independent oftheroleNotchinmaintenanceundifferentiated neuralprogenitors. of aglial-specificationgene.TheseresultsdemonstratethatphysiologicalNotchsignalingisrequiredforgliogenesisinvivo Rbpsuh undifferentiated progenitorsfromthePNSandspinalcorddespitetheirfailuretoformgliainvivo.Inprogenitor in glialspecificationordifferentiation, notprematuredepletionofneuralprogenitors,becausewewereabletoculture spinal cord, binding protein(RBP/J)thatisrequiredforcanonicalsignalingbyallNotchreceptors.InmostregionsofthedevelopingPNS transcriptional effectors al., 1995).Overexpression ofactivated Notch1,oritsdownstream central (CNS)nervous systems(Lindselletal.,1996;Williams et are expressed throughoutthedeveloping peripheral(PNS)and undifferentiated progenitors.MultipleNotchreceptorsandligands components eitherpromotesgliogenesisorthemaintenanceof dependent effects, althoughoverexpression ofNotchpathway Bor andGiangrande,2001). promotes neurogenesisbyinhibitingtheexpression of such as gliogenesis, includingpromotingtheexpression ofgliogenic genes (Umesono etal.,2002).Insomelineages,Notchsignalingpromotes Drosophila the necessityofNotchsignalingduringneuraldevelopment in 1999; Harris,1997;Morrison,2001).However, geneticanalysisof decisions duringneuraldevelopment (Artavanis-Tsakonas etal., gliogenesis invertebrates. Notchsignalingregulates binaryfate the mechanismsthatregulate thetransitionfromneurogenesis to A fundamentalquestionindevelopmental neurobiologyconcerns INTRODUCTION Morrison etal.,2000).Inthiscase,Notchsignalingdoes not (NCSCs) inculturetoundergo gliogenesis(Kubu etal.,2002; the Notchliganddelta-like 1(Dll1)instructsneuralcreststem cells astrocytes inculture(Tanigaki etal.,2001).Inthedeveloping PNS, 2004), andinstructsadulthippocampusprogenitorstobecome of radialgliainthetelencephalon(Gaianoetal.,2000;Yoon etal., 2001). Overexpression ofactivated Notch1promotestheformation retina (Furukawa etal.,2000;HojoScheer Accepted 12April 2007 † University, Allendale,MI,49401-9401,USA *Present address: DepartmentofBiomedicalandHealthSciences,GrandValley State 48109-2216, USA. Medicine, andCenterforStemCellBiology, UniversityofMichigan,Ann Arbor, MI Howard HughesMedicalInstitute,LifeSciencesDepartment ofInternal KEY WORDS:Notch,Gliogenesis,Neuralstemcells,crest, Centralnervoussystem,Spinalcord, Mouse conditionally deleted progenitors. ThisraisesthequestionofwhetherphysiologicalNotchsignalingregulatesgliogenesisinvivo.To testthis,we Constitutive activationoftheNotchpathwaycanpromotegliogenesisbyperipheral(PNS)andcentral(CNS)nervoussystem Merritt K.Taylor*, KellyYeager andSeanJ.Morrison developing peripheralandcentralnervoussystems Physiological Notchsignalingpromotesgliogenesisinthe (2007)doi:10.1242/dev.005520 Development 134,2435-2447 Author forcorrespondence (e-mail:[email protected]) Notch signalinginvertebrates alsohascomplex andcontext- gcm was requiredtomaintainSox9expressionduringgliogenesis,demonstratingthatNotchsignalingpromotesthe (Udolph etal.,2001).Inotherlineages,Notchsignaling Rbpsuh suggests acomplex andcontext-dependent role deletion causedonlymilddefectsinneurogenesis,butseveregliogenesis.Theseresultedfrom Rbpsuh Hes1 or ( Rbpj Hes5 frommousePNSorCNSprogenitorsusing ) , promotesgliogenesisinthe gcm (Van De † Notch1 neural development areregulated byNotchinvivo. Deletionof ligands inmammalshasmade itdifficult totestwhataspectsof understand itsphysiologicalrole inneuraldevelopment. Notch signalingisnecessaryforgliogenesisinvivo inorderto vivo. For allofthesereasons,it is crucialtodeterminewhether environment mightleadtooutcomesthat would notbeobserved in progenitors, itispossiblethatunphysiologicalaspectsoftheculture Furthermore, whentheNotchpathway isactivated incultured hematopoietic stemcellfrequency orfunction(Mancinietal.,2005). conditionally deletedfrommicetherewas noeffect on cell self-renewal anddifferentiation, but whenthesegeneswere Varnum-Finney etal.,1998)overexpression onhematopoieticstem Genome Informatics)(Jonesetal.,1998;Karanu2000; al., 2002;Varnum-Finney etal.,2000)orjagged1( (Bigas etal.,1998;Carlesso1999;Milner1996;Stier et et al.,2003).Anumberofstudieshave reported effects of lack ofobvious neuralphenotypesin adult hippocampusprogenitors(Tanigaki etal.,2001)despitethe Overexpression of overestimate thephysiologicalroleofNotchsignaling. Notch signalingalsoregulates gliogenesisinvivo. in vitroanalyses,raisingthequestionofwhetherphysiological virtually allofthesedatawereobtainedingain-of-functionand/or indicate thatincreasedNotchsignalingcanpromotegliogenesis, 2005; Geetal.,2002;Tanigaki etal.,2001).Althoughthesestudies promoters ofglialgenesandactivate transcription(Anthony etal., activated Notchtoregulate transcription)candirectlybindthe binding proteinthatinteractswiththeintracellulardomainof promotes gliallineagedeterminationinvertebrates, RBP/J (aDNA- (Morrison etal.,2000). Dll1 causesanirreversible commitmenttoglialdifferentiation simply inhibitneurogenesis,becauseeven transientexposure to ( (Xue etal.,1999), Dll4 The presenceoffourNotchreceptors andatleastfive Notch Overexpression ofNotchpathway componentscansometimes Consistent withtheideathatNotchcansendapositive signalthat – MouseGenome Informatics)(Krebsetal.,2004) leadsto (Swiatek etal.,1994), Wnt1-Cre Dll1 Notch3 or Haed nei ta. 97 rdlalk 4 (Hrabe deAngelisetal.,1997)or delta-like Nestin-Cre promotes astrocyte differentiation from Notch2 . RESEARCH ARTICLE Rbpsuh Notch3 (Hamada etal.,1999), -deficient mice(Krebs encodes aDNA- Jag1 , – Mouse and s, Notch1 2435 Jag1

