as inthe cortical plate(CP)(Rakic,2003).Ifradialglialprocessesarealtered, or (SVZ)towards theirfinalpositioninthe structures byneuronstomigratefromtheirplaceofbirthintheVZ membrane (BM).Theseradialprocessesareusedasguiding the neuraltube,attachingviatheirendfeettobasement zone (VZ)andextend longradialprocessesthroughout thewall of characteristics. Radialglialcellshave theirsomata intheventricular The morphologyofradialglialcellsisonetheirdefining INTRODUCTION KEY WORDS:Mouse,Basementmembrane,Laminin,, binding siteofthelaminin to radialglialcellsinthreemousemutantswheregliaattachmenttheBMisdisrupted.Thiscaseifnidog attached tothebasementmembrane(BM)duringcelldivision.Here,weexaminedapotentialroleofdirectsignallingfromB Radial glialcellshavebeenshowntoactasneuronalprecursorsinthedevelopingcortexandmaintaintheirradialprocesse Nicole Haubst neuronal subtypes glial cellfateandforproliferation,butaffectspositioningof Basement membraneattachmentisdispensableforradial Development 133,3245-3254(2006)doi:10.1242/dev.02486 Accepted 12June 2006 3 2 1 Norwich, UK. and Health,IngolstädterLandstr.1, D-85764Neuherberg/Munich,Germany. for adequateneuronalcompositionofthecerebralcortex. *Author forcorrespondence ataddress 1(e-mail:[email protected]) Schillerstr. 46,D-80336,Munich,Germany. cortex ofthelaminin parenchyma. Notably,however,neuronalsubtypecompositionwasseverelydisturbedatlatedevelopmentalstages(E18)inthe migration, orientationofcelldivisionandneurogenesis.Onlyasmallsubsetprecursorswaslocatedectopicallywithinthe Surprisingly, corticalradialglialcellslackingcontacttotheBMwerenotaffectedintheirproliferation,interkineticnucl been examined. process forthefate andproliferationofradialgliacellshas never potential importance,but theroleofBMsignallingvia theradialglia asymmetric inheritanceoftheradialgliaprocesshighlights its process anddevelop intopostmitoticneurons. Thus,thesupposed and colleaguesalsoobserved somecellsthatmaintaintheradial inheriting theradialprocessisandremainsaglialcell,Miyata Kriegstein (FishellandKriegstein, 2003)suggested thatthecell the radialprocess(FishellandKriegstein, 2003).WhileFishelland signals fromtheBMwould beperceived onlybythecellinheriting to onlyonedaughtercellmaybeimportantinfate decisions,as 2001; Miyataetal.,2004),andtheinheritanceofradialprocess maintain theirradialprocessduringcelldivision (Miyataetal., of theradialprocessinthisregard islessclear. Radialglialcells et al.,2003;Malatesta2000;Noctor2001),but therole al., 1998),neuronalmigrationisaffected. and Goffinet, 2003)or Biomedical Research Centre, SchoolofBiologicalSciences,UniversityEastAnglia, IGBMC, CNRS/INSERM/ULP, BP 163,67404Illkirch, CU deStrasbourg,France. Institute forStemCellResearch, GSF, NationalResearch CenterforEnvironment However, radialglialcellsalsoactasprecursor(Malatesta reeler 4 Department ofPhysiology, Ludwig-MaximiliansUniversity, Munich, (Frotscher etal.,2003;HartfussTissir 1 , ElisabethGeorges-Labouesse ␥ Pax6 1III4 –/– mutant mice(Caricetal.,1997;Götz ␥ 1 chainismutated,intheabsenceof mice. Thus,althoughBMattachmentseemsdispensableforprecursorcells,anintactisrequired 2 , AdeleDeArcangelis ␣ Lamc1 6 integrinorofperlecan,anessentialBMcomponent. developing cortex (Campos etal.,2004;Liesi,1985; Sheppardetal., present withintheintermediate andventricular zonesofthe al., 1998).However, as ␤ (Colognato andffrench-Constant, 2004);targeted deletionofeither signalling viacomponentsoftheECMaswellgrowth factors ffrench-Constant, 2004;MottandWerb, 2004).Integrins integrate is enrichedwithavariety ofgrowth factors (e.g.Colognatoand (Erickson andCouchman,2000;Paulsson, 1992;Timpl, 1996),and and heparansulphateproteoglycans,suchasperlecanagrin mainly oftypeIVcollagen,nidogen,membersthelamininfamily laminin functional roleofBMcontactforradialglialcells. defects intheglialendfeetattachmenttoBMassess the cells themselves. Here,wehave used several mousemutantswith endfeet andtheBMaffects cellproliferationorfate ofradialglial examined towhatextent thelackofcontactbetween radialglia Hartmann etal.,1999;Herms2004).However, ithasnotbeen et al.,2005)(seealsoBlackshear1997;Costa2001; 1998; Graus-Portaetal.,2001;Halfter2002;Niewmierzycka subarachnoid space(Beggs etal.,2003;Georges-Labouesse etal., cobblestone , withcorticalneuronsprotrudingintothe integrin-mediated binding.RuptureoftheBMthencauses typeII endfeet contribute totheformationandmaintenanceofBMby Niewmierzycka etal.,2005).Thus,radialgliaandlaterastrocyte Graus-Porta etal.,2001;Halfter2002;Mills2006; integrity (Beggs etal.,2003;Georges-Labouesse etal.,1998; BM endfeet totheBMandtherebyalsodisruptsmaintenanceof integrin-linked kinase(ILK)abolishestheattachmentofradialglial observed inthecortex of (Halfter etal.,2002).Asimilar phenotype ofrupturedBMwas also longer connectedtotheBMin thecortex ofthismousemutant by DiI-tracingofradialglialcells, mostoftheirprocessesareno brain (Halfteretal.,2002;Willem etal.,2002). Aspreviously shown with disintegration andruptureoftheBMinlung,kidney and 1 or The BMisathinsheetofextracellular matrix(ECM)composed The targeted deletionofthe nidogen-bindingsitewithinthe , ␣ Itaga6 6 integrin orthe ␥ 1 chain, , 2 Hspg2 , UlrikeMayer ␥ 1III4, resultsinnidogendepletionfromtheBM ␤ ␣ ␣ 1 integrin cytoplasmic tailbindingprotein 6 6 integrin ␤ 1 integrin bindstolaminin thatisalso 3 and MagdalenaGötz RESEARCH ARTICLE –/– mice (Georges-Labouesse et ear en- cortical 1,4, s 3245 * M

