneuronsthatderived fromtheganglioniceminence, theseareas,wedetectedCdk5- hippocampal cortices.Throughout includingthecerebraland expressing Crerecombinase, revealed alossofCdk5immunoreactivity inthespecific brainareas P nst yrdsto withlayer markers in situhybridisation weconducted mice, the inverted natureofthecortex ofCxCdk5KO confirm mice (Fig.7A).To in’) inthecerebralcortex ofCxCdk5KO neurons withthesamebirthdate was completelyinverted (‘outside- However, thedistribution of (Fig.7A). ‘inside-out’ manner an in adistribution peak andformed neurons ateachbirthdatesegregated embryos ofE12.5toE16.5and analysedatP10.Inthecontrol, carrying BrdUwas injectedintopregnant females BrdU (Fig.7A). date,weperformedserialbirth-datingby according totheirbirth structures (Fig.6D).To analysethepositioningofcorticalneurons were scatteredthroughoutthehippocampuswithoutforminglayer formpyramidal celllayersofCA1toCA3 neurons thatnormally Inthehippocampus, inthesupplementarymaterial). Fig. S3B,C (see mice observed intheCxCdk5KO thecerebralcortex was also of Limitedposteriorextension neurons was observed (datanotshown). not changed(Fig.6C,D)andnosignificant loss of neocortical inCxCdk5KO mice,althoughthecerebralwall thicknesswas cortex Nissl stainingrevealed adefective layerstructureofthecerebral mice CxCdk5KO Inverted cortexin (s.d. 0.08), CxCdk5KO withameanof0.189 (s.d.0.14); 1 withmeanof miceatP10[Fig.6B;Cdk5/actincontrol CxCdk5KO protein inthehippocampusaswellcerebralcortex of We alsodetectedlow levels ofCdk5 mice atE15.5andP2(Fig.6A). and hybridisationinthecoronal sectionsfrom Fig. 5.Insitu disturbed in premigratory zoneof doublecortin probes. 00] muoitceia nlsso h xd5Obrain ImmunohistochemicalanalysisoftheCxCdk5KO <0.01]. Cdk5 +/+ embryos, indicatingthatinitialneuronal differentiation isnot –/– Cdk5 mouse embryosatE16.5using –/– mice. P, pia;V, ventricle.Scalebars:100 Cdk5 The expression ofthesegenesinthe –/– n 5.However, CxCdk5KO micehad =5). embryos wascomparabletothatin RESEARCH ARTICLE Foxp2 Neurod1 , Er81 ( Etv1 and ␮ Cdk5 m. – Mouse 2277 +/+ n =4,

DEVELOPMENT confined layerinCxCdk5KOmice.Scalebar:100 (W.M.) observedinthecontrol were notevidentinthecerebral cortexinCxCdk5KOmice.Inthehippocampus,pyramidalneurons sagittal sectionsrevealed abnormallaminarstructure inCxCdk5KOmice.Inthecerebral cortex,cell-sparsestructures oflayer expressed; theseare alsoGABApositive(datanotshown).Scalebar:200 hand panelreveals thepresence ofCdk5-positiveinterneurons,whichderivefrom theganglioniceminencewhere Cre recombinase was missinginthecerebral cortexandhippocampusfrom CxCdk5KOmice.Highermagnificationofthearea indicatedbythearrow CxCdk5KO miceatP10withanti-Cdk5antibody. Cdk5stainingwasobservedinallregions ofthecontrol mousebrainatP10.This inverted intheCxCdk5KO cortex.Scalebar:100 the control, motor cortex(a)andsomatosensory cortex(b)ofCxCdk5KOmice.( resulting inanou generation ofthelaminarstructure inthecerebral cortexofcontrol wasinverted, miceatP10. Thispattern Birth-datingBrdU labellingfrom E12.5to E16.5revealed atypicalinsid percentage ofBrdU-positive cellsineachbinisshown. Thecorticalmantlewasdividedintotenequallyspaced bins,and (Cont.) andCxCdk5KOmice(CxCdk5KO) atindicatedtimepoints. confirmed incerebral cortexatP10.Mean±s.d.; Cdk5 protein level(Cdk5/actin)was i.7 InvertedcortexinCxCdk5KO mice. Fig. 7. positive neuronswerepositionedinlayerV, and whereas layer, were distributed inlayerVI,thedeepest 2003; Arberetal.,2000;Nieto2004), Inagreementwithprevious reports(Ferlandetal., Informatics). and Genome Informatics) blot.