Spatiotemporally specificknockout,mouse *Author forcorrespondence (e-mail:[email protected]) University ofTexas MedicalCenter, Southwestern Dallas,TX75390,USA. inthedevelopingspinalcord(Luetal.,2002; is essentialfortheformationofmotoneurons and distinct transcriptionalregulatorymechanisms. morphologically heterogeneousastrocytesissubjecttoidentical or present itisunclearwhethertheformationofspatially and (Mehler, 2002;SauvageotandStiles, Sunetal.,2003),at involved inastrocytedifferentiation invitrohavebeenidentified and Wolburg, 2005;Walz, 2000).Althoughmanysignalingpathways glial fibrillaryacidicprotein(GFAP) (Kimelberg, 2004;Reichenbach distinct astrocytesubclassesarecommonlydefinedbyexpression of many branchedprocessesandfewglialfilaments.Thesespatially filaments intheirprocesses,whereasgraymatterastrocytesexhibit based ontheirlocation.Whitematterastrocytesharborabundantglial into atleasttwosubpopulations–whiteandgraymatterastrocytes the cortexexhibitheterogeneousmorphologiesandaresubdivided adulthood (SauvageotandStiles,2002;Temple, 2001).Astrocytesin cortex, wheretheydifferentiate andbecomematureastrocytesat (Goldman, 2004;MillerandReynolds,2004)thatmigrateintothe (SVZ) ofthedevelopingbrainatembryonicandearlypostnatalstages radial gliapresentintheventricularzone(VZ)andsubventricular Cortical astroglialcellsarederivedfromneuralprogenitors,including 1997; ShuandRichards,2001;Sofroniew, 2005;Ullianetal.,2001). in brainintegrityandremodelingafterinjuryordisease(Ridetetal., crucial rolenotonlyinneuronaldevelopmentandfunction,butalso Astrocytes, themostnumerousneuralcelltypeinbrain,playa INTRODUCTION Accepted 7March 2007 1 KEY WORDS:Astrocyte differentiation andheterogeneity, lineage,bHLHtranscriptionfactors, morphologicallyandspatiallydistinctastrocytesubpopulations. anddevelopmentalsourcesof, requirements for, Thus, ourstudiesuncoveracrucialrolefor that abnormalastrocyteformationisatleastinpartattributabletothelossof but withsustainedGFAP upregulationin thesuperficiallayers.Celltype-specificmutagenesisandfate-mappinganalysesindica required forastrocytedifferentiation inthecerebralwhitematter. Bycontrast,astrocytesinthecorticalgraymatterarefo astrocytes inthewhitematterisseverelycompromised.Temporally controlledmutagenesisrevealedthatpostnatal conditionally ablated progressively downregulatedinastrocytesatlatepostnatalstages.To assessthefunctionof obscure. Duringcorticaldevelopment, factor Olig2isessentialformotoneuronandoligodendrocyteformation;however, itsroleinastrocytedevelopmentremains The mechanismsunderlyingastrocyteheterogeneityinthedevelopingmousebrainarepoorlyunderstood.bHLHtranscription Jeff Cai development A crucialroleforOlig2inwhitematterastrocyte (2007)doi:10.1242/dev.02847 Development 134,1887-1899 Basic Research onNerveGrowth andRegeneration, Psychiatry, Q. Richard Lu Department ofDevelopmentalBiologyand KentWaldrep Foundation Centerfor The basichelix-loop-helix(bHLH)transcriptionfactorOlig2 3 1 Department ofPediatricsand , Ying Chen 1,4, * Olig2 1 , Wen-Hui Cai in aspatiotemporallycontrolledmanner. Inthe 4 Department ofMolecularBiology, Olig2 2 , Edward C.Hurlock Olig2 is transientlyexpressedinimmaturedevelopingastrocytesatneonatalstagesand in whitematterastrocytedevelopmentandrevealdivergenttranscriptional 2 Department of 1 , HengWu is atleastinpartattributabletothe lossof analyses indicatethatabnormal developmentofcorticalastrocytes cortical layers.Celltype-specificablationandfate-mapping upregulation inapopulationofastrocyteslocatedsuperficial in thegraymatter, astrocyte differentiation inthe brainandthespinalcord.Bycontrast, postnatal CNS.We uncover acrucialroleofOlig2inwhitematter approach toexamineOlig2functioninastrocytedevelopment the stages. We utilizeaspatiotemporally specificconditionalablation downregulated progressivelyinmatureastrocytesatlatepostnatal immature developingastrocytesneonatallyanditslevel is distinct astrocytesubpopulationsinthedevelopingbrain. present, itisnotclearhowOlig2regulatestheformation of 2002; Takebayashi etal.,2002;ZhouandAnderson,2002).At by theearlyembryoniclethalityof postnatal stages(SauvageotandStiles,2002),hasbeenhampered Olig2 incorticalastroglialdifferentiation, whichoccurslargely at Gabay etal.,2003).Invivocharacterizationofthefunction Olig2 inhibitsastrocytedifferentiation (Fukudaetal.,2004; (Marshall etal.,2005),whereasinvitroevidencesuggeststhat from neuralprogenitorsintheSVZofneonatalbrain Olig2 indicatethatdirectscorticalastrocytedifferentiation Kondo, 2004).Recentstudieswithadominant-negativeformof (Furusho etal.,2006;Malatesta2003;Setoguchiand and inGFAP neural progenitorsincludingradialglialcellsatembryonicstages the developingbrain.Olig2expressioncanbedetectedincortical functions inastrocytedevelopmentremainelusive,particularly Takebayashi etal.,2002;ZhouandAnderson,2002);however, its development. formation ofdiverseastrocyte subpopulationsduringCNS development andrevealdistinct transcriptionalrequirementsforthe dual roleofOlig2inregulating grayandwhitematterastrocyte studies usingconditionalinvivo mutagenesisindicateanimportant In thisstudy, weshowthatOlig2istransientlyexpressedin Olig2 Olig2 1 , StevenG.Kernie -ablated cortexandspinalcord,theformationof + in developingastrocytesandtheirprecursors. progenitor/stem cellsinthepostnatalSVZ Olig2 Olig2 ablation leadstoabnormalGFAP in astrocyteformation,we 1,3 RESEARCH ARTICLE , LuisF. Parada Olig2 Olig2 Olig2 -null mice(Luetal., Cortex, , function. Thus,our Olig2 1 function is rmed, and te 1887