DEVELOPMENT to examine wild-type,heterozygous( mice [thedayofthevaginal plugisconsideredembryonicday(E)0]allowed (Costell etal.,1999)onC57Bl/6background.Crossingofheterozygous al., 1998)onC57Bl/6/SV129backgroundandperlecanheterozygousmice SV129 background, Student’s al., 2006).Allvalues aregiven ±standarddeviation (s.d.)andunpaired ventricular surface usingImageJasdescribedbyStricker etal.(Stricker et (CP:Ctx) was calculated. (CP; Fig.3C)was measuredwiththeneurolucidasystemandratio and thebandofMath2-positive neuronsdelineatingthecorticalplate ( 2004). Math2 andEr81wereobtainedfromC.Schuurmans(Schuurmansetal., described (Chapoutonetal.,2001),andtheprobesforCux2,Rorb(ROR by propidium-iodidestaining(PI).Insituhybridizationwas performedas Neurolucida SystemandtheApotomSystem.Cellnucleiwerevisualized (Leica TCS4NT; OlympusFV1000)andwiththeZeissAxiophotusing (Polysciences, Northampton,UK)andanalysedwithaConfocalMicroscope Jackson ImmunoResearch.SpecimensweremountedinAquaPoly/Mount secondary antibodieswerefromSouthernBiotechnologyAssociatesand mouse IgG1,1:500,kindlyprovided byAndréGoffinet). Therespective Heintz), nestin(mouseIgG1,1:4,Dev. HybridomaBank)andreelin(E4, provided byP. Leprince),BLBP(rbt,1:1500,kindlyprovided byNathaniel Price), GFAP (mouseIgG1,1:200,Sigma),RC2IgM,1:500,kindly IgG2a, 1:100,Sigma),O4(mouseIgM,1:1000,kindlyprovided byJack Tec-3, 1:50,Dako), pan-Laminin(rbt,1:50,BD), calbindin (rbt,1:2000,SWANT), calretinin(rbt,1:2000,SWANT), Ki67(rat (rbt); Biomol;1:200),BrdU(mouseIgG1,1:10,BioscienceProducts), antibodies againstthephosphorylatedformofHistoneH3(PH3)(rabbit after cryoprotection.Sectionswereimmunostainedusingtheprimary buffered-saline (PBS)and12 Embryonic brainswerefixed in4%paraformaldehydephosphate- Immunohistochemistry andin-situhybridization Laminin Animals ␣ 1995), defectsmayalsoarisewithinthecorticalparenchymaof 3246 mutants atembryonic day(E)14,thepeakofradial -mediated endfeet attachmentinthecortex ofthethreedifferent mouse First, weexamined thedegree ofBMruptureandlossradialglia detachment Degree ofBMdisruptionandradialgliaendfeet RESULTS and calculatedasPH3-positive cellsper100 at theventricular surface andatabventricular positionswerethenquantified size ofthequantificationarea.Inthisarea,numbersPH3-positive cells neocortical areawas outlinedusingtheneurolucidasystemtodetermine at abventricular positions(SVZ)(Fig.2C).Inasecond setofexperiments, the per sectionandcalculatedthepercentagesofPH3-positive cellsintheVZand located five ormore celldiametersfromtheVS)(seealsoHaubstetal.,2004) ventricular surface (VS)and atabventricular positions(PH3-positive cells one setofexperiments, wecountedthenumberofPH3-positive cellsatthe levels. respective mutant littermatetelencephaliatrostral,intermediateandcaudal All quantificationswereperformedonfrontalsectionsofwildtypeandthe Quantification with deletionofamoleculerestrictedtotheBM,perlecan MATERIALS ANDMETHODS et al.,1999)(seealsoArikawa-Hirasawa etal.,1999). –/– 6 integrin The anglesofcelldivision inlateana-andtelophaseweremeasuredatthe For theanalysisofneurogenesisradialwidthentire cortex (Ctx) Two different approacheswereusedtoquantifythePH3-positive cells:In ) littermates. RESEARCH ARTICLE ␥ t -test was usedtotestforsignificance. 1III4 (Willem etal.,2002)heterozygousmicewerekept ona –/– mice. We thereforeexamined afurthermousemutant ␣ 6 integrin heterozygousmice(Georges-Labouesse et ␮ m frontalsectionswerecutwithacryostat +/– ) andhomozygousmutantembryo ␮ m 2 (Fig. 2D)atE14. ␤ III-Tubulin (mouse –/– (Costell ␤ ), integrin somata withinthecorticalplateoflaminin this mutant.We firstnoted BLBP-andRC2-immunopositive cell (see below) weredetectedwithintheCPoflaminin immunoreactive forBLBPorRC2ofectopicclustersprecursors pial celllayers(Fig.1F,H). Onlyfew shortdisorganized processes cells thatarealsoRC2-immunoreactive werestillvisiblewithinthe contact withthepialsurface (Fig.1H),even thoughmesenchymal large regions withfew ornoRC2-positive radialglia processesin Double-labelling withRC2(Hartfussetal.,2001)furtherrevealed Ectopic precursor clusters inthecortexoflaminin visible (Fig.1J). laminin the pialmembranes(Fig.1E,G),whereasinlarge regions oftheE14 visualizes theradialgliaendfeetasacontinuousbandunderlying (Hartfuss etal.,2001).Inwild-typecortex, BLBPimmunoreactivity (BLBP), amoleculecontainedinthecytoplasm ofradial glialcells endfeet byimmunolabellingofthebrain-lipid-bindingprotein al., 1999). disruptions intheBM(Georges-Labouesse etal.,1998;Costell are consistentwithprevious electronmicroscopic observations of precursors (Gerlach etal.,1997)(Fig.2A,B). Theseectopic antigen presentinallphasesof thecellcycle inactively dividing phase ofthecellcycle (Hendzel etal.,1997),andagainstKi67,an phosphorylated formofhistone H3(PH3)presentintheG2/M- continued todivide, asevident