( by western (BS) andanalysedforCdk5actin (Hipp), thalamus(Th),andbrainstem cerebral cortex(CC),hippocampus brains were separatedintothe regions (ex)atE15.5andP2.AtP10, compared withthoseinotherbrain cerebral cortex(cc)were low . Cdk5protein levelsinthe blotanalysisforCdk5 western indicated stagesandsubjectedto or postnatalmousebrainsatthe region were dissectedfrom embryonic analysis. Samplesfrom eachbrain blot was confirmedbywestern the cerebral cortexandhippocampus knockout mice. Fig. 6.Cortex-specific 2278 oto ie(i.7) However, intheCxCdk5KO cerebralcortex, control mice(Fig.7B). neurons wereinsuperficial layersIIandIIIinthecerebralcortex of RESEARCH ARTICLE Cux2 -positive, ( B A ) Reductioninthe ) LossofCdk5in Cdk5 Er81 -positive and Cux2 ( Cutl2 Foxp2 ( A Foxp2 n ) BindistributionsofBrdU-positive cellsshownasmeanpercentage ofthree mice forcontrol ␮ =6; *, – MouseGenome ␮ m. -positive neurons accumulatedinlayerII/III, layerVandVI,respectively. was Thispattern m. -positive neurons P Cux2 <0.01. ( -positive C Er81 ) Immunostainingofthecoronal sectionofthecerebral cortexfrom control and B ) Insituhybridisationstudywithlayer markers - ␮ m; inset,100 n 3 AtE17.5,GFP-positive neuronsinCxCdk5KO embryoshad and P3. introduced atE14.5,andfixed sectionswere examined atE17.5,P1 TheCAG-EGFP plasmidwas cortex ofCxCdk5KO mice. analysed thepositionandmorphologyofneuronsincerebral of thecerebralcortex layerstructureinCxCdk5KO mice. aninversion Theseresultsconfirmed occupied thedeeperposition. Foxp2 Using inuteroelectroporationandGFPimmunostaining,we -positive neuronsoccupiedasuperficial positionand ␮ .( m. D ) Comparisonofcerebral cortexinNissl-stained Cux2 , Er81 Development 134(12) I(I)andwhitematter tside-in pattern inthe tside-in pattern e-out pattern of e-out pattern and stainingpattern failed toforma Foxp2 is not the in theright- at P10.In Cux2

DEVELOPMENT was observed: asingleapicaldendrite extending toward thepiawith the controlmice,atypicalpyramidal morphologyoflayerVneurons In cortex ofCxCdk5KO mice. pyramidal neuronsinthepostnatal themorphologicalfeaturesofCdk5-deficient this strategy toanalyse We applied soma andaxonmorphologiesof theseneuronsindetail. observe dendrite, YFP-H lineafterP10(Fengetal., 2000),wecould cortex, aswellthosein thehippocampus,areYFP-positive inthe pyramidal neuronsofasubsetlayerVinthe cerebral Becausethe hippocampal pyramidal neurons(Ohshimaetal.,2005). the detailedmorphologyof double-transgenic micetovisualise etil ieo h otx We previously generated ventricle sideofthecortex. towards thepiasurface andasingleaxonthatextends towards the have abipolarshapewithsingleapicaldendritethatextends Pyramidal neurons,typicallylarge pyramidal neuronsinlayerV, neurons Lack ofCdk5results inabnormaldendritesoflayerV identical abnormalitiestothosein Cdk5-dependent acquisitionofpyramidalmorphology with aconfocalmicroscope. Scalebar:20 positive cellsinthecerebral cortexofCxCdk5KOmiceatP3obtained ␮ positioned deepdownandhadmultipolarmorphology. Scalebars:100 antibody (green). GFP-positiveneurons inCxCdk5KOmicewere plasmid. Brainswere fixedatE17.5,P1orP3andstainedwithanti-GFP littermate controls whichwere electroporated at E14.5withCAG-EGFP immunostaining ofcoronal sectionsfrom CxCdk5KOmiceandtheir cerebral cortexofcontrol andCxCdk5KOmice. Fig. 8.MorphologiesofGFP-labelledneurons atE14.5inthe of thedendriticstructures. migration resultedindefective pyramidal morphology, particularly inability ofneuronstoacquirethebipolarshapeduringradial Theseresultsindicatethatthe (Fig.8). the multipolarmorphology CxCdk5KO micewerelocalisedatthebottomofcortex andhad Bycontrast,GFP-positive neuronsin pyramidal morphology. typical the littermatecontrolswerepositionedatlayerII/IIIwith AtP1andP3,GFP-positive neuronsinthesectionsfrom (Fig. 1). m. Thebottom-rightpanelisahighermagnificationimageofGFP- Cdk5 ␮ –/– m. mice asdescribedabove GFP p35 –/– ;YFP-H bv dt o hw) oee,lble Cnuoshdan CCneurons had However, labelled above (datanotshown). and theseneuronshadtypicalpyramidal morphologyasdescribed byDiI, the control,mainlylayerII/IIIpyramidal neuronswerelabelled In labelling. neurons inCxCdk5KO miceatP10usingretrogradeDiI layer Vneurons. mice arenotasecondaryconsequence ofectopiclocalisation result indicatesthatabnormaldendriticstructuresintheCxCdk5KO This inthesupplementarymaterial). ectopic localisation(seeFig.S4 morphology inthecerebralcortex of Importantly, layerVpyramidal neuronspreserved pyramidal layer Vneurons,similartoprevious reports(Terashima etal.,1983). anabnormalorientationofapicaldendrites We observed mice. we analysedthemorphologiesoflayerVneuronsin abnormalities andtheectopicpositionoflayerVpyramidal neurons, To elucidatetherelationship betweendendritic cortex (Fig.9B). the axonwas notconsistentandaxonsranobliquelywithin the but theexit siteof CxCdk5KO mice;eachneuronhadasingleaxon, abnormal axonaltrajectorywas alsoobserved inthecortex of Furthermore,an extended directlyfromthesoma(Fig.9B). dendrite was difficult toidentify; instead,multipledendrites InthemajorityoflayerVneurons,apical cortex (Fig.9B). inadditiontotheirinverted positioninthe was largely abnormal, in CxCdk5KO mice,thedendritemorphologyoflayerVneurons However, the ventricular sideandrunningstraightdown (Fig.9A). secondary branches,basaldendritesandasingleaxonexiting from (arrowheads) are present inCxCdk5KOmice.Scale bar:50 Abnormal dendriticstructures (arrows) andaxonaltrajectories control (Cont.;YFP-H)andCxCdk5KO(CxCdk5KO;YFP-H)miceatP14. Confocalimagesofthecerebral cortexof black; CxCdk5KO,grey). (B) P14. BindistributionsofYFP-positivelayerVneurons are shown(control, cerebral cortexincoronal sectionsfrom control andCxCdk5KOmiceat CxCdk5KO;YFP-H double-transgenicmice.(A)Typical examplesofthe ( Fig. 9.DefectivedendriticdevelopmentinCxCdk5KOmice. A Positionand( ) efrhraaye h opooyo alslcmisrl(CC) We furtheranalysedthemorphologyofcallosalcommissural B ) detailedmorphologiesoflayerVneurons in RESEARCH ARTICLE reeler mice, despitetheir reeler ␮ m. ;YFP-H 2279