DEVELOPMENT was completelyabsentfromGS lineage cells(Fig.1H,I,arrows;datanotshown).Olig2expression level ofOlig2expressionwereonlyidentifiedasoligodendrocyte the developing cortex bygenerating deleter line(Zhuoetal.,2001)to ablate used ahGFAP-Cre To assessthefunctionof ablated cortex Abnormal astrocyte developmentinthe astrocytes atlatepostnatalstages andadulthood. postnatal stages,whereasitundergoes downregulationinmature (IACRAC) attheUT SouthwesternMedicalCenteratDallas. by theInstitutionalAnimalCareandResearchAdvisoryCommittee McMahon, 2002).Allprotocolsinvolvingtheuseofanimalswereapproved neonatal pupsandtheirfostermotherasdescribedpreviously(Hayashi ablation, 0.3mgTMper4gbodyweightwasinjectedintraperitoneallyinto expressed inimmaturedevelopingGFAP adulthood (datanotshown).Thus,Olig2appearstobetransiently astrocytes atP5(Fig.1L-O),butwasabsentinmature by scattered GFAP stages. Atearlyneonatalstagessuchaspostnatalday5(P5), of Olig2andtheastrocyticmarkerGFAP atdifferent developmental differentiation inthepostnatalcortex,weexaminedexpression To determinetherelationshipbetweenOlig2andastrocyte white matterastrocytes atearlypostnatalstages Transient Olig2 expression indevelopinggrayand RESULTS confocalmicroscope. Zeiss LSM510laserscanning were usedfordouble-labelingexperiments.Microscopywasperformedona secondary antibodiesconjugatedtoCy2andCy3(JacksonImmunoResearch) per kgbodyweight4hourspriortosacrifice.Goatanti-mouse,ratandrabbit mutant littermateswereinjectedwith100mgBrdU(Sigma,StLouis,MO) 5 (Sigma), Ki67(1:500,Swant)andratPDGF NG2 andNeuN(Chemicon),BrdU,GFAP, S100 from ChuckStiles,HarvardMedicalSchool,Boston,MA);GFAP (Dako), with therelevantantibodies.Thefollowingantibodieswereused:Olig2(gift al., 2002).Doubleimmunostainingwasperformedbysimultaneousincubation with tissuesectionsfrommousebrains,wereasdescribedpreviously(Luet RNA insituhybridizationandprobes,aswellimmunostainingmethods RNA insituhybridizationandimmunohistochemistry Olig2 Generation oftissue-specific MATERIALS ANDMETHODS 1888 expression wasdetectedinapopulationofdevelopingGFAP developing cerebralwhitemattertract–ofwild-typemice,Olig2 mice byP21(Fig.1J,K).Similarly, inthecorpuscallosum–a was detectableinGS stages suchasP14(Figs2and3),alowlevelofOlig2expression GFAP expressionbecameundetectableinthecortexatperinatal synthetase (GS)(Stanimirovicetal.,1999)(Fig.1C-E).Whereas astrocytes (Fig.1F,G). Atthisstage,Olig2 compared withintenseOlig2expressioninearly-developing GFAP (Fig. 1A,B-B the majorityofwhichexpressedahighlevelOlig2innucleus CAGGS-CreER knockout mice, specific mice werebredwithaCrelineand/orreportertogeneratetissue- were maintainedonaC57BL6/Jand129SVJhybridbackground. hGFAP-Cre, CNP-CreandSyn1-Cre,aswelltheRosaYFP reporterline, Ј -bromo-2 conditional knockout( + RESEARCH ARTICLE Olig2- astrocytes expressedanotherastrocytemarker, glutamine Ј -deoxyuridine (BrdU)immunostaining,perinatalcontroland Љ knockout mice.To generatetemporallyregulated ). Theiridentitywasconfirmedbythefactthatthese TM Olig2Cko + astrocytes weredetectedinthecorticalgraymatter, mice (HayashiandMcMahon,2002).To induce + cortical astrocytes(Fig.1H,I,arrowheads),as mice werebredwithtamoxifen(TM)-inducible Olig2 Cko Olig2 miceandCredeleterlinesincluding ) + in corticalastrocyteformation, we cortical astrocytesofyoungadult conditional mutantmice Olig2Cko;hGFAP ␣ R (1:500,BDBioscience).For + ␤ + GS , NF200,Tau-1, Gap43 astrocytes atearly – cells withahigh Olig2 Cre Olig2Cko Olig2 ( Cko - Olig2 Olig2 G in + ) - marked downregulationnotonlyof In thecerebralwhitematterof the Astrocyte formationdeficitinthewhitematterof cortex. S100 in thisregion,weexaminedexpressionofanotherastrocytemarker, To determinewhethertheformation ofastrocytesiscompromised of cellnumberinthecorpuscallosum(Fig.3FcomparedwithE). Eosin stainingforcellmorphology, therewasasubstantialreduction When protein level,ascomparedwiththecontrol(Fig.3A-D,arrowhead). of Nonetheless,theseobservationssuggestthattheeffect in eachcell. fewerastrocytesorreducedGFAP expression to signal couldbedue 2F,H comparedwithE,G,arrowheads).Thelowerlevelofinsitu downregulated in matter, 2D,H). Strikingly, incontrastto of thecortex(layersV-VI) wasessentiallyundetectable(Fig. (Forster etal.,2006),whereas region anatomicallyakintothesuperficiallayersofcortex stratum lacunosum-moleculareofthehippocampus(Fig.2D,F),a expression wasseeninsuperficialcorticallayersII-IV andthe and controlhGFAP-Cre ( 3M,N) wasmarkedlyreducedtoapproximately21%ofwild-type and hGFAP-Cre-expressing controlmice( the astrocytemarkersGSandGFAP inthewhitematterofwild-type from postnatalweek2(Fig.2D).High the cortexatneonatalstageP7(Fig.2B),andincreaseddramatically Gfap not uniforminthe throughout adulthood(Fig.2H).Thisincreaseinexpressionwas 1985). matter astrocytescontainlittle,ifany, GFAP (ConnorandBerkowitz, observations areconsistentwithpreviousthatmostgray cells wasdetectedinthecortexatearlyneonatalstages(Fig.1).These expression of matter, thehippocampusandatsubpialsurface.However, precursors atpostnatalstages(Malatestaetal.,2003;Zhu2005). addition, theCreactivitypersistsindevelopingastrocytesandSVZ astrocytes inthecortex(Malatestaetal.,2003;Zhuo2001).In give risetoneuralcelltypesincludingneurons,oligodendrocytesand majority, ifnotall,embryonic multipotentcorticalprogenitors,which mice. ThehGFAP promoterdrivesCrerecombinaseexpressioninthe cells ascomparedwithGS GS-expressing cellsinthecorpuscallosumof expression ofanadditionalastrocytemarker, GS.Thenumberof population ofoligodendrocytelineagecells,weexamined cells inthecontrol(Fig.3G,H,O).SinceS100 cells wasseverelyreduced,incontrasttothehighdensityofthese 1995) thatarelost inthe oligodendrocytes (Deloulmeet al.,2004;RickmannandWolff, population ofS100 fwiemte srcts A disproportionate reductioninS100 of whitematterastrocytes. hGFAP-Cre doesnotexertdetectabletoxic effects ontheformation with K,L)wascomparable(Fig. 3O),suggestingthatexpressionof stage P7toadulthood(Fig.2A,C,G),althoughapopulationofGFAP essentially undetectableinthecorticalgraymatterfrompostnatal By contrast,inthe In controlmice, Olig2 Olig2 ␤ expression wasinitiallyobservedincellsscatteredthroughout Olig2 . Inthewhitematterof Gfap ablation on -ablated cortex -ablated corticeswereexaminedbyHematoxylinand expression inthewhitematterwassignificantly Gfap Olig2 Olig2- Gfap , asassessedbyinsituhybridization,was Olig2 Gfap ␤ mutants atperinatalandadultstages(Fig. was readilydetectedinthecerebralwhite Olig2 + ablated cortex.Thehighestlevelof Ctrl expression isregion-specificwithinthe conditional mutant( + cells markingearly-differentiated cells (Fig.3O)islikelytobedue toa Olig2 G ) mice(Fig.3I-L,O).Expressionof Gfap mutant (Yue etal.,2006).Thus,the Olig2- Gfap mutants, thenumberofS100 expression inthedeeplayers Gfap ablated mice,weobserved upregulation inthegray Gfap Ctrl Development 134(10) mRNA butalsoatthe G ) (Fig.3I,Jcompared Olig2 Cko expression persists ␤ G could reflecta ) mice,intense mutants (Fig. Gfap ␤ ␤ + + +