DEVELOPMENT al., 2003).Deletionof increased neurogenesisanddefectsingliogenesis(Grandbarbeet progenitors culturedfrom subsequent reductioningliogenesis(Lutolfetal.,2002).Neural cerebellum leadstoprematureneuronaldifferentiation anda stem cells(Hitoshietal.,2002).Deletionof Notch1- 1997). Deletionof mediate theirtranscriptionaleffects (Katoetal.,1996; Katoetal., intracellular domainsofallfourNotchreceptorsandisrequiredto RBP/J (delaPompaetal.,1997).interactswiththe neuronal differentiation occursinembryosdeficient for signaling regulates CNSprogenitormaintenance.Premature a physiologicalroleinmany aspectsofneuraldevelopment. result, ithasbeendifficult toassesswhetherNotchsignalingplays may leadtofunctionalredundancy invivo (Krebsetal.,2000).Asa cases, theoverlapping expression ofmultiplereceptorsandligands little physiologicalfunctioninvivo (Krebsetal.,2003).Inother 1998) leadstomilderphenotypes.Somereceptors/ligandsmayhave 2002), orjagged2( 3( like premature depletionofundifferentiated progenitors. gliogenesis, orwhetherthedefectsingliogenesisaresecondarytoa maintain undifferentiated progenitorsandsubsequentlytopromote whether Notchactsatmultiplestagesofneuraldevelopment, first to (Anthony etal.,2005).Itisnotclearfromtheseobservations levels ofBFABP expression inprogenitorsanddifferentiated cells uncertain whetherthisreflectedreducedgliogenesisorjust – MouseGenomeInformatics)expression invivo, but itwas progenitors reducedbrainfatty acid-bindingprotein(BFABP; Fabp7 (Hatakeyama etal.,2006).Conditionaldeletionof as toanatomicaldefectsincranialnerves andsensory ganglia differentiation inthespinalcord(Hatakeyama etal.,2004),aswell leads toalossofneuroepithelialcellsandprematureneuronal target genesthatactdownstream ofRBP/J(Ohtsukaetal.,1999), deletes genesin neuroectodermalprogenitors inthedeveloping of the effects ofthesemutationsongliogenesis.By contrast, deletion to embryonicday(E)11.5,beforethereisanopportunitystudy severe developmental defectsandthedeathofmouseembryosprior 2436 Rbpsuh tubes of undifferentiated neural progenitors.We also examined theneural is requiredtopromotegliogenesis beyond simplymaintaining undifferentiated NCSCs suggeststhatphysiologicalNotchsignaling reduction ingliogenesisdespite theongoingpresenceof throughout allregions ofthelategestationPNS.Thesevere some locations.However, atleastsomeNCSCsremainedpresent signaling doesplayaroleinthemaintenanceofNCSCsatleast regions ofthePNSinabsence the PNS.We observed areductioninthenumberofNCSCssome neurogenesis, but severely reduced gliogenesisthroughoutmostof Rbpsuh Tanigaki etal.,2004). We observed that conditionaldeletionof and lossof were previously generatedandshown topermitconditionaldeletion conditionally delete 2000; Josephetal.,2004;Zirlinger2002).The neural crestcells(Chaietal.,2000;Hari2002;Jiang al., induces efficient recombinationthroughoutcephalicandtrunk Loss-of-function experiments indicatethatphysiologicalNotch To examine this wegenerated Notch3 RESEARCH ARTICLE Dll3 ( or Rbpj from neuralcrestcellshadonlyminoreffects on Nestin-Cre (Krebs etal.,2003), Rbpsuh Rbpsuh MouseGenomeInformatics)(Dunwoodie etal., – – MouseGenomeInformatics),thegenethatencodes Rbpsuh Jag2 -deficient embryosalsohave many fewer CNS function (Hanetal.,2002;Tanigaki etal.,2002; + Rbpsuh Rbpsuh MouseGenomeInformatics)(Jiangetal., – thus abolishescanonicalNotchsignaling. Notch1 Delta1 fl/fl from neuralcrestcells. Notch4 mice. -deficient embryosalsoexhibit Wnt1-Cre and Rbpsuh (Krebs etal.,2000),delta- Nestin-Cre Notch3 Hes1 , indicatingthatNotch + Rbpsuh and from forebrain Rbpsuh Notch1 conditionally Hes5 fl/fl Notch1 Wnt1-Cre mice to , Notch fl in the mice or acetate, 10mMimidazole, 100mMNiSO times for1houreachwash andthenincubatedinNi-DAB (125mMsodium sodium acetate,10mMimidazole,pH 7.2with30%glacialaceticacid)three each wash. Theembryoswerewashed inacetate imidazolebuffer (175mM overnight at 4°C, andthenwashed againfive timesinPBSblockfor1hour rabbit peroxidase(Vector laboratories, Burlingame,CA,PI-1000,1:200) five timesinPBSblockfor1hourperwash andincubatedwithgoatanti- of anti-Sox10antibody(Chemicon AB5774) at4°C.Embryoswerewashed for 1houreachwash. Embryoswereincubatedovernight with1:50dilution containing 5%goatserum,0.2%Triton X-100,1%DMSOand0.5%BSA) washed inPBSbeforeblockingtwo washes ofPBSblock(PBS ProLong antifade solution(MolecularProbes,Eugene,OR). 