DEVELOPMENT enreportedin been expression ofMap2has reduced CxCdk5KO mice(Fig.11A,B),as Informatics) protein,adendritemarker, inthecerebralcortex of ie(i.1A.Themorphologyof layerVIneurons,whichextend mice (Fig.11A). wherelayerVIneuronsexisted inCxCdk5KO the superficial layer, Instead,Map2-positive stainingwas detectedin CxCdk5KO mice. was missinginthecerebralcortex of radial stainingofMap2 Such were evident inthecerebralcortex ofthecontrols(Fig.11A). Map2-positive apical dendrites cerebral cortex ofCxCdk5KO mice. inthe pyramidal neuronsareassociatedwithalower level ofMap2 pyramidal neurons,i.e.thatdefectsinthedendritestructuresof reflect thedevelopmental deficit ofthedendriticstructure 0.06), (s.d. 0.78 withmeanof CxCdk5KO versus 0.12) control withmeanof1(s.d. was notchanged[NF-M/actin Nefm –MouseGenomeInformatics) (neurofilament, medium polypeptide;alsoknown as level ofNF-M 0.16), (s.d. with ameanof0.04 CxCdk5KO withameanof1(s.d.0.10)versus Map2/actin control [Fig.11B; withthedecreased proteinlevel ofMap2 11A), consistent staining ofMap2 decreased inthecerebralcortex ofCxCdk5KO miceatP10(Fig. Overall, 2005). neurons (CiceroandHerrup, contralateral cerebralcortex in abnormal dendriticmorphologyandaxonaltrajectoryinthe wealsofoundan ByDiIlabelling, cortex inCxCdk5KO mice. the throughout abnormal morphologyofpyramidal neuronsoccurred Theseresultsindicatethatthe in CxCdk5KO;YFP-H mice(Fig.10A). abnormal morphologyinCxCdk5KO mice,similartolayerVneurons 2280 eas tde h ee fteMp (Mtap2–MouseGenome We alsostudiedthelevel oftheMap2 RESEARCH ARTICLE n Cdk5 =4, P –/– 005,teseii eraei a2might =0.055], thespecific decreaseinMap2 embryos atE16.5andincultured Cdk5 n =4, –/– P mice atE18.5(Fig.10B). 00] Becausetheprotein <0.01]. Cdk5 –/– observed in (Fig. 2A branchedleadingprocesses Interestingly, someoftheseneuronshad (Fig.2). CP the became bipolar, but theirmigrationwas delayedwithin When weuseda50%(1:1)dilutionofCdk5-DN,someneurons EGFPandCdk5-DNplasmid. usedanequalratioof Hatanaka etal. DN afterthey becamebipolar(Hatanakaetal.,2004). However, expressing Cdk5- neurons found asubstantialdelayinthemigrationof of neuronsexpressing Cdk5-DNdiffers fromaprevious studythat layer, preserved thepyramidal morphologyinthe althoughplacedinthesuperficial we foundthatlayerVIneurons, Asinourprevious report(Gilmoreetal.,1998), withDiI. labelling their axonstoward thedorsalthalamus,was examined byretrograde morphology ofmigrating result isconsistentwithourobservation ofthebehaviour and aswellinvivo (Fig.2).This (Fig.3), study the time-lapseimaging in of multipolar-to-bipolar transitioninneuronsexpressing Cdk5-DN Cdk5usingCdk5-DN,weobserved asevere impairment suppressed multipolar tobipolarduringradialneuronalmigration.Whenwe Cdk5 ismainlyinvolved in thetransitionalphaseofneuronsfrom wereportthat et al.,1996;Gilmore1998).Inthepresentstudy, (Ohshima neuronal migrationofcorticalneuronsinthecerebralcortex Cdk5isrequiredfortheproper We have previously shown that DISCUSSION P10 (Fig.11D). at mice pyramidal morphologyinthecerebralcortex inCxCdk5KO We alsofoundthatlayerVIneuronshadtypical brain (Fig.11C). extended theirapicaldendritestoward thepia.Bycontrast, commissural neurons. abnormaldendritestructures of confocal imageof neurons extendedtheirdendritesinmultipledirections. Inset, and DiIlabellingofcommissuralneurons inP10CxCdk5KO Fig. 10. Scale bar:25 dendrite developmentofcommissuralneurons inCxCdk5KOmice. a fluorescent (A,B)orconfocal(insetinB)microscope. (A)Abnormal littermate controls. sections(100 Vibratome fixed brainsof( extended theirapicaldendritetoward thepiain ( of NF-M/actinwasnotsignificantly altered inCxCdk5KOmice. mice was also confirmed by western blotting. *, mice wasalsoconfirmedbywestern CxCdk5KO miceatP10.Thereduction inMap2/actinCxCdk5KO were examinedinthedissectedcerebral corticesfrom control and the CxCdk5KOcortex.