DEVELOPMENT inducible Creline( postnatal stagesbybreeding cerebral whitematterastrocyteformation,weablated To determinethecriticalstage atwhich have distincttemporalmaturationpatterns(MillerandRaff, 1984). D). Ithasbeensuggestedthatastrocytesubtypesintheopticnerve 2006), butalsopersistspostnatallyinGFAP progenitors atembryonicstages(Malatestaetal.,2003;Yue etal., hGFAP-Cre activityisnotonlydetectedinradialgliaandcortical matter astrocyte formation Postnatal Olig2functionisrequired forwhite matter. differentiation ofanastrocytesubpopulationinthecerebralwhite markers suggestthatOlig2isrequiredforformationand reduction incellnumberandtheexpressionofdifferent astrocyte Olig2 regulates astrocyte diversity induced Creactivity (Fig.4F).SinceOlig2 isrequiredfor Olig2 their brainsharvestedatP13 for insituhybridizationanalysis. administrated TMatneonatal stages for4days,fromP2-P5,and by aubiquitouscytomegalovirus promoter. 2002), inwhichtamoxifen(TM)-inducible Creactivityiscontrolled in thewhitematterandGFAP The controlandcompound expression inthecortexwasessentially eliminatedbyTM- CAGGS-CreER Olig2 Olig2Cko;CreER + progenitors intheSVZ(Fig.4A- conditional mutantmicewithan TM ) (HayashiandMcMahon, Olig2 + developing astrocytes function isneededfor TM animals were Olig2 at early formation. that astrocytes. Thus,theresultsfrom inducible stages resultsinaseveredeficit intheformationofwhitematter These resultssuggestthatTM-induced Olig2 was alsoobservedinthecerebralwhitematterofTM-treated with I,K). surface atalevelcomparabletothecontrol(Fig.4J,L compared tract, whereas matter ascomparedwiththeotherwiserobustexpressionin this expression wasseverelydownregulatedinthecerebralwhite Olig2 of developingoligodendrocytesformedpriortoTM-induced the whitematterarelikelytoresultfromcontinuedmaturation significant difference intermsofthenumberGS perturbed inthecorticalgraymatter(Fig.4J)andtherewas no the TM-injectedcontrol(Fig.4G,H,M).Residual and whitematterofthe CC1 wereaccordinglysignificantlydownregulatedinthegray the myelingene adequate postnatalmyelination(Yue etal.,2006),expressionof the corticalareaswhen Downregulated expressionofotherastrocytemarkerssuchasGS Olig2 mutant mice(Fig.4M).Bycontrast, ablation inthesecells.Intheinducible plays animportantroleinpostnatal whitematterastrocyte and Olig2.Arrowheads indicateGFAP P5 wasimmunostainedwithantibodiesagainstGFAP ( (green) atP5.Arrowheads indicateGS positive cells.(H,I)AtP14,Olig2expression inGS edge andbeneath).Arrowheads indicateGFAP level are showninsidepanels(alongtheright-hand Orthogonal reconstructions ofconfocalimagesat level ofOlig2(green) asjudgedbystainingintensity. (GS). (A)GFAP antibodies againstGFAP, Olig2orglutaminesynthetase from wild-typemiceatP5were immunostainedwith compared withtheintenseOlig2expression inGS astrocytes (arrowheads) isreduced toalowlevelas double-positive cells.AGFAP cortical astrocytes. Fig. 1.Transient Olig2expression indeveloping by crosslines inO.Scalebars:50 z M-O. Orthogonalreconstructions ofconfocalimagesat boxed area inLisshownatahighermagnification positive cellsinthisdevelopingwhitematter. The FG GS (F,G) at with antibodiesagainstOlig2andGS.Confocalimages at P5(F,G), P14(H,I)andP21(J,K)were immunostained cortex. ( green) wasobservedintheastrocytes oftheneonatal (C-E) Co-expression (E)ofGFAP (C,red) andGS(D, cross-lines, isshownatahighermagnificationinB-B undetectable inGS (blue arrows). (J,K)AtP21,Olig2expression (green) is Gfap L-O -axis levelare shownforaGFAP Mbp z -axis levelare showninsidepanelsofGandI. The corpuscallosumregion ofwild-typemiceat ) expression persistedintheSVZandsubpial + F-K and ofthematureoligodendrocytemarker astrocytes (red) express ahighlevelofOlig2 Olig2 Olig2 ) Sagittalcorticalsectionsofwild-typemice + cells (red) atthisstageexpressed ahigh -ablated cortex,ascomparedwith was ablatedpostnatally(Fig.4M). + astrocytes (red) inthecortex. ( A-E RESEARCH ARTICLE ) Sagittalcorticalsections Olig2 + Gfap Olig2 Olig2 ␮ + Olig2 deletion atneonatal m inA,F-L. Olig2 expression wasnot + cell, markedby + ablation suggest + Olig2 Olig2 + + Mbp mutant, cell marked astrocytes in + + + double- double- + cells in Olig2 + – 1889 Gfap z -axis cells Љ . +

DEVELOPMENT formation inthebrainof 2006). Inlightoftheprofounddeficitinwhitematterastrocyte could giverisetoastrocytesinthespinalcord(Masahiraet al., P14 ( A recentfate-mappingstudyindicatedthat astrocyte formationin thespinalcord Olig2 functionisrequired forwhitematter hybridization oncoronal brainsectionsofcontrol ( Olig2 Fig. 2.AlterationofGFAP expression inthedevelopingandadult 1890 the right-handsideofD. , respectively. Corticallaminationlayersare indicatedon the whitemattertractandstratumlacunosum-moleculare ofthe arrows inE,Findicate respectively. Arrowheads and Olig2 representing thehippocampusandneocortexfrom control and by bluearrows. Red,blueandwhiteboxedareas inC,DandG,H, S100 expressionoftheotherastrocyte markers, spinal cordatP14,aswas expression wasobservedinthe white matterregionofthewild-type region oftheCNSareaffected intheabsenceofOlig2.Strong mutants todeterminewhetherwhite matterastrocytesinthiscaudal through hGFAP-Cre, weexaminedthespinal cordfromthese Ctrl G ) and C-F -ablated mice,are shownathighmagnificationinE,F, andI,J, ␤ -ablated cortex. RESEARCH ARTICLE and GS(Fig.5). Bycontrast,inthespinalwhite matterof ) andadult(P79; Olig2 mutant mice( Expression of G-J ). Neocortical Olig2 Olig2Cko;hGFAP -ablated mice( Gfap Gfap was analyzedbyinsitu Cre Olig2 expression isindicated or Olig2C/+;hGFAP Gfap Cko -expressing cells Cko G expression in ) atP7( G ) mediated A Cre Gfap , B ), or with H).Asinthewhitematter, areductioninS100 in the population ofS100 the superficialcorticallayers(Fig.6G),weobservedthata oligodendroglial cellscouldacquireectopicGFAP expressionin conditions (Raff etal.,1983),weexaminedwhethercortical nerves canadoptanastrocytic fateinvitroundercertainculture Since oligodendroglial(orO2A) precursorsisolatedfromoptic astrocyte conversioninthe Absence ofdetectableoligodendrocyte-to- ablated cortex. upregulated inastrocytesofthesuperficiallayers GS cortical astrocyteformationintheabsenceofOlig2.Intriguingly, suggesting thatthereisnosignificantalterationintheextentof Olig2 from thelossofasubsetS100 observed inthegraymatter(Fig.6J);however, thisislikelytoresult In the of theproliferationmarkerKi67(Maedaetal.,2001;Torp, 2002). mice atperinatalstageP14forBrdUincorporationandexpression Gfap Olig2 al., 1999).ThenumberofGS is