2.5 nestin (BDPharmingen,611658,1:1000).Slideswerecounterstained in (p75NTR –MouseGenomeInformatics;ChemiconAB1554,1:5000)and from B.Novitch, University ofMichigan,AnnArbor, MI;1:20,000),p75 Informatics) (Chemicon,Temecula, CA,MAB377,1:1000),Olig2(gift Technology, Pasadena, CA; 1:50), NeuN(Neuna60–MouseGenome AB5804, 1:200),Sox10(giftfromD.Anderson,CaliforniaInstituteof 1:1000), BrdU(Caltag,Burlingame,CA,MD5000,1:200)Gfap (Sigma, 1:2000), activated caspase3(BDPharmingen,SanDiego, CA,559565, BFABP (giftfromT. Muller, Max-Delbruck-Center, Berlin,Germany; Genome Informatics)(Covance, Berkeley, CA,MMS-435P, 1:1000), (Tubb3 –Mouse room temperature.AntibodiesincludedthoseagainstTuJ1 secondaryantibody incubationfor1hourat 4°C, followed bywashing then overnight at were dilutedinmodified GSSandincubatedwiththesections Primaryantibodies (PBS containing5%goatserumand0.5%Triton X-100). sections (12 OCT (VWR,West Chester, PA) priortosnap-freezingandsectioning.Tissue washed inPBS,cryoprotected15%sucroseandmountedTissue-Tek necessary tofix theembryosfor2.5hoursat4°C.Thewerethen 4°C. For somemarkers (antibodies againstPdgfr were immediatelydissectedandfixed in4%paraformaldehydeovernight at of BrdU(Sigma,StLouis,MO)andsacrificed 30minuteslater. Embryos To examine BrdUincorporation,pregnant damswereinjectedwith50 Immunohistochemistry andtissuepreparation ATTGCTGTCACTTGGTCGTGG-3 gene incrossesinvolving used ongenomicDNA isolatedfrommousetissue.For detectionofthe Care ofAnimalsguidelines.For genotyping,thefollowing primerswere University ofMichiganaccordingtoUniversity CommitteeontheUseand Mice werehousedintheUnitforLaboratoryAnimalMedicineat Mice MATERIALS ANDMETHODS CNS. Notch signalingpromotesgliogenesisinthedeveloping PNSand more oligodendrocytes. Thesedatademonstratethatphysiological gliogenesis includingsignificantly fewer astrocytes andsignificantly the numbersofneuronsthatformed,but profoundeffects on et al.,2006).We againdetectedlittleeffect of CNS, includingwithintheneuraltube(Tronche etal.,1999;Yang with 5:1PBS:30%H E9.5 embryoswerefixed overnight in4%paraformaldehyde,thenbleached Whole-mount immunohistochemistry Anderson (WhiteandAnderson,1999). Wayne StateUniversity, Detroit, MI)was adaptedfromthatof Whiteand Rbpsuh AATGC-3 GTCGGA-3 wild-type TCTGTCCGTTTGC-3 bp PCRproduct. Rbpsuh The insitumethodfordetectionof For immunohistochemistry, tissuesectionswereblocked inmodified GSS ␮ g/ml DAPI for10minutesatroomtemperature,thenmountedusing allele: fl R,5 Ј Rbpsuh , yieldinga487bpPCRproduct.For detectionofthefloxed ␮ Ј Ј m) werecollectedusingaLeicacryostat. Rbpsuh and -GAAGGTCGGTTGACACCAGATAGC-3 Rbpsuh 2 allele: O fl F, 5 2 Ј at roomtemperaturefor3-5hours.Embryoswere , yieldinga210bpproduct.For detectionofthe Ј wt R,5 Rbpsuh -GCAATCCATCTTGTTCAATGGCC-3 Wnt1-Cre Ј -GCTTGAGGCTTGATGTTCT GT- Ј wt F, 5 Mbp and or 4 , 0.3mg/mlDAB) for20minutes. (probe was agiftfromA.Gow, Nestin-Cre Cre Ј -GTTCTTAACCTGTTG - Development 134(13) R, 5 ␣ Rbpsuh and Sox10),itwas Ј mice: -GAAAATGCT - Ј , yieldinga598 deletion on Cre F, 5 F, Ј ␮ Cre and g/g Ј -

DEVELOPMENT treated withpoly- the neurosphereswerereplatedtoadherentsix-wellplatesthathadbeen plate (Corning)andallowed togrow for14-16days.To testmultipotency, cell cultures,2000cellswereplatedperwellofasix-wellultra-low-binding culture medium(1%CEE,10ng/mlbFGFandIGF1).For CNS then allowed todifferentiate for8daysinlow-mitogen andgrowth factor NRG1- (Bixby etal.,2002).Whenindicated,PNSculturesweretreatedwithhuman Although germlinedeletionof littermate controls(seeFig.S1inthesupplementarymaterial). crest cellsthatappearedtomigratenormallycomparedwith cells (p75 development, weexamined thenumbersofmigratingneuralcrest expected inthesemice. the supplementarymaterial), defects inmigrationmightnotbe had beentreatedwithpoly- adherent PNScultures,500cellswereaddedperwellofsix-wellplatesthat neurons (TuJ1), andastrocytes (Gfap). Studies HybridomaBank,University ofIowa, Iowa City, IA;1:800ascites), (Molofsky etal.,2005)formarkers ofoligodendrocytes (O4,Developmental differentiation, adherentneurosphereswerefixed andstainedasdescribed Rbpsuh Wnt1-Cre RESULTS #291-G1). Allculturesweremaintainedat37°Cin6%CO nM)retinoicacid(Sigma)and20ng/mlhumanIGF1(R&DSystems, (110 culture mediumalsocontained15%chickembryoextract, 35ng/ml described byStempleandAnderson(StempleAnderson,1992)].PNS (R&D Systems,#236-EG))and10%chickembryoextract [preparedas pen/strep (Biowhittaker). CNSculturesalsocontained20ng/mlhumanEGF (Invitrogen), 2% B27(Invitrogen), 50 basic FGF(R&DSystems,Minneapolis,MN;#233-FB),1%N2 medium (Invitrogen, Carlsbad,CA)supplementedwith20ng/mlhuman The culturemediumwas a5:3mixture ofDMEM-low glucose:neurobasal Tissue culture counting cellsinTrypan Bluewithahemocytometer beforeculturing. fresh stainingmedium.Celldensityandviabilityweredeterminedby triturated beforebeingfiltered throughanylon meshandresuspendedin Dissociation was terminatedwithstaining mediumandthetissuewas lightly 0.025% trypsin/EDTA inCaandMg-free HBSSat37°Cfor2minutes. cord intoice-coldD-PBSbuffer. Thecellswerethendissociatedwith density andtoensurecompletedissociation. medium andcountedusingahemocytometer todeterminecellviability, necessary. Beforeaddingtoculture,cellswereresuspendedinstaining NY) withtheexception ofsympatheticchain,forwhichfiltration was not