fromimmunostainingagainstthe 1E). Theseclustersofectopic BLBP-immunopositive cells littermates whereallradialglia somata werelocatedintheVZ(Fig. arrows) thatwerenever observed inthecortex ofwild-type (Fig. 1C,c).Inthe material) andthishemispherehadpartialdisruptionsoftheBM also Costelletal.,1999)(seeFig.S2A,Binthesupplementary perlecan Similarly, radialgliaendfeetwerevirtuallyabsentinthemedial perlecan SV129 background,seeMaterialsandmethods].Amongthe20 mixed background,whereasthemiceinthisanalysiswereonapure al. (Halfteretal.,2002)performedtheiranalysiswithmiceofa analysis, presumablyowing tobackgrounddifferences [Halfteret this studyappearedslightlymorewidespreadthanintheprevious disruption inthelaminin component oftheBM,revealed themostsevere phenotypeinBM . Immunohistochemistryagainstlaminin,amajor ␥ Given theseverity ofthe BMphenotypeinthecortex oflaminin midneurogenesis E14inthelaminin broad disruptionsoftheBMoverlying thecerebralcortex by widespread lackofpialendfeetradialglialcellsisduetothe previous data(Halfteretal.,2002),clearlydemonstratethatthe ventricular zones(Fig.1E-H).Thesedata,together withthe processes was stillmaintainedwithintheintermediateand cortices (Fig.1H),whiletheradialorganization ofradialglia the lateralcortex ofthesemice(datanotshown). IntheE14 These defectsoftheBMinperlecan overlying theneuroepitheliumwas largely absent(Fig.1D,d). had onlysmallpunctuatedisruptions,whereastheBMdirectly partial BMdisruptionsinthelaminin reduced inthecortex ofmutantmice(Fig.1A,B,a,b).Notably, the the BMandlamininstainingalongbloodvessels strongly ␥ 1III4 1III4 We furtherexamined thelocalizationofradialglialprocessesand –/– –/– –/– ␥ –/– –/– we focusedouranalysisofproliferationandcellfate on 1III4 cortex, gapsinradialglialendfeetliningthesurface were cortex (Fig.1I),whereasthey appearedlessdisrupted in embryos, onlyonedidnotsuffer fromexencephali (see –/– cortices, nosuchbandwas detectable(Fig.1F-H). ␣ 6 integrin ␥ 1III4 –/– –/– cortex theouterlayerofBM , withwidespreaddisruptionsof ␥ ␥ 1III4 –/– 1III4 and Development 133(16) –/– –/– ␥ ␣ cortex (Fig.1B,b). 1III4 6 integrin cortex analysedin –/– (Fig. 1F,H, –/– ␥ 1III4 cortex ␣ –/– 6

DEVELOPMENT Basement membraneattachment precursor cellsintheCPoflaminin ( oligodendroglial markers GFAP, O4orNG2,neuronal markers continued todivide untilE18but didnotacquiretheastroglialor as RC2ornestin(seeFig.S1in thesupplementarymaterial).They dividing precursors alsocontainedotherradialglialmarkers, such with theirBLBPandPax6 immunoreactivity, theseectopically precursor cells(Englundetal., 2005; Götzetal.,1998).Consistent neuroepithelial/radial glialprecursorsintheVZbut notinSVZ Pax6 (datanotshown), whichisnormally expressed onlyin ␤ III-tubulin, NeuN). ␥ 1III4 –/– cortices alsocontained ␥ small proportionofectopicallydividing precursorsinthelaminin ␣ phase (BrdUpositive) were co-labelledwithPH3,which is laminin surface oftheVZ,whereS phasetakes placeinbothwild-typeand phase andresultedinabandof BrdU-labelledcellsatthebasal deoxyuridine (BrdU)0.5hour priortosacrificelabelscellsinS cycle. Injection oftheDNA-base analogue5-bromo-2 cell BLBP-positive cellsomatainthelaminin laminin To determinetheextent ofprecursorectopiainthecortex ofthe the cortexoflaminin Cell divisionandinterkineticnuclearmigrationin (arrowhead inE,G)thelaminin BM. ( respective mouseline.Arrowheads inC,Dindicatedisruptionsofthe Insets (a-d)depictrepresentative high-powerviewsoftheBMin (arrowheads inA)andsurrounding thebloodvessels(arrows inA). ( telencephali ofembryonicday(E)14wild-typeandmutantlittermates. immunostained (asindicatedinthepanels)frontal sectionsof cortex atmidneurogenesis. detachment inlaminin Fig. 1.Basementmembranedisruptionandradialglialendfeet position, 22sections,oneanimal)andlaminin dividing attheVS;34.8±8.3%PH3-positive cellsdividing atSVZ observed betweenwild-type(65.2±8.3%PH3-positive cells percentages ofcellsdividing attheVSorSVZpositionswere developmental stages(E16)nosignificantchangesinthe precursor cellswas notaffected (Fig.2C,D). Becausealsoatlater ventricular zones.Scalebars:200 surface (VS).CTX,cerebral cortex;GE,ganglioniceminence;VZ, (arrows inF,H). Thebroken whiteline(E)indicatestheventricular precursors inthelaminin within theCP(Fig.2C;0,6%),they constitute4.6% ofall in wild-typecortex virtuallynodividing cellswereobserved in theSVZfromthosetakingplaceectopicallyCP. Although was requiredtodiscriminatetheabventricular mitosesoccurring see Materialsandmethods)(Haubstetal.,2004).Thisdefinition at leastfive celldiametersdistantfromtheventricular surface (VS, ventricular surface (abventricular), but locatedbelow theCPand SVZ was definedasabandofmitosesnotoccurringatthe CP), attheventricular surface (VZprecursors)orintheSVZ.The dividing atectopicpositions(misplacedproliferatingcellsinthe ␥ we examined interkineticnuclearmigrationinthelaminin crucial asanchoringpointtoallow interkineticnuclearmigration, 2005)]. AsattachmentoftheradialglialprocesstoBMmay be 1935) [forrecentreview, seeGötzandHuttner(GötzHuttner, moving backapicallytoundergo Mphaseandcytokinesis (Sauer, nucleus migratingtowards