independentofGFAP expressioninastrocytes(Stanimirovicet formation inthegraymatter, weanalyzedexpressionofGS,which matter ofthe there wasnosignificantalterationintotalcellnumbersthegray cells. HematoxylinandEosinstainingofcorticalcellsindicatedthat cortical astrocytesortoupregulationofGFAP expressioninglial mutants (Fig.3).Thiscouldbeduetoanincreaseinthenumberof GFAP expressionwasobservedinthecorticalgraymatterof In contrasttothesituationincerebralwhitematter, excessive gray matter expression inthesuperficiallayersofcortical Olig2 matter astrocytesubpopulationsinthespinalcord. mutant suggeststhatOlig2isrequiredfortheformationofwhite astrocyte markerexpressioninthespinalwhitematterof in thecontrols,respectively(Fig.5O).Thus,reductionof (Fig. 5I-J,M-N),representingapproximately18%and25%ofthat and GSinthewhitematterofspinalcordwasmarkedlyreduced cord. In relatively normalGFAP expressioninthegraymatterofspinal cortical GFAP (Deloulme etal.,2004)inthe another astrocytemarker, S100 compared withE).Similarly, whenexaminingtheexpressionof the lowlevelorabsenceofGFAP expressioninthecontrol(Fig.6F GFAP BrdU wassimilartothecontrol,despitepresenceofexcessive cells atearlypostnatalstagesP7andP14aftera4-hourpulse of proliferation ofexcessiveGFAP with anormalnumberofcorticalastrocytes,anabsencelocal proliferation amongSVZprogenitorcells(Fig.6O,P).Together labeling suggestedthattherewasnosignificantalteration of cycling stateatperinatalstages.Inaddition,theKi67andBrdU that thehighGFAP-expressing cellsinthecortexwerenot proliferation, weexaminedthecortexof To furtherdetermineifthereisanalterationinastrocyte + cells inthe mRNA, butalsoofproteinexpression(Fig.5B,E,F),despite -ablated cortexwascomparabletothecontrol(Fig.6C,D,J), Olig2 + mutants, weobservedmarkeddownregulationnotonlyof Olig2 ablation results insustainedGFAP cells (Fig.6L comparedwithK;Fig.6N).Similarly, these Olig2 -ablated cortex,butnotinthecontrol(Fig.6Icompared Olig2 mutant cortex,thenumberofBrdU -mutant mice,thenumberofcellsexpressingS100 + Olig2 cells didnotexpressKi67(Fig.6M),suggesting -ablated cortex(Fig.6A,B).To examineastrocyte ␤ cells acquiredahighlevelofGFAP expression -ablated cortexbecameGFAP – Olig2 ␤ cells inthesuperficiallayersof , whichco-expresseswithOlig2in ␤ + + mutant (Yue etal.,2006). oligodendroglial lineagecells Olig2 cells suggeststhatGFAP is -ablated cortex Development 134(10) Olig2 uat( mutant + + , incontrastto proliferating ␤ + cells was Olig2 Cko Olig2 Olig2 G ␤ - )

DEVELOPMENT control ( atr ( matter. was analyzedbyimmunohistochemistryoncoronal brainsectionsofcontrol (A,C)and average numberofGS acquiring ectopic consequence ofoligodendrocyte lineagecellsofacertainstage so becoming,theylosetheirclassic OPCmarkers. that OPCsareeithernotthesource ofGFAP-expressing cellsorin distinct fromthatofGFAP (Fig.7B-B imaging (Fig.7A-A we didnotdetectGFAP expressioninPDGF GFAP wasperformed inthe expression. WhendoubleimmunostainingofPDGF et al.,2006).TheyarepresentwithinthedomainofexcessiveGFAP al., 1996)aremaintainedthroughoutthe Cspg4–MouseGenomeInformatics)(Nishiyamaet (also knownas (OPCs) thatexpressPDGF oligodendrocytes isvirtuallyabolished,oligodendrocyteprecursors absenceofOlig2.Althoughformationmyelinating the Olig2 regulates astrocyte diversity Fig. 3.Astrocyte differentiation deficitinthe whitematterofthe (I,J), callosum (CC).( (>300 cellscounted, knock-in line(Lappe-Siefke etal.,2003),inwhich Creisexpressed ablated To furtherdeterminewhetherexpressionof Ctrl G Ctrl E Olig2 , (K,L) and F ) HematoxylinandEosin(H/E)stainingofsagittalsectionscontrol ( G ) and using anoligodendrocytelineage-specific CNP-Cre I-N Olig2 ) Doubleimmunostainingofglutaminesynthetase(GS,red) andGFAP (green) inthecorpuscallosum(arrowheads) ofwild-type Olig2 Gfap n =3); barsindicates.d.Scalebars:100 Љ + ). Similarly, expressionofNG2wasclearly and S100 mutant ( mutant ( expression intheabsenceof Olig2 ␣ R (Woodruff etal.,2001)andNG2 Cko ␤ Cko + -ablated cortexatP14(Fig.7A,B), cells perunitarea (0.1mm G G ) miceatP14were immunostainedwithananti-S100 ; M,N)mice.GS Љ ). Theseobservationsindicate Olig2 ␣ R -ablated cortex(Yue + OPCs byconfocal ␣ + R orNG2with and GS Gfap ␮ Olig2 m inA-D;100 2 + ) inthecorpuscallosumofwild-type(wt), is the GFAP , we + Olig2 cells are showninI,K,MandJ,L,N,respectively. ( cells inthewhitemattercontrol and mutant mice,asindicatedbyacomparable numberofGFAP was noapparentalterationinwhite matterastrocyteformationinthe a deficitinoligodendrocytemyelinationthewhitematter, there Olig2 lineage cellsacquiringectopicGFAP expressionintheabsenceof persistent corticalGFAP expressionisnotduetooligodendroglial and adulthoodinthis was, however, anabsenceofGFAP upregulationinthecortexatP14 There formation orcausingsignificantcelldeath(datanotshown). deficit (Fig.7DcomparedwithC),butwithoutaffecting OPC ablation byCNP-Creactivityresultedinseverecorticalmyelination in allstagesofoligodendrocytesincludingtheirprecursors. white matterastrocyte formation. oligodendrocyte lineagecellsdoes notleadindirectlytoadefectin deficit thatresultedfrom (Fig. 7HcomparedwithG).This suggeststhatthemyelination ␮ -ablated cortex. m inG,H;50 In addition,although Ctrl . G ) and Olig2 Olig2 ␮ ␤ -ablated (B,D)miceatP14.Arrowheads indicatewhite m inI-N. antibody. Arrowheads inE-Hindicatethecorpus -ablated ( ( A-D ) Expression ofGFAP (Red)andOlig2(green) Olig2 Olig2 Cko Ctrl ablation byCNP-Creactivityleadsto mutant (Fig.7E,F),suggestingthat G G ) corticesatP14.( Olig2 and RESEARCH ARTICLE Olig2 ablation incommitted O lg Cko Olig2 ) Barchartshowingthe mutant ( G , H Cko ) Corticesof C mutant mice G ) mice + or GS Olig2 1891 +