Cells werefiltered throughanylon screen(45 ME)] thatcontained25 3912), 10mMHEPESatpH7.4,1%pen/strep(BioWhittaker, Rockland quenched withstainingmedium[L15containing1mg/mlBSA(Sigma,A- HBSS (Invitrogen, #14175-095)at37°Cfor4minutes.Thedissociationwas type-4 collagenase(Worthington, Lakewood, NJ,#4186)inCaandMg-free dissociated in0.025%trypsin/EDTA (Invitrogen 25300-054)plus1mg/ml mouse embryosandcollectedinice-coldD-PBSbuffer. Thecellswerethen PNS tissues(DRG,sympatheticchainandgut)weredissectedfromE13.5 Isolation ofneuraltissue H Physiological Notchsignalspromote gliogenesis stored in100%methanoluntilphotographscouldbetaken. washed, dehydratedinareverse seriesofmethanoldehydration stepsand for 5-10minutestoformthedepositionproduct.Theembryoswerethen attributable todefects insomiteformation.Since Angelis etal.,1997),thisappearstobeatleastpartially formation andmigration(DeBellardetal.,2002;Hrabe de 2 CNS tissuewas dissectedfromtheE19.5embryonicupperthoracicspinal O To begin toexamine theeffect of 2 (0.0003%) was addedandtheembryoincubatedatroomtemperature ␤ fl/fl 1 (R&DSystems,#396-HB).Thecellswereculturedfor6days + + mice appeartohave normalsomites(seeFig.S1Din ) thatcolonized thegut,aswellnumbers of Rbpsuh D -lysine andallowed todifferentiate for5-7days.To assay fl/fl ␮ g/ml deoxyribonucleasetype1(Sigma,D-4527). mice formednormalnumbersofneural D -lysine andfibronectin aspreviously described Dll1 leads todefectsinneuralcrest ␮ M 2-mercaptoethanol,and1% Rbpsuh ␮ m, Sefar America,Depew, deletion onPNS 2 /balance air. For Wnt1-Cre + section (Fig.2E)orTuJ1 in gliogenesis,withalmostnoBFABP modest reductioninneurogenesis, therewas aprofoundreduction as many neuronspersection atE18.5(Fig.2F).Incontrasttothis many neuronspersectionascontrolgangliaatE14.5andonethird Wnt1-Cre less neurogenesiswas observed atlaterstagesofdevelopment in TuJ1 (Fig.2F)orperipherin(datanotshown) staining.Significantly in significant reductioninthenumbersofneuronsandgliapersection compared withlittermatecontrols.However, gut, with By E14.5,however, cleardifferences hademerged throughoutthe neurogenesis (gliogenesiswas notdetectedatthispoint)(Fig.1D). did notdetectany statisticallysignificant differences in that migratedthroughthegutatE10.5(Fig.1C).AtE10.5,wealso just adifference inBFABP expression, as were notabletodeterminetheeffect of sensory gangliaof detected noevidence ofprematureneuronaldifferentiation in (Fig. 1H).Thesedatademonstratethat addition ofthegliogenicfactor neuregulin1- with controlembryos(Fig.1H).Thisdifference was notrescuedby of NCSCsthatformedmultilineagecoloniesincultureascompared guts didhave anapproximatelythreefoldreduction inthefrequency neurons (TuJ1 Rbpsuh demonstrating thatvirtuallyno gliogenesisoccurredin sensory gangliabetweenE13.5 andE18.5(Fig.2A-F), Rbpsuh cultured gutNCSCsfrom subsequently forlineagedetermination. maintenance ofundifferentiated progenitorsorisalsorequired do notdistinguishwhether numbers ofneuronsandgliathroughoutthegut,althoughthesedata maintain normalnumbersofNCSCsandtogenerate littermate controlsinthenumberofp75 not detectany difference between hindgut thatarosefromthesemigratingneuralcrestcells.We did examining theconsequencesof required forthemaintenanceofgutNCSCs,but prevented usfrom Rbpsuh infrequent NCSCcoloniesthataroseinculturefrom controls toexamine thedifferentiation ofthesecells.However, the undergoing celldeath(notshown) in with virtuallynogliaobserved in (Fig. 1E).Thedifference ingliaatE14.5was particularlyprofound, fewer neuronsandgliapersection,relative tolittermatecontrols littermate controlsinthenumberofp75 p75 birth. postnatal gutdevelopment becausethemicediedwithinhoursof was nodifference between deletion indeveloping sensory(dorsalroot) ganglia.AtE10.5,there PNS development, weexamined theconsequencesof To gainfurtherinsight intotheroleofcanonicalNotchsignalingin gliogenesis insensoryganglia Rbpsuh differentiation inculture. Wnt1-Cre In anattempttodistinguishbetweenthesepossibilities,we We didnotdetectany difference intherateofproliferation + cells (Fig.1G)orthenumberofactivated caspase-3 fl/fl fl/fl allele. ThissupportedtheideathatNotchsignalingwas Wnt1-Cre + deletion leadstoprofound defectsin embryos. Thisreflectedadifference ingliogenesis,not mice consistentlyretainedatleastoneunrecombined Rbpsuh + Rbpsuh + ) andglia(BFABP Wnt1-Cre fl/fl + fl/fl Rbpsuh ganglia, whichhadapproximatelyhalfas guts persistedthroughE18.5(Fig.1F).We + Wnt1-Cre neurons persection(Fig.2F).We thus + fl/fl Wnt1-Cre Rbpsuh Rbpsuh embryos exhibiting significantly Rbpsuh Wnt1-Cre Wnt1-Cre + RESEARCH ARTICLE + ) intheforegut, midgutand Rbpsuh + Wnt1-Cre fl/fl glia in + is onlyrequiredforthe + deficiency ongutNCSC Rbpsuh embryos basedoneither neural crestprogenitors + Rbpsuh ␤ neural crestcellsper Wnt1-Cre fl/fl + + (Nrg) tothecultures 1 Wnt1-Cre Wnt1-Cre Rbpsuh Rbpsuh Rbpsuh + mice andlittermate Rbpsuh fl/fl is requiredto embryos and fl/fl fl/fl deletion on + + + Wnt1-Cre Wnt1-Cre Rbpsuh fl/fl Rbpsuh Rbpsuh guts. The mice and Rbpsuh guts as + 2437 cells fl/fl fl/fl fl/fl + +