basalpositionsforSphaseandthen only theformerundergo interkinetic nuclearmigrationwiththe not tobeaffected bythelossofBMcontact. animal), weconcludethatproliferationofradialgliacellsseems PH3-positive cellsdividing atSVZposition,22sections,1 (66.9±6.6% PH3-positive cellsdividing attheVS;29.9±7.0% G,H; 100 A-D 6 integrin 1III4 1III4 A crucialdifference betweenVZandSVZprecursorsisthat ) Lamininimmunoreactivitythepialsurface underneath E-J –/– –/– ) TheabsenceofBLBP-immunoreactive radialgliaendfeet ␥ ␥ ␮ 1III4 cortex, thenumberandproportionofVZSVZ 1III4 cortex bylabellingcells inSandMphaseofthe –/– m inI,J. cortex (CTX;J)compared withwildtype(E,G).Ectopic –/– –/– mice, wequantifiedtheproportionofprecursors cortex (Fig.2E,F).Atthis time,nocellsinS ␥ 1III4 ␥ 1III4 Fluorescent micrographs of ␥ –/– 1III4 , perlecan ␥ ␮ 1III4 m inA-D;50 –/– –/– RESEARCH ARTICLE cortex (Fig.2C).Besidesthis –/– ␥ 1III4 (F,H), perlecan –/– –/– and cortical plate(CP) ␮ m inE,F;25 ␣ 6-integrin ␥ 1III4 –/– cortex (I)and –/– ␮ –/– m in cortex 3247 Ј -

DEVELOPMENT normally inthe absenceofradialgliaattachment totheBM. 2G,H). Thus,tooursurprise,interkinetic nuclearmigrationoccurs 3248 the corticesofwild-typeand laminin progressed towards theapicalsurface toundergo Mphaseinboth positive 6hours aftertheinjectionandBrdU-labellednucleihad However, mostPH3-positive cellsinG2/M-phasewerealsoBrdU contained incellsG2/M phase (Fig.2E,F, arrowheads). RESEARCH ARTICLE ␥ 1III4 –/– littermates (Fig. two animals;laminin and perpendicularly(60-90°)inE14cortex(wildtype: dividing horizontallywithrespect totheVS(0-30°),obliquely(30-60°) separating chromatids. ( assessed bymeasuringtheanglebetweenalineatVSand anaphase labelledwithpropidium iodide.Theangleofcelldivisionwas (wild type: positions assessedbythequantificationofPH3-positivecellsatE14 ventricular surface(VZinC),subventricularzone(SVZ)oratectopic laminin abventricular positions.Ectopicclustersofprecursor cellswithinthe positive) attheventricularsurface(VS,arrowheads inA)orat reveal similarnumbersofprecursors (Ki67-positive)inmitosis(PH3- type: cell divisions inwild-typeandlaminin Notably, nodifferences wereobserved intheorientationofapical 1995; Haydaretal.,2003;Kosodo etal.,2004;Noctor2002). in symmetricorasymmetriccelldivision (ChennandMcConnell, an anglevertical totheVS(60-90°),anorientationthatmayresult VS, stillconsideredasasymmetricallydividing cells;and(3)with withrespecttothe cell division; (2)withanobliqueangleof30-60° parallel, withrespecttotheVS,normallyresultinginanasymmetric classified inthreegroupsdividing: (1)withanangleof0-30°,i.e. As depictedinFig.2J,cellsdividing attheapicalsurface were Estivill-Torrus etal.,2002;Heins2001;Stricker etal.,2006). methods, example inFig.2I)(seealsoChennandMcConnell, 1995; in thespindlerotation(Haydaretal.,2003)(seeMaterialsand late anaphaseandtelophaseofthecellcycle toavoid furtherchanges orientation ofcelldivision was determinedat theendofMphasein the orientationofcelldivision attheventricular surface. The Next, weexamined iftheabsenceofBMattachmentmayrandomize cortex area (100 animals). ( moved towards theventricularsurface(G,H).( BrdU-injection; E,F)and6hoursafterS-phaselabellingwhentheyhave BrdU-immunostaining (red) reveals cellsinSphase(0.5hoursafter type andlaminin cortex atmidneurogenesis. orientation ofcelldivisioninwild-typeandlaminin Fig. 2.Proliferation, interkineticnuclearmigrationand of neuronswere visiblebetweenthecorticesofwild-type andmutant and MAP2,but noobvious differences inthethicknessofband , weimmunostainedfor theneuronalantigens influence thefate ofradial gliaprogeny. To assessthenumberof Next, weexamined whetherthelossofBMattachmentmay mice S toMphaseinthelaminin number ofcellsinSphase(Fig.2E,F)northeprogressionfrom Moreover, thisanalysisshowed thatthereisnochangeinthetotal ( laminin Orientation ofcelldivisioninthecortex absence ofBMattachment. cycle progressionofradialgliacellsoccursnormally, despitethe Neurogenesis inthecortexoflaminin required forproperorientationofcelldivision. 2J), suggestingthatanchoringofthebasalprocessatBMis not C Histogramdepictingthepercentages ofprecursors dividingatthe ) n =43, twoanimals;laminin ␥ 1III4 D n ␥ ) HistogramdepictingthenumberofPH3-positivecellsper =40 sections,twoanimals,laminin 1III4 –/– cortical plate(CP)are indicatedbyarrows inB. ␮ ␥ m 1III4 –/– 2 ) dividingatVZorSVZpositionsrespectively (wild ␥ mice 1III4 –/– J ) Histogramdepictingthepercentages ofcells cortex immunostainedasindicated( –/– : n Fluorescent micrographs ofE14wild- =110, twoanimals).Scalebars:100 ␥ 1III4 ␥ 1III4 –/– –/– ␥ : cortex, suggestingthatcell 1III4 n =51, twoanimals).( I ) Adividingcellin Development 133(16) ␥ –/– 1III4 cortices atE14(Fig. –/– n ␥ : =103 mitoses, 1III4 n ␥ =45, two ␤ 1III4 III-Tubulin A –/– –/– , E-H B ,E-H) ) ␮ m