DEVELOPMENT Olig2Cko;CAGGS Olig2 Furthermore, thereisalackof markedapoptosisinthecortexof within thedomainofGFAP upregulation (Fig.8C,D). , whicharepresent insuperficiallayersofcortex we wereunabletodetectrobust changesincalbindin-expressing appeared tobenormalinthe reflecting GABAergic interneuronsandsubcorticalafferents, ablated cortex.ImmunoreactivityfortheneurotransmitterGABA, further examinedinterneuronsubtypedevelopmentinthe in thedevelopingcortex(Furushoetal.,2006;He2001), we certain neuronalpopulations,includingGABAergic interneurons, multipotent were comparablewiththatofcontrolmice(Fig.8A,B).Since NeuN (alsoknownasNeuna60–MouseGenomeInformatics) cortical laminationandtheexpressionofpan-neuronalmarker and function.Forcorticalneuronsinthe GFAP expressioncan beinducedbyabnormalneuronalformation cortical ,asreactiveastrocytesexhibitingelevated (Yue etal.,2006),we examinedtheeffects of In viewoftheneurologicalabnormalitiesin Olig2 Absence ofsignificantneuronal deficitinthe GFAP matter (A,B)andSVZ(C,D)ofhGFAP-Cre miceatP14wasexaminedbyimmunohistochemistry. Arrowheads indicateCre Fig. 4.Effects ofneonatal 1892 Arrows andarrowheads indicatethewhitematterandSVZ,respectively. ( (I,J) wasexaminedbyinsituhybridizationoncoronal sectionsofthecortex.Boxedareas inI,Jare shownathighmagnificati administrated withtamoxifen(TM)for4daysfrom P2toP5,andtheirbrainswere harvestedatP13.Expression of GS + cells inthecortex(Ctx)andwhitematter(Wm)CC1 + cells (A,C).LV, lateralventricle;wm,whitematter. ( -ablated cortex RESEARCH ARTICLE mutant miceas assessedbyTUNEL andcaspase 3 Olig2 CreErTM + progenitor cellsarecapableofgenerating animals ( Olig2 Olig2 n =3); barsindicates.d.Scalebars:100 ablation onwhitematterastrocyte development. mutant (Fig.8C,D).Inaddition, Olig2- Olig2- Olig2 bae mutants ablated ablated cortex, E-L deletion on ) Control ( Olig2- + cells inthewhitematterofTM-treated control ( Olig2C/+;CAGGS ␮ m inAforA,B,andCC,D. not leadtoGFAP upregulation oralterthenumberofNeuN Olig2 neurogenesis (Luikartetal.,2005;Zhu2001).Ablation of asearlyembryonicday(E)12.5,theonsetofcortical I promoterdrivesCrerecombinaseexpressioninearly-postmitotic transgenic line(Luikartetal.,2005;Zhu2001).Thesynapsin neurons usingasynapsinIpromoter-driven Cre (Syn1-Cre) astrocytes, weablated the cortex,potentiallyaccountingforincreasedGFAP expressionin neurogenesis. in the in boththegrayandwhitematter of . Thus,thereisanabsence ofsignificantaxonalproteindeficit observed normalformationof axonalfilamentsin control mice. indicate that immunoreactivity (datanotshown).Thus,ourobservations cortex ofwild-typeand Tau-1 (Ifne1–MouseGenomeInformatics)andGap43, inthe 200 (NF200;alsoknownasNefh –MouseGenomeInformatics), Furthermore, whenexaminingaxonalproteinssuchasneurofilamin M To examinewhether ) Barchartshowingtheaveragenumberperunitarea (0.1mm in early-postmitoticneuronalcellsdirectedbySyn1-Credid Olig2 CreErTM ( A-D mutant ( Olig2 ) and ) Cre andGFAP expression inthecerebral white ablation doesnotobviouslyaltercortical Olig2 Olig2Cko;CAGGS Cko Olig2 Olig2- S specifically inearly-postmitoticcortical ) cortex(Fig.8FcomparedwithE). ablation altersneuronalfunctionsin ablated Olig2C/+;CAGGS Olig2 Cko CreErTM Cko G + (E,F), cells (B,D)andCre G mice ascomparedwith Development 134(10) on inK,L,respectively. pups were mice (Fig.8G-N),we Mbp CreErTM (G,H) and Olig2 ) and + -ablated neurons + Gfap 2 ) of

DEVELOPMENT detectible inthewhitematterof 9B,D, arrowheads).Thisreciprocalpatternof layers (Fig.9D,F),butmarkedlyreducedinthewhitematter Intriguingly, appearance of with thecontrol(Fig.9DcomparedC),coincident the in thesuperficialcorticallayerswasdownregulatedcompared perinatal stageP8(Fig.9A,B).AtP14,however, gray matterandinnumberscomparabletocontrolmice at crossed hGFAP-Cre micewith thefloxed the absenceof hybridization labeling(Fig.9F). Theseobservationssuggestthat expression inthecortexwasconfirmedbydouble situ GS and ( C,E isshowninD,F, respectively. GFAP isstrongly expressed inthespinalwhitematterofcontrol, butonlyweaklyinthe ( . ( Since upregulation isderivedfrom A subsetofcorticalastrocytes withsustainedGFAP Olig2 regulates astrocyte diversity GFAP, S100 Fig. 5.Whitematterastrocyte formationdeficiencyinthe GFAP upregulationarederivedfrom regulating corticalastrocytedevelopment. directly orindirectly, andthatOlig1maycooperatewithOlig2 in the same cells(Zhouetal.,2000),weexamined G-N C-F To furtherexaminewhetherthecorticalastrocytesdisplaying + Olig2 Expression ofOlig2andGFAP wasanalyzedbyimmunohistochemistryinthecontrol (C)and ) Olig2 cells perunitarea (0.06mm Expression ofOlig2andS100 ) Olig1 -ablated cortex mutant (I,J,M,N)mice.StainingofS100 ␤ A n Swere performedonfrozen spinalsectionsof and GS has beenshowntobeco-expressedwith Olig1 , B ) mRNAexpression of Gfap Olig2 expression wasmaintainedinthedeepcortical upregulation (Fig.9FcomparedwithE). affects theexpressionof Olig1 . 2 Olig2 ) inthespinalwhitematterofcontrol and + ␤ cells werepresentthroughoutthe (G,I), andOlig2GS(K,M),were analyzedbyimmunohistochemistryinthespinalcord ofcontrol (G,H,K,L) Gfap mutants (J,N)ascompared withcontrols (H,L).( Olig2 was examinedinthecontrol (A)and Olig2- Olig2 -ablated cells ␤ Olig1 and GSisshowninH,JL,N,respectively. S100 ablated cells,we mutant miceon a Olig1 Gfap Olig1 expression in Olig2 expression and , either Olig2 Olig1 in the Ctrl G mutant spinalcord. and Olig2 astrocytes (Gabay etal.,2003).Alternatively, itispossiblethata downregulation ofthe Gfap presumptive cortical astrocyteswith deep layers(Fig.9I,J),suggestingthatthispopulationof in expressed cells cortex oftriple-transgenicmice( ablation andactivatesYFP expression(Fig.9G).Indeed,inthe mediated RosaYFP reporterbackground(Srinivasetal.,2001).Cre- the the cellsinwhichendogenous GFAP expressionare derivedfrom indicating thatcorticalastrocyteswithGFAP upregulationorhigh at P14,GFAP expression wasobservedinYFP-expressingcells, the of YFP reporter expression(Fig.9H).Furthermore,tomapthefate Olig2-3 floxed Cko Olig2 G Olig2 Olig2 Olig2 + mice atP14.Arrows indicatethewhitematterregion ofthe mutant mice( cells express Olig2 Ј -ablated cellsinthecortex,weexaminedexpressionof O UTR B uatsia odb insituhybridization. (B) mutantspinalcord by mutant cortexatP14,essentiallyall mRNA 3 loxP ) BarchartshowingtheaveragenumberofS100 Olig2 , transcribedfromthe In situhybridizationandimmunostainingofOlig2, coding sequence(Fig.9G).Expressionofthe Gfap site excisionsimultaneouslyleadsto n =3); barsindicates.d.Scalebar:100 -expressing cells.We observedthatnotall Olig2 Ј untranslated region(3 Olig2-3 in thesuperficialcorticallayers butnot ␤ - andGS-expressing cellsare barely mutant (E)spinalcord. GFAP expression in Olig2 Gfap Ј UTR. Olig2 Olig2Cko;hGFAPCre;RosaYFP upregulation isderivedfrom promoter activityinmature RESEARCH ARTICLE Olig2 Olig2 This mightbeduetothe is normallyexpressed.In -ablated cellsexhibiting mutant(arrows). promoter, represents Ј UTR) outsidethe Olig2-3 ␮ m inA-N. ␤ + Ј and Olig2 UTR 1893 + )