DEVELOPMENT from 2G) orthefrequency ofapoptoticcells(Fig.2H)insensoryganglia as wedidnotobserve any differences intherateofproliferation(Fig. gliogenesis didnotreflectdifferences inproliferationorcelldeath, (see Fig.S2inthesupplementarymaterial).Thisdifference in embryos (Fig. 3A). Incontrasttothisreduction ingliogenesis, sensory gangliaalsoexhibited asimilarreductioninS100 2438 Gfap colonies andsignificantly fewer coloniesofalltypesthatcontained conditions, weobserved significantly ( glia-only coloniesandothercolonies) from progenitors persistedafter clonal densitytotestwhether NCSCsorotherneuralcrest gliogenesis invivo. to theonsetofgliogenesisorthatthey wereunabletoundergo that neuralcrestprogenitorswereeitherprematurelydepletedprior We cultureddissociated E13.5dorsalrootganglioncellsat + Wnt1-Cre glia (G-containing;theseincluded multilineagecolonies, RESEARCH ARTICLE + Rbpsuh fl/fl and controlembryos.Thesedatasuggest Rbpsuh P deletion. Understandard <0.05) fewer multilineage Wnt1-Cre + Rbpsuh ␤ + cells fl/fl from E13.5control (A)or percentage ofcellsfrom E13.5 no difference intherateofproliferation ofp75 were stainedforneurons (TuJ1) andglia(BFABP). Scalebar:10 (data notshown).( (foregut, midgutandhindgut)basedonstainingforactivatedcaspase3 development (E10.5,E14.5andE18.5)oratanylevelofthegut rate ofcelldeathbetweencontrol andmutantmiceatanystageof per genotype).Additionallythere wasnosignificantdifference inthe three tosevensectionspergutregion permouse,three tofourmice control andmutantembryos(BrdU wasadministered for30minutes; seven independentexperiments).Allerror barsindicates.d. medium andsupplementedwithneuregulin (+Nrg)(three to multilineage NCSCcoloniesinclonal-densitycultures inbothstandard significantly (*, embryos (thenumberofp75 the gutafter ThenumbersofNCSCs,neurons andgliaare reduced in Fig. 1. Wnt1-Cre ganglia of with glialpotentialpersistatleastthroughE13.5inthesensory Nonetheless, thesedatademonstratethatneuralcrestprogenitors progenitors thatformnociceptive neurons insensoryganglia. neurogenesis reflectsareductioninthesecondwave ofneurogenic deletion, althoughitispossiblethatthelate-onsetreduction in progenitors withglialpotentialinsensorygangliaafter only 23±12%ofwild-typelevels byqRT-PCR (datanotshown). rescue gliogenesisby culture mediumtoascertainwhetherstimulationbyNrg might depletion ofprogenitors,weaddedthegliogenicfactor Nrg tothe gut didnotdiffer betweenE10.5control and Wnt1-Cre (Fig. 3A). myofibroblast-containing coloniesandneuron-containing owl yein to wildtype numbers ofneurons (TuJ1 levels ofthedevelopinggut(foregut, midgutandhindgut),the ( region permouse,three micepergenotype). deficient neuralcrestprogenitorsinculture. that Nrg was abletobypasstheblockingliogenesis in maintenance ofstem/progenitor cells.Ourresultsfurthersuggest signaling isrequiredforgliogenesis beyond simplypromotingthe gliogenesis instandardmedium, demonstratesthatcanonicalNotch progenitors toundergo gliogenesisinvivo, ortoexhibit normal ( complete lossof Rbpsuh variable. Insomeexperiments, allcolonies exhibited complete genotyping individual colonies, (Dong etal.,1995;Morrison1999;Shah1994).Upon survival andgliallineagedeterminationbyneuralcrestprogenitors myogenesis byuncommittedprogenitors;Nrg promotesboth as increasedgliogenesisattheexpense ofneurogenesis and Nrg islikely toreflectincreasedsurvival byglialprogenitors, aswell containing andmyofibroblast-containing coloniesinthepresenceof (Fig. 3B).Theincreaseinglialcoloniesandthedecreasesneuron- of Nrg, weobserved normalnumbersofglia-containingcolonies dissected sensorygangliafrom C Thenumbersofp75 ) We thusfoundnoevidence foradepletionofneuralcrest To testwhetherthepaucityofglia-containingcoloniesfrom excision, but onaverage, 65% ofcoloniesexhibited a + + Rbpsuh Wnt1-Cre Rbpsuh Rbpsuh Wnt1-Cre P <0.05) loweratE14.5(E)andE18.5(F).( H fl/fl Rbpsuh ) However, asignificantly(*, fl/fl Rbpsuh + embryos reflectedadefectingliogenesisor deletion. + progenitors formednormalfrequenciesof + neural crest cellsthatinitiallycolonizedthe Rbpsuh Wnt1-Cre + Rbpsuh ) andglia(BFABP . + cells persection;three sectionspergut Rbpsuh Wnt1-Cre dfcetpoeios Inthe presence -deficient progenitors. Wnt1-Cre fl/fl fl/fl ( Rbpsuh A + , embryos atE10.5(D)but Rbpsuh B embryos. Thefailure of these ) Transverse sectionsoftheguts expression levels infreshly + Rbpsuh excision was extensive but + + fl/fl ) persectionwere similar Rbpsuh Wnt1-Cre D-F + Development 134(13) (B) mouseembryos cells atE14.5between P ) Atrepresentative fl/fl <0.05) lower guts gaveriseto fl/fl + embryos were Rbpsuh G ) There was ␮ Rbpsuh Rbpsuh m. fl/fl -