DEVELOPMENT ␥ space, arrows inBindicate ectopicclustersofprecursors inthelaminin and red arrows inH indicateneuronal ectopiasinthesubarachnoidal CP tothetotalradialwidthofcortex (Ctx)(Fig.3C)didnot (Fig. 3C,D).TheratiooftheradialthicknessMath2-positive 2004), abHLHgeneexpressed inglutamatergic corticalneurons Math2 (Neurod6–MouseGenomeInformatics)(Schuurmansetal., methods) (seealsoHaubstetal.,2004)byinsituhybridizationfor thickness ofthebandneuronsformingCP(Materialsand order todetectsubtlechangesinneurogenesis,wequantifiedthe ectopic clustersofprecursorsdescribedabove (Fig.3B,arrow). In expression isabsentinthe upperlayersofthelaminin cortical platethicknessinrelation tothecortexthickness.Math2 ( free areasintheCPoflaminin littermates atE14(Fig.3A,B),withtheexception ofsomeneuron- Basement membraneattachment Fig. 3.Neurogenesis inwild-typeandlaminin for A,B,E,F;200 (H), whiletheseneurons still contain for theneuronal antigen A-H 1III4 ) E14orE18wild-typeandlaminin –/– cortex. Thebroken yellow and red linesinCdelineatethe ␮ m forC,D,G,H. ␤ III-Tubulin orMath2mRNA.Arrowheads inB ␥ 1III4 ␤ III-Tubulin (F).Scalebars:100 ␥ –/– 1III4 mice correspondingtothe –/– cortex sectionsstained ␥ 1III4 ␥ 1III4 –/– cortex. –/– cortex ␮ m of thelaminin calbindin-positive cellswereconcentratedintheoutercorticallayers our surprise,wenotedthatGad65-(datanotshown), calretinin-and immunolabelling (Fig.4G-J)todetectGABAergic .To of thecerebralcortex inlaminin examined thinner, the E18laminin significantly thinnerthaninwild-type mice( ␥ cortex; E18wild-type thinner thanitswild-typecounterpart(11%reductionatlateral calretinin. large extent GABAergic interneuronscontainingalsocalbindinor cortex, whilethemedial cortex oflaminin defects werestrongestinthe caudal andlateralregions ofthe wild type delineating cortex andGE tothemedialsulcus(60%increase;E18 by thetotallengthofventricular surface fromthesulcus ␥ reveal any significantdifference betweenwild-typeandlaminin stages inthecortexoflaminin Neuronal migrationanddifferentiation atlate even afterlossofradialgliaattachmenttotheBM. medial)=0.06], suggestingthatneurogenesisstilloccursnormally 0.41±0.06, Calretinin-positive neuronsthatalsocontain (Fig.4G were detectedatthispositioninthewild-typecortex (Fig. 4G,I). laminin animals, medial=0.47±0.06, [wild-type ratioCP:CTXlateral=0.47±0.09, ratio ofthetotalcortex widthwas significantlyreduced atthisstage identity ofthe with BMdisruptions(seeDiscussion),they stilldidnot reveal the with thedefectsinradialmigrationcortex ofmouse mutants labelled byEr81(Fig.4E,F).Althoughthesedataareinagreement fact, they seemtolocateatthesamepositionaslayerVneurons, neurons fail tomigratetowards theirappropriatelayerposition.In type cortex (Fig.4A-D,redarrow) suggestingthatupperlayer ␥ a lossofMath2expression intheoutercorticallayersoflaminin However, whenweexamined neuronalmarkers atE18,weobserved laminin lateral=0.44±0.24, medial=0.48±0.09, neurons) and analysed themRNA for they mayhave otherfeaturesofcorticalpyramidal neurons,we neurons thatnormallyexpress Math2.Inordertoexamine whether some neuronslocatedbelow thepialsurface arenotpyramidal markers suchas P two animals,medial=0.38±0.12, neurons intheouterpartofE18laminin ␥ neurons werestillfew innumberandscatteredthelaminin early stagesincorticaldevelopment (Stoykova etal.,2003).These located inlayer1ofthewild-typecortex andaregeneratedatvery 2004). Interestingly, expressed inlayerVneuronsofthecortex) (Schuurmansetal., deeper positionsinthecortex ofthelaminin 1III4 1III4 1III4 1III4 (lateral)=0.00046; In afurtherattempttoidentifythesubtypeoftheseneurons,we Notably, atthisstagethemutantcortex was alsosignificantly –/– –/– –/– –/– ␥ cortex (Fig.4G,G mice (Fig.3G,H).ThewidthoftheMath2-positive CPas 1III4 ␥ n n cerebral corticesatE14[wild-typeratioCP:Ctx Gad65 1III4 =21 sections,two animals;E18laminin n =53 sections,two animals; =7 sections,oneanimal; Er81 ␤ –/– ␥ III-tubulin-positive neuronslocatedintheouterpart 1III4 –/– ␤ ratio CP:CTXlateral=0.42±0.09, III-Tubulin werestillpresent(Fig. 3E,F).Thus, mRNA, aswellcalbindin-andcalretinin- (Etv1 –MouseGenomeInformatics)(whichis ratio CP:CTXlateral=0.30±0.5;medial= Cux2 –/– P n (medial)=0.0029], whilepan-neuronal cortex, whereasfew oftheseinterneurons =40 sections,two animals;E18laminin ␥ Ј Cux2 1III4 and ,H,H n Rorb , –/– Ј =27 sections,two animals;E18 ␥ ). Thesedatathereforesuggestthat Rorb 1III4 cortex was longer, asmeasured RESEARCH ARTICLE n mRNA signalwas detectedat =43 sections,two animals; P –/– n (ratio lateral)=0.17, (which labelsupperlayer =13 sections,oneanimal; ␥ mice. 1III4 P ␥ ␥ ␥ =1.85 1III4 1III4 1III4 n P =40 sections,two –/– =0.21). Although –/– –/– ϫ –/– mice n ␥ cortex aretoa 10 mice was not =53 sections, 1III4 than inwild- –5 ). These –/– P Ј (ratio 3249 n ) are =32