DEVELOPMENT development. Nonetheless,co-expressionofGFAP with ventrally wouldneverhaveexpressed cell-autonomous functionof UTR subset ofGFAP 1894 astrocytes inthe developingbrain. mechanisms anddevelopmental sourcesforgrayandwhitematter known, whileilluminatingdivergent transcriptionalregulatory Olig2 inwhitematterastrocyte formation, aboutwhichverylittleis mutagenesis, ourpresentstudies unveilanovelandcrucialrolefor understood. With -,celltype-,andtemporal-specificinvivo behind grayandwhitematterastrocyte developmentarenotfully Wolburg, 2005;Walz, 2000).Atpresent,theregulatorymechanisms are relatedtotheirfunctions(Kimelberg, 2004;Reichenbachand structural, biochemicalandelectrophysiologicalproperties,which in theCNS.Thisdiversityisunderscoredbytheirdifferent Astrocytes comprisealarge and heterogeneousgroupofneuralcells DISCUSSION development. and theYFP reporterinthe RESEARCH ARTICLE + cortical astrocytessuchasthosebeinggenerated Olig2 Olig2 -ablated cortexsuggestsa in corticalastrocyte Olig2 during their Olig2-3 Ј progressively as astrocytesmatureinthecortex (Fig.9K). immature astrocytesatearlypostnatal stagesbutisdownregulated Thus, Olig2appearstobeexpressed transientlyindeveloping stage andiscompletelyabsentfrom matureastrocytesatadulthood. subsequently decreasesinastrocytes beginningattheperinatal neonatal stagesexpressahigh levelofOlig2.Olig2expression astrocytes inthedevelopingcerebral grayandwhitematteratearly In thepresentstudy, however, wefoundthatimmatureGFAP population ofimmaturecorticalastrocytesatearlypostnatalstages. of GFAP intheadult cortex,GFAP expressioncanbedetectedina 2000). Althoughmostastrocytesdonotexpressadetectablelevel astrocytes ofthecortexinadulthood(Luetal.,2000;Zhou al., astrocytes, suggestingthatOlig2isnotexpressedinmature Olig2 iscolocalizedwithmarkersforoligodendrocytesbut not Takebayashi etal.,2000;Zhou etal.,2000).Intheadultbrain, gives risetooligodendrocytesandmotoneurons(Luetal.,2000; specialized domain(pMN)oftheembryonicventralspinalcord that Previous studiesindicatethatOlig2isinitiallyexpressed in a astrocyte development Cell-autonomous functionofOlig2incortical of sagittalsectionscontrol ( of BrdU of GS Scale bars:100 the control (O)and ( ( 01mm (0.1 layers. ( Arrows indicatethesuperficialcortical ( cells inthe S100 control and Olig2 ablated ( superficial layersofcontrol and ( n before sacrifice.Arrows indicateBrdU a 4-hourpulseofBrdU administration GFAP (K-M),Ki67(M)andBrdU (K,L)after animals atP14were immunolabeledwith indicate GS antibodies againstGSandGFAP. Arrows mice atP14were immunolabeledwith layers ofthe GFAP upregulation inthesuperficial Fig. 6.Formationofastrocytes with Arrows inH,IindicateS100 cells co-labeledwithOlig2andS100 cortex are shown.Arrows inGindicate GFAP. Regionsfrom superficiallayersofthe immunostained withOlig2,S100 s.d. ( >0 cellscounted, (>400 control and expressing cells(F).( Ki67 (G,H) and A N J O =3) atP7andP14;barsindicates.d. Barchartshowingtheaveragenumber ) , Bar chartindicatingthegross number ) , B P HematoxylinandEosin(H/E)staining ) Immunostaining ofKi67intheSVZ ) + K-M -ablated ( + cells inthecortex,respectively. and S100 C-F + 2 Cko cells perfield(0.4mm ) Corticesofcontrol and ) inthesuperficiallayersof Cko Corticesofcontrol and ) Olig2 + Olig2 ) cortices(>200cellscounted, cells (C,D)andGSGFAP co- G Olig2 Cko ␮ (I) miceatP14were ␤ mutant, respectively. ␤ m. mutant ( + Development 134(10) G GFAP co-expressing Olig2 cells perunitarea ) corticesatP14. G-I -ablated cortex. n =3); barsindicate ) Corticesofcontrol mutant (P)brains. Cko ␤ + Ctrl in the ) mice 2 Olig2- ) inthe ␤ G Cko ) and and Cko ␤ + . G and G +

DEVELOPMENT ablated corticesare shown.Expression ofGFAP isdistinct from thatofOPCmarkers.( ( suggest acell-autonomousfunctionof GS (red). Olig2 regulates astrocyte diversity population ofGFAP developing ,ourfate-mappingstudyfurtherindicatesthata developing astrocytesandtheirprogenitorsintheSVZ gray matter(Fig.9K).Together withOlig2expressioninimmature sustained upregulationofGFAP inapopulationofastrocytesthe severely compromisedinthewhitematter, whereasthereisa ( Fig. 7.AbsenceofectopicGFAP expression inoligodendroglial precursors ofthe mouse cortexatP14wasimmunostainedwithGFAP andOPCmarkersNG2(A-A astrocytes in cord. Themechanismsunderlying thedeficitofwhitematter astrocyte formationinthewhite matterofthebrainandspinal By contrast,ourdataindicate that Olig2functionisrequiredfor a GFAP repressionfunctionofOlig2suggestedby invitrostudies. developing astrocytesofthe throughout adulthood(Fig.9K). AbnormalGFAP upregulationin ablated cellsexpressingthe specific withinthebrain.Ingraymatter, differentiation doesnotappear tobeuniform,butratherregion- analysis, however, indicatesthatthefunction ofOlig2inastrocyte (Fukuda etal.,2004;Gabay2003).Ourinvivomutagenesis In vitrostudiesindicatethatOlig2inhibitsastrocytedifferentiation in thecortex development ofgrayandwhitematterastrocytes Differential requirement ofOlig2function inthe astrocytes andtheirprogenitors. part, attributedtothedirectlossof development andthatabnormalastrocyteis,atleastin Olig2 to alleviatelikelyGFAP repressioninapopulationofpresumptive G Cko , In theabsenceof H C ) Thecorpuscallosumofcontrol ( ) mutantcorticesatP14were subjectedtoinsituhybridizationlabelingfor + cortical astrocytesandleadstopersistentGFAP expression Olig2 mutants arenot entirelyclearatpresent.Itis + Olig2 cortical astrocytesisderivedfrom Olig2-3 , astrocyteformationinthecortexis Olig2 mutant cortexisconsistentwith Ј Ctrl UTR Olig2 C ) and Olig2 . Thus,theseobservations function indeveloping Olig2 Olig2 in corticalastrocyte ( ablation appears Cko C ) mutantcorticesatP14were immunostainedwithantibodiesagainstGFAP (green) and Olig2 - caused byCNP-Cre-mediated astrocyte formation, thereremainsthe possibilitythat formation. Conversely, giventhat insufficient toaccountforthedeficitinwhitematterastrocyte presence inthewhitematter. Thus,hypomyelinationaloneis hypomyelination doesnotpreclude formationofastrocytesandtheir GFAP upregulationoralterastrocyteformation,indicating that lineage cellsincludingoligodendrocyte progenitorsdoesnotcause progenitor/stem cellsbecauseGFAP astrocyte formationisunlikelytobeduealossofneural lineage tracingstudies.Therequirementfor somehow endupinthecortex.Thiswillberesolvedfuturecell possible