DEVELOPMENT were stainedforneurons (TuJ1 similar numbersofp75 Neural crestmigrationintosympatheticgangliaappearednormalas sympathetic ganglia gliogenesis despitenormalneurogenesis in Rbpsuh Physiological Notchsignalspromote gliogenesis reduced in detected atthisstage),neurogenesis wassignificantly(*, difference inneurogenesis wasobservedatE10.5(gliacouldnotbe sections permouseandthree micepergenotype).( neural crest progenitors thatinitiallymigratedintothe ganglion(three ( Fig. 2. activated caspase 3).Error barsindicates.d. vivo atE14.5),orcelldeath(H,the percentage ofcellsexpressing percentage ofcellsthatincorporated a30-minutepulseofBrdU in did notdetectanydifference intherateofproliferation (G,the could bedetectedin Gliogenesis wasevenmore profoundly reduced, asvirtuallynoglia (four toninesectionspermouse, seven toeightmicepergenotype). ganglia of Cre bar: 50 Wnt1-Cre A-D + ) Transverse sectionsofdorsalroot gangliafrom control and Rbpsuh Rbpsuh ␮ m. ( + Wnt1-Cre deletion leadstoprofound defectsin Rbpsuh Wnt1-Cre fl/fl E ) There wasnodifference betweencontrol and embryos atE10.5intermsofthenumberp75 is required forgliogenesisinsensoryganglia. fl/fl + Wnt1-Cre mouse embryosatE14.5(A,B)andE18.5(C,D) Rbpsuh + Rbpsuh + cells wereobserved inthesympathetic fl/fl + + , green) andglia(BFABP Rbpsuh sensory gangliaatE14.5andE18.5 fl/fl mice andcontrollittermatesat fl/fl sensory ganglia.( F ) Althoughno + , red). Scale P <0.05) G , Wnt1- H + ) We frequency ofactivated caspase-3 proliferation orcelldeath,asBrdUincorporation(Fig.4E)andthe 4D). Thisdifference ingliogenesisdidnotreflect differences incell observed atE14.5andeightfoldfewer gliaobserved atE18.5(Fig. cultured from (Fig. 5B).Infourindependentexperiments, 89±21% of colonies that containedgliainnumbersweresimilartocontrolembryos locations ofChx10 certain p2-domainprogenitors,whichnormallygive risetoGata2 Rbpsuh supplementary material).Thesedataraisedthepossibility that material). Moreover, incontrasttocontrolmice,theS100 sympathetic chain(seeFig.S3Aversus Einthesupplementary grossly reducedin Genome Informatics)cellsorNgn2 on TuJ1 stainingorNeuN stainingwas notaffected by number ofneuronspercross-section throughthespinalcordbased pMN andp2domainsofthe developing spinalcord.Thetotal differences tocontrol embryosinglialfate determinationwithinthe Wnt1-Cre embryos andlittermatecontrols.Instandardmedium,cellsfrom ganglion cellsatclonaldensityfromE13.5 gliogenesis. the sympatheticchaininabsenceof 1999), thesedatasuggestthatundifferentiated progenitors persistin material). SinceNCSCsexpress p75andS100 mice alsoexpressed p75(seeFig.S3Bversus Finthesupplementary Cre development fromE10.5toE18.5inthesympatheticchainof E10.5 (Fig.4C).Neurogenesiswas alsonormalatallstagesof interneurons, Chx10 reduction inthenumberofGata2 normal. We didhowever observe asmallbut statisticallysignificant suggested thatoverall patterningwithinthespinal cordwas grossly Informatics) cells(seeFig.S4inthesupplementarymaterial). This Rbpsuh sympathetic chainof that undifferentiated neuralcrestprogenitorspersistinthe complete Cre controls. AtE11.5,wedidnotdetectany difference between developing spinalcord(Jessell,2000;Muroyama etal.,2005). difference inBFABP expression after that werepresentinsympatheticgangliafrom Wnt1-Cre or any Gfap supplemented withNrg, sympatheticchaincellsfrom control embryos(Fig.5A).However, whenthemediumwas a requirementfor depletion ofneuralcrestprogenitorsandmustinsteadbecausedby defect ingliogenesisobserved invivo cannotbecausedbya gliogenesis invivo orintheabsenceofNrg inculture.Thus,the Cre Cre development in To examine the roleof the CNS Rbpsuh differentiation inthesympatheticchain. To testthisproposition,wecultureddissociatedsympathetic By E14.5, + + + + Rbpsuh Rbpsuh Rbpsuh Rbpsuh fl/fl is requiredforthegenerationofnormalnumbersatleast Rbpsuh + + embryos formedmultilineagecoloniesandother is alsorequired fornormalgliogenesisin Rbpsuh Rbpsuh + fl/fl fl/fl fl/fl fl/fl glia-containing colonies,incontrasttocellsfrom Wnt1-Cre Nestin-Cre mice alsohadfewer S100 embryos andlittermatecontrolsinthenumbersor mice (Fig.4A,B,D).Bycontrast,gliogenesiswas spinal cord(seeFig.S4Cversus D,Kinthe Nestin-Cre excision (datanotshown). Thesedatademonstrate Wnt1-Cre fl/fl Rbpsuh fl/fl + + Wnt1-Cre embryos rarelyformedmultilineagecolonies cells, Olig2 interneurons, andBFABP embryos. Thisalsodidnotsimplyreflecta Rbpsuh + + Rbpsuh in gliallineagedeterminationor + + Rbpsuh Rbpsuh Rbpsuh + in theCNS,westudiedspinalcord + Rbpsuh cells (Fig.4F)appearednormalin + + fl/fl cells persectioninthe RESEARCH ARTICLE cells, HB9 fl/fl + fl/fl Rbpsuh sympathetic chainexhibited (Neurog2 –MouseGenome fl/fl Rbpsuh fl/fl mice, withnoBFABP embryos exhibited clear ␤ embryos andlittermate mice, but fail toundergo + cells persectioninthe Wnt1-Cre deletion, asthe Wnt1-Cre , but fail toundergo + ␤ + (Hlxb9 –Mouse (Morrison etal., astrocytes inthe + + Wnt1-Cre Rbpsuh Rbpsuh ␤ Rbpsuh Nestin- Nestin- + Wnt1- Wnt1- 2439 + cells glia fl/fl fl/fl + +