( stainingofMap2immunoreactivity was reduced in arrows. Overall, Insets are highermagnificationsoftheareas indicated bythe the superficiallayerofCxCdk5KOcortex(inset,arrowheads). missing, withonlyafewMap2-positiveradialdendritesobservedin Map2-positive inthecontrol (inset,arrowheads).was This pattern mice. Apicaldendritesofpyramidalcorticalneurons were typically defective dendritestructures ofpyramidalneurons in CxCdk5KO arrowheads indicatetheiraxons. Scalebars:100 CxCdk5KO (D)mice.Arrows indicateapicaldendritesand Cdk5-deficient brains. and unaltered pyramidalmorphologyoflayerVIneurons in ReducedMap2expression intheCxCdk5KOcortex, Fig. 11. C , D Cdk5 Cortico-thalamicneurons, corresponding tolayerVIneurons, ) Ј b, andmildermorphologicalabnormalitiesthathave been p35 –/– –/– mice. ␮ m. (B)In A ern Gpae l,20)(T.O., T.A. andK.M., neurons (Guptaetal.,2003) ) P10CxCdk5KOand( DiI crystalswere placedinthecerebral cortexof Cdk5 Cdk5 B ( ) Protein levelsforMap2,NF-Mandactin A ) Map2immunostainingrevealed –/– +/+ ern Fg ,4.Ourobservation (Figs1,4). neurons mice, corticalpyramidalneurons B ) E18.5 Development 134(12) ␮ m) were imagedusing Cdk5 Cdk5 Cdk5 P ␮ <0.01. Thelevel Cdk5 m. –/– –/– –/– –/– mice and embryonic (C) and Cdk5 –/–

DEVELOPMENT irto.Whenweculturedthe migration. ofmigrationarrestatthemultipolarstageradial simply theresult utpednrts Becausethisdefectwas observed in multiple dendrites. neurons failed todevelop anapicaldendritestructureandextended These CxCdk5KO mice. pyramidal neuronsinthecerebralcortex of 2006). LoTurco andBai, Baietal.,2003;Tsai2005; by this transition aswell(reviewed disrupts andDcx (Pafah1b1 –MouseGenomeInformatics) RNAi formigration-relatedproteinsincludingLis1 disruption, as process isconsideredastageofneocorticaldevelopment vulnerableto Recent studiesindicatethatthistransitional require Cdk5activity. rearrangement ofthecytoskeleton ofmigratingneurons andmight transition frommultipolartobipolarmightrequiredynamic Morphologicalchangeduring acquire thepyramidal morphology. loss ofCdk5doesnotseverely affect theabilityofcorticalneuronsto inthesupplementarymaterial),indicatingthat morphology (seeFig.S2 seen inthe justlike that activity causesimpairedmultipolar-to-bipolar transition, ofCdk5 Theseresultsindicatethatstrongsuppression unpublished). Cdk5-dependent acquisitionofpyramidalmorphology opooisweobserved in morphologies Theseobservations alsosuggestthatthemultiple processes. have branchedleading although thesebipolarneuronssometimes radial migrationwithintheCPafteracquisitionofbipolarshape, migration ofcerebral corticalneuronsinthemultipolar morphology premigratory zone(Baietal.,2003). InhibitionofRac1arreststhe prematurearrest ofmigratingmultipolarneuronsinthe causes (Tanaka etal.,2004a).siRNA ofDcx association withmicrotubules organise thelinkbetween centrosomeandnucleusthroughtheir to Dcx andFak1 atCdk5-phosphorylation sitesisconsideredimportant Niethmmer etal.,2000;Xie 2003).PhosphorylationofNudel, (Tanaka etal.,2004b;Sasaki