thattheastrocytesoriginallydestinedforwhitematter of axonal proteinexpressionordegenerationinthewhitematter 2006). However, wedonotobservesignificant, ifany, deficitin number ofproliferativecells(Ki67 In addition,wedonotobserveanysignificantalterationinthe Olig2 ablation suggeststhat of well-definedastrocyteprecursormarkers.Temporally specific significant numberofproliferativeOPCsandhamperedbythe lack precursor proliferationdeficit,whichmightbeobscuredamidsta stages, althoughthisdoesnotexcludethepossibilityofastrocyte cortex andthewhitematterof myelination deficitinthewhitematterof incompatible withwhitematterastrocyteformationasthere isa Alternatively, hypomyelinatedaxonsmaycreateanenvironment at neonatalstagesintomatureastrocytesinthecerebralwhitematter. neural progenitorcellsordevelopingimmatureastrocyteprecursors Cko Mbp Љ ) andPDGF mutants aremaintainedatacomparableleveltothecontrol. G C-F (C,D) and mice. Inaddition,dysmyelinationinthewhitematter Olig2 ) Coronal sectionsofcontrol ( ␣ R (B-B -ablated cortex. Gfap Љ Olig2 ). Regionsofthesuperficiallayers (E,F). Arrows indicatethecorticalgraymatter. is requiredforthedifferentiation of Olig2 Olig2 Olig2 ( + A-B RESEARCH ARTICLE or BrdU + precursor cellsintheSVZof Љ ablation inoligodendrocyte ) The Ctrl mutants atearlypostnatal is requiredforwhitematter C Cko ) and + Olig2 ) orincelldeaththe Olig2 G Olig2Cko;CNP ( mice (Yue etal., Cko in whitematter G ) mutant Olig2- 1895 Cre

DEVELOPMENT expression isobservedinapopulationofdevelopingGFAP with astrocyte-specificdeficits.Nonetheless,becauseOlig2 in thefuturebyexaminingoligodendrocyteformationanimals prior tooligodendrocytes.Thispossibilitywillbeaddressedfurther whicharenormallygenerated by thelossofwhitematterastrocytes, oligodendrocyte deficitinthe 1896 equally inthedeveloping CNS. transcriptional regulation,suggesting thatastrocytesarenotcreated not onlyintheirspatiallocations andmorphologiesbutalsointheir observations indicatethatgray andwhitematterastrocytesdiffer null mutations(ZhouandAnderson, 2002).Therefore,our GFAP expressionwasonlyobservedwithboth with observationsintheembryonic spinalcordwhereincreased promoter activityinthesecells(Xinetal.,2005).Thisisconsistent expression ofOlig1,which,likeOlig2,couldrepress upregulation inthedeepcorticallayersmaybeattributableto the superficial layersandnotinthedeepof of allcorticalastrocytes,weonlyobserved in developingastrocytesandtheirprogenitors. astrocytes is,atleastinpart,attributedtothelossofOlig2function matter astrocytes,thedifferentiation deficitofwhitematter different layersdiffer intheirresponsesto cortex. Ourobservationsuggeststhatastrocytespopulating Gfap expression of precursors. Inaddition, downregulation ofboth downregulation of layers. The Although upregulation inthesuperficiallayers.Theabsenceof RESEARCH ARTICLE Gfap Olig2 Gfap upregulation appearstooccurinparallelwiththe loss atearlystagesmayaffect thedevelopment repressors suchas Olig1 Olig1 Olig2 in the Olig2 and ablation maydifferentially affect Olig2 mutant mightbecausedinpart Olig2 Olig1 is likelytocontribute Gfap -ablated cortex.The Olig2 in different cortical upregulation inthe Olig1 ablation intheir Olig2- and - ablated + Olig2 white Gfap Gfap - early postnatalstages(Goldman,2004). radial glialcellsatearlyembryonicstagesandSVZprogenitors Cortical astrocytesformfromatleasttwodifferent origins–namely, matter astrocyte subpopulations developmental sources forcorticalgrayandwhite Temporally specific of genetic reasons forthisdiscrepancyareunclear. Itispossiblethattheeffects that matter astrocyteformation,incontrasttoourpresentstudyshowing provided evidencethat et al.,2005).However, Marshalletal. (Marshall etal.,2005) was introducedintoSVZprogenitorsatpostnatalstages(Marshall astrocyte differentiation when adominant-interferingformof observation isinkeepingwiththeinhibitoryeffects of formation ofwhitematterastrocytesinthedevelopingbrain.This and thedevelopingwhitematter, contributesignificantlytothe that postnatalprogenitorcells,whicharelikelytoresideintheSVZ differentiation deficitspecificallyinthewhitematter. Thissuggests Cre activityatearlypostnatalstagesleadstosevereastrocyte might bedifferent sourcesforcorticalastrocyte development,with specific alterationofastrocyte subpopulations suggeststhatthere a subpopulationofcorticalastrocytes. Bycomparison,region- white matterastrocyteformation, butalsoinGFAP upregulationin early embryonicandpostnatalstages, resultsinnotonlyadefect hGFAP-Cre activity, whichispresentincorticalprogenitorsatboth upregulation inthegraymatter. Bycontrast, Olig2 functionisperturbeddiverge betweenthesetwoapproaches. interfering formofOlig2,orthatthetimingandcelltypeswhere Interestingly, postnatalablationof Olig2 Olig2 is requiredforwhitematterastrocytegeneration.The ablation mightdiffer fromthose ofthedominant- Olig2 Olig2 Olig2C/+;Syn1 hybridization intheforebrain ofcontrol insitu expression wasexaminedby (green), asindicated.( to NeuN(red), calbindin(red) andGABA P14 were immunostainedwithantibodies and intense staininginthe Olig2Cko;Syn1 accessible totheantibodies. inthe be duetothefactthatdysmyelinating compared withthewild-typemice,might ( cortex. neuronal deficitinthe Fig. 8.Absenceofsignificant 1 (red) inthecortexofcontrol ( and axonalproteins NF200(red) andTau- ( mutant ( (CC) ofwild-type(K,M),and NF200 andTau-1 inthecorpuscallosum ( Ctrl G-J K-N function isrequiredforcorticalgray ImmunostainingofOlig2(green) ) G Olig2 Immunostainingofaxonalproteins ) ) and ablation reveals diverse ( Cko A-D mutant ( Olig2 Olig2- G Olig2 Olig2 ) Thecorticesfrom control ; L,N)mice.Themore Cre Cre Development 134(10) ablated ( ( does notleadtoGFAP ( Ctrl Cko mutant are more Cko ablation byinducible Olig2 S E ) and S Cko G ) mice. , F ; H,J)mice. Olig2 ) Cko Gfap G ablation with mice, as Olig2 G -ablated ) miceat Ctrl Olig2 G Olig2 ; G,I) on