DEVELOPMENT labeling in progenitors (Fig.6A,B,G-I)andasignificant increase inOlig2 were muchmorestrikinglyaffected in interneurons (seeFig.S5inthesupplementarymaterial).Glialfates significant ( in theabsenceof preferentially expanded andastrocyte lineagecellsfailed toexpand cannot ruleoutthepossibility that oligodendrocyte lineagecells a fate normallyassociated withthepMNdomain.Nonetheless,we normally acquireanastrocyte fate insteadbecameoligodendrocytes, in theabsenceof oligodendrocyte progenitors(Fig.6C,D,G-I).Thissuggestedthat, embryos, withasignificant reductioninBFABP Rbpsuh deletion (Fig.6E-G).ThetotalnumberofHB9 2440 whereas Olig2 exhibited greaterBrdUlabeling incontrolmice(Fig.6Lversus M), neuronal identitywithinthep2domainwas affected, as deletion (seeFig.S5E-Ginthesupplementarymaterial).However, arise fromthepMNdomain)was alsonotaffected by in thenumberofChx10 RESEARCH ARTICLE fl/fl embryos hadamodestbut significant ( Nestin-Cre P + <0.05) reductioninthenumberofGata2 oligodendrocyte progenitors exhibited greaterBrdU Rbpsuh Rbpsuh + Rbpsuh , p2-domainglialprogenitorsthatwould . Indeed,BFABP + V2a interneurons,andamodestbut fl/fl mice (Fig.6Nversus O). Nestin-Cre + astrocyte progenitors + motor neurons(that P <0.05) increase + Nestin-Cre + Rbpsuh astrocyte Rbpsuh + V2b fl/fl + + numbers ofNeuN cultured dorsalroot ganglioncellsfrom E13.5control and significant increaseinOlig2 development thatwestudied. the generationofoligodendrocytes duringthewindow of spinal cordbypromotingthegenerationofastrocytes and inhibiting signaling isnecessarytoregulate gliogenesisinthe developing added toculture thatformedeachtypeofcolony. were alsocounted.Thedataare expressed asthepercentage ofcells standard medium(C-E), view from withinmultilineagecoloniescultured from control cellsin with control cells(B).(C-N)Representative photosofsinglefields multilineage, glia-containingandothertypesofcoloniesascompared and F,G) inthe neurospheres inculture(Fig.7I-Q). Althoughthese of Mbp cultures allowed colonies (A).Additionofthegliogenicfactorneuregulin (Nrg)tosister their abilitytoformneuron-containing ormyofibroblast-containing littermate controls.We observed noeffect of to undergogliogenesisinculture. gliogenesis invivo,butrequire stimulationbyagliogenicfactor astrocyte-only colonies(Fig.7Q).These resultsareconsistentwith BFABP neurospheres thataroseintheseculturesfrom oligodendrocytes. Fromthreeindependentexperiments, 86±12%of before stainingformarkers ofneurons,astrocytes and then transferredtheresultingneurospherestoadherentcultures thoracic spinalcordcellsatclonaldensityinnon-adherentcultures, form multilineagecoloniesinculture,wecultureddissociatedE19.5 [M, Sma contained neurons (N,peripherin under adherent conditions.(A,B)Multilineagecolonies(N+G+M) cells inNrg(L-N)showthat P other coloniesthatcontainedglia)ascompared withcontrol cells(A;*, containing) colonies(whichincludedallmultilineage,glia-only, and cells formedsignificantlyfewermultilineageandglia-containing(G- Rbpsuh Wnt1-Cre Fig. 3.Neuralcrest progenitors withglialpotentialpersistin independent experiments). (H, Gfap,red), exceptinthepresence ofNrg(N)(three tosix Rbpsuh Rbpsuh mice exhibited excision ofboth spinal cord.We againobserved decreasednumbersofGfap oligodendrocytes intheabsenceof decreased numbersofastrocytes andincreasednumbers of progenitors from form astrocytes (Gabayetal.,2003).To testwhetherspinalcord a subsetofcellsinthesecoloniestoloseOlig2expression andto colonies incultureundertheinfluenceofbasicFGF, whichcauses frequency of (F-H), control cellsinthepresence ofNrg(I-K)and Nestin-Cre relative towhat was observed incontrolcolonies(Fig.7Kversus O). number ofsuchcellsandtheirlevel ofGfap stainingwerereduced <0.05). Olig2 To testwhetherthesedifferences observed atE14.5translatedinto fl/fl + fl/fl fl/fl + + + Wnt1-Cre and Sox10 (Acta2/Actg1 –MouseGenomeInformatics)].Othercolonies astrocytes (Fig.7Aversus B,G,H)andincreasednumbers mouse embryosatclonaldensity(500cellsper35mmdish) + spinal cord,wealsoobserved asignificant increaseinthe progenitors inthespinalcordgeneratemultilineage multilineage coloniescontained Gfap Rbpsuh + Nestin-Cre Rbpsuh Nestin-Cre Wnt1-Cre Nestin-Cre + + fl/fl Rbpsuh neurons (Fig.7G).ThesedataindicatethatNotch + Pdgfr fl/fl sensory gangliadespitethelackof Wnt1-Cre + + cells alsoformedsignificantly fewer Rbpsuh fl/fl + Rbpsuh ␣ Rbpsuh Rbpsuh + + cells didnotdiffer from control cellsin + oligodendrocytes (Fig.7Cversus D,E Rbpsuh + progenitors withinthe ), glia(G,Gfap + Rbpsuh -deficient NCSCsrarely formedglia Rbpsuh fl/fl fl/fl Rbpsuh fl/fl ( A-N cells toformnormalnumbersof cells thatformedmultilineage fl/fl spinal cordascomparedwith ) We dissociatedand alleles. Consistentwiththe fl/fl mice retainedtheabilityto , weexamined theE19.5 cells instandard medium Development 134(13) Rbpsuh Nestin-Cre + ) andmyofibroblasts Wnt1-Cre Wnt1-Cre + astrocytes, the deletion onthe Wnt1-Cre Nestin-Cre Nestin-Cre + + + Rbpsuh Rbpsuh Rbpsuh + and + fl/fl fl/fl fl/fl + +