DEVELOPMENT interneurons ( (red inI,J)were detected intheouterpartoflaminin GABAergic interneuronscontaining calretinin (red inG,H)orcalbindin red arrows indicate the layerspecificgeneexpression. Conversely, positive (G reelin-immunoreactiveEarly born cells(green inG-J)are mostlycalretinin (H,J) incontrasttotheirscattered positioninthewild-typecortex(G,I). compared withwildtype(G).Scalebars:200 Er81-positive deeplayerneurons inthelaminin and 3250 Fig. 4.Neuronal subtypesintheE18laminin Cux2, Rorb(ROR Micrographs ofinsituhybridizationforthelayer-specific mRNAs of Rorb RESEARCH ARTICLE -expressing layerII-IVneurons are misplacedtothepositionof Ј ,H Ј G-J ) andare notincreased inthelaminin ␤ ) inE18wild-typeandlaminin ) andEr81( A-F ), andimmunostainingfor ␮ m inA-F;50 ␥ ␥ 1III4 ␥ 1III4 1III4 ␥ –/– –/– 1III4 –/– cortex (E,F).The cortex. ␥ –/– cortex. 1III4 ␮ cortex (H) m inG-J. –/– Cux2 cortex - of perlecan Neurogenesis andcell proliferation inthecortex division, proliferationorneurogenesis. mediated signallingtotheradialglialcellsdoesnotaffect cell Math2 (Fig.5G,H).Thesedatathereforesuggestthat division was comparablebetweenwild-type and material) weredetectable intheperlecan orientation ofcelldivision (seeFig.S2Einthesupplementary of neurons(seeFig.S2C,Dinthe supplementarymaterial)orinthe no obvious defectsinthe bandofproliferatingcells,inthenumber in thiscortex (arrows inseeFig.S2Dthesupplementarymaterial), neurogenesis. Despitesevere cobblestonetypeIIneuronalectopia we couldanalyseonlyonecortical hemisphereforproliferationand exencephaly itselfmayexert many influencesonbraindevelopment, material). Aswehadtoexclude thesefromouranalysisbecause previous observations; seeFig.S2A,B inthesupplementary exhibited exencephali (19of20E14embryos,consistentwith of Neurogenesis andcellproliferation inthecortex one animal; wild type:0.38activated caspase3-positive cellspersection, appeared comparablebetweenwild-typeandthe ventricular zoneslabelledbyKi67orBrdUimmunostaining 0.67 activated caspase3-positive cellspersection, related totheoverall smallersizeofthelaminin failure ofradialmigrationlategeneratedneurons,ormaybe pial BMcounteractingthepressureofventricular fluidortothe the cortex maybeduetoareducedforcenormallyexerted bythe sections; two animals; immunostaining for normally intheabsenceof as describedabove (Fig.5A,B).Neurogenesisalsooccurred to significantchangesintheinterkineticnuclearmigrationassessed littermates (Fig.5D)andthespecificlossof in contrasttothelaminin clusters ofproliferatingcellswerevisibleintheCPthismutant, precursors arenotaffected bytheabsenceof suggesting thatproliferationandtheproportionofVZSVZ the VSorinSVZwild-typeandmutantcortex (Fig. 5A-C), substantiated bytheequalnumberofPH3-immunopositive cellsat littermate cortex (Fig.5A,B,E,F).Thissimilaritywas further ␣ also examined radialgliacellproliferationandneurogenesis inthe In ordertoensurethegeneralrelevance oftheabove findings,we P cells persection; 3-positive cellspersection, n section, laminin no significantchangesincelldeathweredetectableatE18the resulted insevere bleeding(seealsoHalfteretal.,2002).However, glia attachment. at theendofneurogenesisafterdisruptionBMandradial migration andtheoverall cortex architectureareseverely distorted other inwild-typecortex (Fig.4A,C,E).Taken together, neuronal (Fig. 4B,D,F),althoughthey arenormallylocatedontopofeach Indeed, layerII-IVandVneuronscolocalizeatthesameposition As describedabove, mostperlecan =6, oneanimal)comparedwithwildtype(1.25activated caspase 6 integrin (TUNEL)=0.12), norattheearlierstageE14(laminin At theselateembryonicstages,thelossofBMintegrity also ␣ 6 integrin ␥ n 1III4 =6, oneanimal;1.66TUNEL-positive cellspersection, –/– P =0.26). –/– –/– cortex asdescribedabove. Thethicknessofthe cortex (1.33activated caspase 3-positive cellsper mice –/– n =6, oneanimal; ␤ mice III-Tubulin (Fig.5E,F),Map2(notshown) and P ␥ =0.0002). Thisthinningandextension of 1III4 n =8, oneanimal;0.67TUNEL-positive –/– . Inaddition,theorientationofcell ␣ –/– 6 integrin, asrevealed by P embryos (Costelletal.,1999) (activated caspase3)=0.90; –/– cortex. ␣ Development 133(16) ␣ 6 integrin didnotlead 6 integrin. Noectopic ␥ n 1III4 =18, oneanimal; ␣ ␣ 6 integrin 6 integrin ␣ –/– 6 integrin- ␥ embryos. 1III4 n =16, –/– –/– –/– :

DEVELOPMENT three animals, positive cellsinMphasepercortex area (C;wildtype: H animal, orientation totheventricularsurface (D;wildtype: percentages ofcellsdividing withhorizontal,obliqueorperpendicular divide withnormalorientations,suggestingthatBM attachmentof BM anchoringperformnormalinterkineticnuclearmigrationand developing cerebralcortex. Furthermore,radialglialcellswithout radial gliaproliferationnortheirneurogenicprogeny inthe the lackoflaminin-receptorscontaining processes totheBM.Ourdatashow thatlossofthis contactand/or Here, weexamined theroleofdirectcontactradialglial DISCUSSION Basement membraneattachment Fig. 5.Proliferation andneurogenesis in Micrographs ofE14wild-type and integrin ) stainedasindicated.( –/– ␣ 6 integrin cerebral cortex. Scalebars:100 ␣ 6 integrin –/– : n =150, oneanimal)inwild-typeand C –/– , D : ) Quantificationofthenumber PH3- n =50, three animals)andof the ␣ 6 integrin ␮ m. ␣ –/– 6 integrin ␣ 6 integrin donotaffect cortex sections( n =89 mitoses,one n =39 sections, –/– ␣ cortex. 