etal.,2000; Genome Informatics) (Ndel1 –MouseGenomeInformatics)andFak1 (Ptk2–Mouse Nudel includingDcx, arerelatedtoneuronalmigration, substrates that Recentstudies identified Cdk5 migration ofcorticalneurons? neuronal migration. related tothetransitionaldefectfrommultipolarbipolarduring CxCdk5KO miceiscell-autonomousandtightly abnormality in Therefore,weconsiderthatthemorphological mice (datanotshown). inpostnatal-stageCxCdk5KO any significant lossofcorticalneurons notdetect However, wedid exhibit dendriteabnormalities. neurons andtheremaining Cdk5 causesneuronalapoptosis(Lietal.,2002), of abnormal dendritestructureinCxCdk5KO miceisthattheabsence Anotherpossibleexplanation for inthesupplementarymaterial). S4 layer Vneuronseven intheirectopicpositionsthismutant(seeFig. ispreserved in withasingleapicaldendrite, pyramidal morphology, However, ouranalysisof above. responsible fortheabnormalitiesofdendritestructuresdescribed of Cdk5-deficient pyramidal neuronsmightbeatleastpartially Therefore, wecannotexclude thepossibilitythatectopic position important roleinthesculptingofmatureneuronaldendritictree. afferent inputplaysan Itisknown thatintrinsic unpublished). brain areconsistentwiththeseresults(T.O., T.A. andK.M., preliminary resultsfromGolgistainingoftheCxCdk5KO mouse Our correspondingtolayerII/IIIandV (Fig.10A). neurons throughout thecortical abnormal pyramidal morphologyoccurred indicatedthat Ouranalysis withDiIlabelling radial migration. inability ofneuronstoacquireabipolarmorphologyduringtheir 8, 9),itislikely thatthisabnormalityoccursasaconsequenceofthe aswellinCxCdk5KO micepostnatally(Figs at E18.5(Fig.10B), cortex, however, The mostinterestingfinding istheabnormalmorphologyof Why doesCdk5deficiency causesubstantialdefectsintheradial Cdk5 –/– Cdk5 ie n oeaesprsinofCdk5delays mice, andmoderatesuppression –/– ern eeabletoacquirethepyramidal neurons were Cdk5 reeler Cdk5 –/– ieidctsthatthetypical mice indicates –/– irtn ern eenot migrating neuronswere neurons fromthecerebral Cdk5 –/– mice ern inaCdk5-dependentmanner. neurons this radialaxisisbroughtabout bytheactive radialmigrationof andthat and extend singleaxons towards thebottomofcortex, in whichneuronsextend theirsingleapicaldendritetoward thepia, into abipolarshapeisessential toformthepyramidal morphology mouse models,weproposethat thetransitionofmigratingneurons defective migration. Basedonourfindings intheCdk5-deficient Cdk5-deficient neurons(Fig.1B;Fig.9),probablybecause oftheir (Fig.1A)andaxonaltrajectoriesareimpairedin migrating neurons from ofaxons the presentstudy, wealsoobserved thatextensions 2002).In (Noctoretal.,2004;HatanakaandMurakami, their axons recent observations suggestthatmigratingneuronsalreadyextend Inaddition, development inCdk5-deficient pyramidal neurons. bipolar morphologyduringmigrationresultedinabnormaldendrite toacquirea evidence tosupportthisview, becausetheinability Ourresultsprovide 2002). morphology (HatanakaandMurakami, pyramidal neuronstoobtaintheircorrect requirement forthe migratingneuronsislikely tobeafundamental bipolar shapein for atransitionfromtheleadingprocesstoapicaldendrite, the et al.,2003). Cdk5 ofcorticaldevelopment; therefore, intheearlystages short distance alternate explanation isthatneuronsneedtomigrate a relatively An radial