DEVELOPMENT adulthood. Bycontrast, expression inadulthood.Intheabsenceof astrocyte maturationinbraindevelopment.GFAP downregulation inmature astrocytes suggeststhatother factorsalsonegatively expression isabsentinmature astrocytes, suggestingthattheOlig2expression levelisdownregulated incorticalastrocytes d Beginning atpostnatalweek2,theOlig2expression levelisreduced inastrocytes andGFAP expression becomesundetectable.In expressed incorticalneuralprogenitor cells(NP).Atearly postnatalstages,Olig2isexpressed inimmature developingastroc Olig2-3 Cre-mediated excisionof showing fate-mappingof Gfap superficial corticallayersof other sourcesof astrocytesmightcontributeto thedevelopmentof formation ofasubsetastrocytes inthewhitematter. Alternatively, mutant micesuggeststhat progenitor cellstowhitematter astrocytes. matter astrocytesandasignificant contributionofpostnatal a majorcontributionofearlyembryonic corticalprogenitorstogray Olig2 regulates astrocyte diversity UTR Fig. 9.Apopulationofcorticalastrocytes withGFAP upregulation isderivedfrom examined byinsituhybridizationthecoronal sectionofcontrol and the RosaYFPreporter gene(Srinivasetal.,2001)generatedoffspring with and belowthedashedlineinindicatesuperficiallayerswhitemattercortex,respectively. ( expressing cellsinthedeepcorticallayers.Boxedarea inIisshownathighmagnificationJ.Arrowheads inJindicate cel The presenceofresidualastrocytes inthewhitematterof and in thegraymatter. Arrows aboveandbelowthelongerdashedlineindicatesuperficialdeepcorticallayers,respective Ј UTR Gfap and . ( K ) Schematicdepictingcorticalastrocyte developmentinrelation toOlig2expression. Inthenormaldevelopingbrain, Olig2is Gfap a xmndb obei iuhbiiainonsectionsofthe was examinedbydoubleinsituhybridization Olig2 loxP Olig2 Olig2 ablation results inadefecttheformationofwhitematterastrocyte subpopulations(lowerpanel). sites (blacktriangles).Redlineindicates Olig2 -ablated cellsafterhGFAPCre-mediated recombination. Breeding ofhGFAP-Cre micewith -ablated triple-transgenicmiceatP14.Arrowheads indicatecellsco-labeledwithYFPandGFAP. ( might onlyberequiredforthe Olig2 , GFAP expression issustainedorupregulated inapopulationofcorticalastrocytes throughout Olig2 Olig2 Olig2 Olig2 - -ablated and whitematter astrocytesmightexistinthedeveloping CNS. observations suggestthatdiverse developmentalsourcesforgray contribute toastrocyteformation inthewhitematter. Together, our cells (Yue etal.,2006),ventrallyderivedastrocytes maymigrateand hGFAP-Cre activityismainlyactiveindorsal corticalprogenitor remain inthewhitematteris presentlyunknown;however, as white matterastrocytes.Theexact sourcefortheastrocytesthat 3 Ј UTR ablation andactivationofYFPexpression inthesamecellsafter . ( Cko H ) ImmunostainingwithantibodiestoYFPandGFAP inthe Olig2 G Olig2 cortices atP8(A,B)andP14(C,D).Arrowheads above -ablated cortexatP14.Arrows inIindicate -ablated cells. E , F ) Insituhybridizationfor ( A-D RESEARCH ARTICLE ) Expression of Olig2 ls co-expressing ytes (A)atahighlevel. uring theprocess of -floxed micecarrying adulthood,Olig2 regulate GFAP I , J ly. ( ) Expression of Olig1 G Olig1 ) Schematic Olig2 was Olig2-3 - and 1897 Ј

DEVELOPMENT the regulate anintrinsicfateswitchforglialsubtypes.Intriguingly, in progenitor cellsinvitro,thisraisesthepossibilitythat Since Olig2negativelyregulatesastrocytedifferentiation inneural astroglial lineagedevelopment requirements forcorticaloligodendroglial and Distinct andconvergentdevelopmental in thedevelopingspinalcordwhereOlig2playsanessentialrole. the presentablationconditions,incontrasttomotoneuronformation does notsignificantlyalterneurogenesisinthecortex,atleastunder Olig2 Nonetheless, thelackofanyobviousneuronalabnormalityin addition toinhibitingOlig2,therebyalteringcorticalneurogenesis. with othertranscriptionalregulatorssuchasneuralbHLHfactorsin dominant-negative formsofOlig2usedcouldpotentiallyinteract or thealterationofneuralpatterningincortex.Alternately, the We donotobserve,however, anyappreciableincreaseincelldeath neurons subsequentlyeliminatedthroughprogrammedcelldeath. might leadtoanincreaseinneuronalformation,withnewlyformed discrepancy isthat (Buffo etal.,2005).Onepotentialexplanationforthis (Marshall etal.,2005)andtonewneuronformationinresponse of Olig2leadstoanincreaseinneurogenesisduringdevelopment recent studiesindicatethatexpressionofdominant-negativeforms alteration incorticalneuronalformationisrathersurprisingbecause abnormality incorticalneurogenesis.Thelackofanyovert and early-postmitoticneuronsdoesnotleadtoasignificant funded bygrantsfrom theNationalMultiple Sclerosis Society, March ofDimes helpful suggestions andKendyXainfortechnicalassistance. Thisstudywas mice, respectively. We alsothankDrsRobertMillerandMahendraRaofor Costantini forproviding hGFAP-Cre, Foxg1-Cre, CNP-Cre andRosaYFPreporter We thankDrsAlbeeMessing,SusanMcConnell,KlausA.Nave andFrank fact that astrocytes ifculturedundercertainconditions(Raff etal.,1983).The from theneonatalratopticnervetractcandifferentiate intofibrous differentiation inthewhitemattertract.Intriguingly, OPCsisolated there isacommonrequirementfor oligodendrocytes andastrocytesinthewhitematter, suggestingthat (Yue etal.,2006). into maturemyelinatingoligodendrocytes fate intheabsenceof oligodendroglial lineagecellsinthecortexdonotadoptanastrocytic of GFAP inthecortex.Together, thesedatasuggestthatcommitted oligodendrocyte-specific CNP-Crelinedoesnotleadtoupregulation Olig2 GFAP expressiondonotacquireGFAP expression.Furthermore, cord of Although thereisadeficitinmotoneuronformationthespinal Olig2 functionincorticalneurogenesis 1898 highlight importantrolesof the developmentofgrayandwhite astrocytesubpopulationsand specific the whitematterduringbraindevelopment.Thus,spatiotemporal- regulatory mechanismbetweenoligodendrocytesandastrocytes in developmental mechanisms,althoughtheremightbeaconvergent astrocytes inthegrayandwhitematterareregulatedbydistinct absence of be obtained.Sinceastrocytesinthegraymatterareformed the although furtherevidenceforthiscelllineageconnectionneeds to astrocytes inthewhitemattermightdevelopfromsamesource, astrocyte differentiation suggests thatoligodendrocytesand Conversely, Olig2- -ablated cortexsuggeststhateliminationofOlig2function RESEARCH ARTICLE Olig2 ablation inoligodendrocytelineagecellswiththe Olig2 Olig2 ablated cortex,OPCspresentwithinthedomainofectopic Olig2 -null mice, ablation revealsdivergent regulatory mechanismsfor is requiredforbotholigodendrocyteandwhitematter Olig2 , ourresultssuggestthatthedevelopmentof Olig2 Olig2 ablation leadstoadecreaseinboth Olig2 althoughtheyfailtofurtherdifferentiate , ablation inthecorticalprogenitorcells Olig2 ablation incorticalprogenitorcells in glialsubtypedevelopment. 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DEVELOPMENT