6 A , B , E- disruptions. radial glialcellswas affected inthemousemutantswithsevere BM 2005). However, noneoftheseproposedfunctionsBMcontact crucial roleofsuchacontactforradialglialcells(Colognatoet al., the BM(Colognatoetal.,2002),furthersupportingpotentially signalling, e.g.tooligodendrocytes, canbealtereduponcontactwith renewal (Camposetal.,2004).Moreover, growth factor-mediated maintenance ofprecursorproliferationoreven stemcell-like self with dataimplicating 2003; Miyataetal.,2001).Thishypothesiswould alsobeconsistent and proliferatefaster thanotherprecursors(FishellandKriegstein, maintaining theircontactwiththeBMmayremainprecursorcells 2003). FishellandKriegstein hadsuggestedthatradial glialcells apicobasal polarityinmany celltypes(forareview, seeLietal., by Fujita(Fujita,1963). from theapparenthomogeneityatearlierstages,asfirstdescribed heterogeneity ofprecursorsduringneurogenesisisnotablydifferent pial surface (Hartfussetal.,2003;Gal2006).This precursor cellswithsubsetsofprecursorsdevoid ofcontacttothe Nevertheless, thereissomedegree ofheterogeneityamongthe glial cellsthatareconnectedbytheirradialprocessestotheBM. proliferating precursorcellsintheVZduringneurogenesisisradial (Hartfuss etal.,2001;Noctor2002)themajorityof 2003).Thus,consistentwithimmunocytochemical evidence al., radial processesreachingabove thecorticalplate(Hartfusset cells labelledfromtheVSrevealed thatmostprecursorspossesslong Gadisseux etal.,1992).However, 3Dreconstructionofprecursor precursor cellsduringneurogenesiswould alsoendthere(e.g. previous studiessuggestedthattheradialprocesses ofsome restriction ofinterkineticnuclearmigrationbelow the corticalplate, migration (forareview, seeGötzandHuttner, 2005).Owingtothe al., 1997)],aswellareducedextent ofinterkineticnuclear side [forjunctionsbetweenpialendfeetseeBalslev etal.(Balslev et glial cellsthatexhibit reducedtightjunctionalcouplingattheapical onset ofneurogenesis,neuroepithelialcellsdifferentiate intoradial cells (forreviews, seeGötzandHuttner, 2005;Fujita,2003).Atthe the entirethicknessofwall oftheneuraltube,neuroepithelial Prior toneurogenesis,precursorcellswithepithelialpropertiesspan proliferation The role oftheBMonradialglialfateand possibly maturation. demonstrating theneedofanintactBMforneuronalmigrationand GABAergic neuronsintheoutercorticallayersaroundbirth, ectopic positionswithinthecorticalparenchymaand defects inradial gliacellproliferation,inthe generation ofbasal caused byBMdisruption.None ofthesemutantsexhibited any et al.,1999),therebyensuringthat wedidnotmissany phenotype included inouranalysis(Georges-Labouesse etal.,1998;Costell disruptions weredemonstrated byelectronmicroscopy were material). Moreover, two additionalmouselineswhere theBM lissencephaly (Figs1,2,5;see Fig.S1inthesupplementary neurons inthesubarachnoidspace,cobblestone-(type II) markers scatteredintheCPandasevere accumulationofectopic endfeet withonlysomeectopiccellspositive forradialglial immunoreactivity, inthefrequentabsenceofsubpialradialglial evident inlarge areasofthecorticalsurface devoid ofany laminin the laminin besides thecobblestoneneuronalectopiabelow the pialsurface in the basalprocessisnotrequiredforany oftheseprocesses.However, Severe BMdisruptionsinthelaminin BM contacthasbeenshown toactasacrucialfactor for ␥ 1III4 –/– cortex, wealsoobserved someprecursorsat ␤ 1 integrin-mediated signallinginthe RESEARCH ARTICLE ␥ 1III4 –/– cortex were 3251

DEVELOPMENT and ␥ deletion ofcomponentsintegral totheBM,suchasinlaminin above) resultinBMdisruptionssimilartothe phenotypeupon migration. Allmutationsaffecting signallingfromtheBM(see functionally relevant forthemaintenance oftheBMandforneuronal relevant forradialgliaproliferation and neurogenesis,but itis Thus, theattachmentofradialgliaendfeettoBMmaynot be differentiation The role oftheBMfor neuronal migrationand cell proliferationorneurogenesis. predominantly exhibit cobblestoneectopiabut noobvious defectin McCarty etal.,2005;Niewmierzycka etal.,2005),also neuroepithelium (Beggs etal.,2003;Graus-Porta2001; of mediating signallingfromtheECM,suchasconditionaldeletion analysed here.However, micewithdeletionsinthecomponents parenchymal ECMcomponentsisstillpresentinallthemutantmice deposited laminins(DeArcangelisetal.,1999).Thus,signallingvia may stillbepresenttomediatesignallingviaparenchymally laminin receptorscontaining radial glialcellswerealsonormalinthecortex of located BM(Miyataetal.,2004).Inthelaminin apical contactsbut oftenmaintaintheiranchoringtothebasally precursors dividing atabventricular positions,thelatterloosetheir earlier developmental stages.WhenVZprecursorsgenerateSVZ result fromprecursorsloosingbothbasalandapicalanchoringat stillexpressing radialglialantigens,we speculate thatthey were ectopic precursorcellsformedclustersofdividing cellsand Hartfuss etal.,2003), suggestingthatBMdisruption affects radial process extension supposedly viatheBLBP(Försteretal.,2002; not shown). Reelin signallingtotheradialglialcellspromotestheir of reelin-secretingcells(alsoobserved inthisstudy, Fig. 4anddata (Beggs etal.,2003),cases accompaniedbydisruptionofthelayer in thecorticalparenchymaoflaminin regulating theirpolarity, proliferationandcellfate. direct signallingfromtheBMtoradialglialcellsisnotinvolved in BM-deficient cortex (datanotshown). Therefore,weconcludethat of themarkers forthesecelltypeswas observed untilE18inthe prematurely intoastrocytes oroligodendrocyte precursors,asnone absence ofanchoringattheBM,radialgliadidnottransform should manifestbyE14toE18,thestagesanalysedhere.In analysed, possibleinfluencesoncellproliferationorneurogenesis As BMdisruptionsoccurredalreadyaroundE12inthemutants progenitors, intheorientationofcelldivision or in neurogenesis. 