migration(Nadarajahetal.,2001;Hatanaka2004). neurons usedistinctmolecularandcellularmechanismsfortheir current studysupportstheproposedideathatearly-andlater-born Inthissense,our pyramidal morphologyintheabsenceofCdk5. Thus,they canmigrateproperlyandobtaina (Noctor etal.,2004). the transitionalprocessfrommultipolartobipolarmorphology neurons usesomaltranslocationastheirmigrationmodeandskip deficiency haslittleimpactonlayerVIneurons isthatlayerVI Ourexplanation forwhyCdk5 obtain apyramidal morphology. (Fig. 11C,D)suggestthatlayerVIneuronsmigrateproperlyand experiments (Fig.7B)andDiIlabelling from insituhybridisation Ourresults migration isCdk5-independent(Nadarajahetal.,2001). correspond tolayerVI,migratebysomaltranslocationandthatthis remains tobeelucidated. al., 2005)andthepossibleinvolvement ofCdk5inthisprocess rear ofmigratingneurons(SchaarandMcConnell,2005;Bellionet involvement ofnon-muscletypemyosinIIinthenucleokinesisat Recentstudiesindicate (Tanaka etal.,2004a). and nucleus betweenthe properdistance whichmaintains a nucleokinesis, transitional phasefrommultipolartobipolar. Cdk5alsoregulates dynamics ofmicrotubules andofthecytoskeletal proteinactininthe andregulates the substrates multiple clear thatCdk5phosphorylates cells inthepremigratoryzone(Naganoetal.,2004).Therefore,itis resultsintheaccumulationofmultipolar offilamin A the expression filamin A,andadecreasein phosphorylates theactin-bindingprotein Cdk5also radial neuronalmigration(Kawauchi etal.,2006). mediated phosphorylationofp27regulates RhoAactivity during Because p27inhibitsRhoAactivity (Bessonetal., 2004),Cdk5- in thecytoplasm ofmigratingneurons(Kawauchi etal.,2006). (CdKn1b –MouseGenomeInformatics)atSer10andstabilisesp27 Interestingly, Cdk5phosphorylatesp27 al., 2005;Geet2006). radialneuronalmigration(Handet inhibited withintheIZforproper multipolar tobipolar. OtherstudiessuggestthatRhoAactivity is in migratingneuronsmightimpairtheirtransformationfrom RhoGTPase of improperregulation (Kawauchi etal.,2003);therefore, stebace hp,previously reportedin as thebranchedshape, morphology orintheincompletelytransformedbipolarshape,such Although thereisasyetnodirectevidence inmigratingneurons A previous studydemonstratedthatearly-bornneurons,which –/– ern ol eabletoreachthepiaeven inthemultipolar neurons would be RESEARCH ARTICLE p35 –/– ern (Gupta neurons 2281

DEVELOPMENT Besson, A.,Gurian-West, M.,Schmidt,A.,Hall,A.andRoberts,J.M. Bai, J.,Raddy, L.,Ackman,J.B.,Thomas,A.M.,Lee,R.V. andLoTurco, J. Cicero, S.andHerrup,K. Bellion, A.,Baudoin,J.P., Alvarez,M.andMetin,C. C.,Bornens, Arber, S.,Ladle,D.R.,Lin,J.H.,Frank,E.andJessell,T. M. Angevine, J.,JrandSidman,R.L References http://dev.biologists.org/cgi/content/full/134/12/2273/DC1 Supplementary materialforthisarticleisavailableat Supplementary material K.N.). Foundation andtheTokyo BiochemicalResearch Foundation(to theJapanSocietyforPromotion Science,theJapanBrain and byNEXT, toT.O.), MolecularBrainSciencefrom NEXTofJapan(18022050 Priority Area paper. ThisworkwassupportedbyGrants-in-AidforScientificResearch on CAG-GS plasmid;andEliasUtreras andHarryGrantforcriticalreading ofthis T. JessellandC.Walsh forinsituprobes; DrJ.Miyazakiforthe We thankDrs 2282 Hashimoto, M.andMikoshiba,K. 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