3252 endfeet attachmenttotheBMthanlaminin mice. Althoughthesemicehadmuchmilderdefectsinradialglia lack thelamininreceptorscontaining intermediate zoneinthelaminin the BManormalbandofSVZprecursorswas locatedbelow the context, itisinterestingtonotethat,despitethelackof anchoring at dispersion oftheprecursorsthroughoutparenchyma.Inthis the lossofapicalcontactsviaadherensjunctionswould resultin where anchoringofradialprocessestotheBMisvirtuallyabsent, precursors weremis-positionedinthelaminin to positionbothVZandSVZprecursors,asfewer than5%ofall absence ofBManchoringsuggeststhatthisplaysonlyaminorrole the normalarrangementofvast majorityofprecursorsinthe some othersignalsmayalsocontribute tolocalizeSVZcells.Infact, 1III4 Notably, cellproliferation,neurogenesisorlatergliogenesisof However, asmallproportionofprecursorswas ectopicallylocated ␤ ␣ 1, 6 –/– ␣ ␤ RESEARCH ARTICLE v integrin, ILKorthefocaladhesionkinase(FAK) inthe 4 (forareview, seeColognatoetal.,2005)].However, the or perlecan –/– mice. BMdisruptionisinmost, but notall ␣ 3 and ␥ 1III4 ␣ 7 integrins, anddystroglycan –/– ␣ 6 integrin subunit [ cortex, suggestingthat ␥ 1III4 ␥ ␥ 1III4 1III4 ␥ –/– 1III4 –/– ␣ –/– mice. Asthe 6 integrin cortex. mice, they –/– cortex, ␣ 6 ␤ –/– 1 largely dispensablefortheprecursor rolesofradialglialcells. for neuronalmigration–bothradialandtangentialwhereas itis together, ourdatasuggestthatcontacttoan intactBMisimportant where corticallayeringisalsodisturbed(Yabut etal.,2006).Taken the mispositioningofGABAergic interneuronsinthe to andcorticallayersdonotdevelop normally. Thisisconsistentwith ␥ abnormally concentratedintheouterpartofcortex oflaminin altered, interneuronscontainingcalbindinorcalretininwere Although thenumberofreelin-positive neuronswas notobviously GABA-, calretinin-orcalbindin-positive neuronsweredetected. In theoutercorticalregions, onlyreelin-positive neuronsand located deepinthecortex atthesamepositionasdeeplayer neurons. Math2 atE18.Neuronswithupperlayermarker expression were 2004), whileneuronslocatedclosertotheBMdidnotexpress marker forglutamatergic pyramidal neurons(Schuurmansetal., in thedeeperpartsofcorticalgrey matterexpress Math2,a in theouterpartoflaminin this regard, itisofinterestthatGABAergic neuronssettledmostly cerebral cortex (forareview, seeMarínandRubenstein,2003).In pursued byGABAergic interneurons,originatingoutsidethe parallel totheventricular orpialsurface. Thesepathways aremostly et al.,2005). 2005; Georges-Labouesse etal.,1998;Halfter2002;McCarty BM disruption(thiswork) (Beggs etal.,2003;Chiyonobu etal., mode ofmigration,mostneuronswillbedisplacedinacortex with and Silver, 2003;Nadarajahetal.,2001).Thus,independentoftheir at thepialsurface (Miyataetal.,2001;Miyata2004;Morest the somatowards thepialsurface bytheirapicaldendriteanchored migration ofneurons,bysomaltranslocationwithneuronspulling manner. ThelackofBMwillalsoaffect theothermodeofradial plate, andhencecannotguidemigratingneuronsinanorganized cortex, radialgliaprocessesaredisorganized withinthecortical glia processextension directlyandindirectly. Inthelaminin http://dev.biologists.org/cgi/content/full/133/16/3245/DC1 Supplementary material forthisarticleisavailableat Supplementary material Trust (U.M.)andARC(E.G.L.). bank. Thisworkwassupportedbythe DFG(M.G.),BMBFWellcome The antibodyagainstnestinwasobtained from thedevelopmental hybridoma P. Leprince forantibodies,andtoM.Körbsexcellenttechnicalassistance. deficient mice,toC.Schuurmansfor in-situ probes, toA.Goffinet, N.Heintz, We are particularlygratefultoR.FässlerandK.Rodgersfortheperlecan- laminin the cortex maychangetheirfate towards GABAergic neuronsinthe normal, consistentwithamisroutingoftheseneurons. fewer GABAergic, calbindinorcalretinin-positive neuronsthan normal guidanceinformationinthelaminin type cortex. Thus,tangentiallymigratinginterneuronsmay lacktheir lower layersoftheE18laminin hence accumulateintheouterpartofcerebralcortex. Indeed,the laminin with amisroutingofGABAergic interneuronstotheouterpartof (Marín andRubenstein,2003).Thus,thesedataaremostconsistent anyhow migratewithinlayer1ontheouter surface ofthecortex reach theouterlayers,whilesometangentiallymigratingneurons difficult formis-specifiedneuronsgeneratedwithinthecortex to neurons aredisplacedratherthanabsent.Moreover, itmayalsobe present atlower positionsinthemutantcortex, suggestingthatthese neurons, includingthosedestinedforuppercorticallayerswere 1III4 A thirdmodeofneuronalmigrationisorientedtangentially, in Alternatively, glutamatergic pyramidal neuronsoriginatingwithin –/– ␥ ␥ mice incontrasttotheirscattereddistribution inthewild- 1III4 1III4 –/– –/– cortex, whereglutamatergic neuronsfail tomigrate mice. However, allneuronalsubtypesofpyramidal ␥ 1III4 ␥ 1III4 –/– cortex. Onlyneuronslocated –/– cortex seemtocontain Development 133(16) ␥ 1III4 –/– reeler cortex and ␥ 1III4 cortex –/–

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