DEVELOPMENT Nestin-Cre reductions inastrocyte formation(Stoltetal.,2003).ThroughE12.5, from thedeveloping spinalcordusing differentiated glia(Stoltetal.,2003).Conditionaldeletionof developing spinalcordaswellbyglial-restricted progenitorsand neuroepithelial progenitorswithintheventricular zoneofthe deletion on determination. To address this,weexamined theeffect of is requiredfortheexpression ofgenesthatregulate gliallineage The foregoing dataraisedthequestionofwhetherNotch signaling expression duringgliogenesis Notch signalingisrequired tomaintainSox9 development) forthemaintenanceofundifferentiated progenitors. cord, withoutbeingrequired(atleastduringthiswindow of generation ofnormalnumbersastrocytes inthedeveloping spinal the invivo resultsinindicatingthat Physiological Notchsignalspromote gliogenesis treatment atthesetimepoints. However, byE19.5,Sox9 numbers ofSox9 only rareSox9 explained bythedeathof Sox9-expressing cellsbecausewedetected The lossofSox9expression atE13.5andE14.5didnotappeartobe Whereas underwent increasedcelldeath in theabsenceof Cre spinal cordsections(Fig.8A,B,F).However, fromE13.5on, detect aneffect of + Rbpsuh Rbpsuh + Sox9 Rbpsuh fl/fl + cells thatstainedforactivated caspase3ineither mice hadincreasinglysevere reductions inthe expression. Sox9isexpressed byundifferentiated + was requiredforSox9expression, wedidnot cells relative tolittermatecontrols(Fig.8C-F). Rbpsuh fl/fl mice hadnormalnumbersofSox9 deletion onthe expression ofanother Rbpsuh Nestin-Cre is requiredforthe Rbpsuh leads tosevere (Fig. 8G). + Rbpsuh cells in Nestin- + Sox9 cells E19.5 diencephalon.Thediencephalonfrom gliogenesis throughout thePNSandCNSin absenceof physiological roleingliogenesis invivo. We observed defectsin Our datademonstratethatcanonical Notchsignalingplaysa DISCUSSION littermates, makingitdifficult tocomparehomologousbrainregions. mutant brainswas somewhat different fromthatofcontrol hemorrhaging (datanotshown). Asaresult,themorphologyof deletion ledtoanexpansion oftheventricles aswellsome compare thefrequency ofgliathroughoutthebrainbecause least certainregions ofthebrain.However, itwas difficult toprecisely demonstrates thatNotchsignalingalsoregulates gliogenesisinat oligodendrocytes (seeFig.S6inthesupplementarymaterial).This from thesamemicehadsignificantly increasednumbers ofOlig2 Sox9 (seeFig.S6inthesupplementarymaterial).Thediencephalon (Slc1a3–MouseGenomeInformatics), S100 stained forGlast mice hadsignificantly reducednumbersofastrocytes, whetherwe increased numbersofoligodendrocytes after significantly reducednumbersofastrocytes andsignificantly gliogenesis isnotlimitedtothespinalcordaswealsoobserved by glialprogenitors. spinal cordpartlybecauseitisrequiredtomaintainSox9expression that Notchsignalingisrequiredforgliallineagedeterminationinthe Informatics) (Muroyama etal.,2005)(Fig.8H).Thesedatasuggest regulator ofglialspecification, This requirementforcanonicalNotchsignalinginCNS s.d.; *, Error barsrepresent mouse atupperandlowerthoraciclevels. stages, betweenfiveandtwelvesectionswere countedper caspase 3)(three micepergenotype).Foralldevelopmental cell death(F, thepercentage ofcellsexpressing activated incorporated a30-minutepulseofBrdU invivoatE14.5)or rates ofproliferation (E,thepercentage ofcellsthat of thecontrol and (BFABP migratory neuralcrest cells(p75 at E10.5.( Numbers ofBFABP (four tosevenmicepergenotype)sympatheticchain. genotype), E14.5(fourtosevenmicepergenotype)andE18.5 glia (BFABP control ( sympathetic chainwere significantlyreduced relative to from control or Fig. 4. (A) orE18.5(B)were stainedforneurons (TuJ1 ganglia. gliogenesis despitenormalneurogenesis insympathetic + Rbpsuh P ) persectionthrough theE10.5(three miceper P <0.05. <0.05). ( ( A D + , ) Thenumbersofneurons (TuJ1 , red). Scalebar:20 B Transverse sectionsofthesympatheticchain ) Wnt1-Cre deletion leadstoprofound defectsin E + Wnt1-Cre , F glia inthe ) We didnotdetectanydifference inthe + Rbpsuh + Scl RESEARCH ARTICLE Rbpsuh Wnt1-Cre+ Rbpsuh + ␮ ) inthesympatheticganglion m. ( ( fl/fl Tal1 mouse embryosatE13.5 fl/fl C Nestin-Cre Rbpsuh ) Thenumberof embryos didnotdiffer – MouseGenome + ) andglia + deletion inthe , green) and fl/fl + Rbpsuh Rbpsuh Rbpsuh 2441 ␤ fl/fl or + .

DEVELOPMENT progenitors, oligodendrocytes (Figs6,7).ConsistentwiththeincreaseinOlig2 the numberofastrocytes andasignificant increaseinthenumberof neurogenesis werenormal,but therewas asignificant decreasein gestation (Fig.5).Inthe supplemented withNrg persistedinnormalnumbersintolate NCSCs andotherprogenitorsthatcouldformgliaincultures despite analmostcompletelackofgliogenesis(Fig.4).Again, Rbpsuh could formgliainculturessupplementedwithNrg (Figs2,3). persistence ofnormalnumbersNCSCsandotherprogenitorsthat neurogenesis but analmostcompletelackofgliogenesis,despitethe deficient sensorygangliaexhibited amodestreductionin even afterthedefectsingliogenesiswereevident invivo. progenitors capableofundergoing gliogenesisinculturepersisted Yet defectsinneurogenesisweremodestorundetectable,and 2442 frequency ofmultilineagecoloniesinculture(Fig.7).Thesedata RESEARCH ARTICLE -deficient sympatheticchainexhibited normalneurogenesis Rbpsuh -deficient spinalcordcellsformedanincreased Rbpsuh -deficient spinalcord,levels of Rbpsuh + - whether of suchgenes.Additionalwork willberequiredtodetermine suggesting thatNotchisnotgloballyrequiredfortheexpression specification genes,suchas observed. We didnotdetectreducedexpression ofotherglial- consistent withthedramaticreductioninastrocytogenesis thatwe 2003), thelossofSox9expression after formation ofastrocytes inthedeveloping spinalcord(Stoltetal., spinal corddevelopment (Fig.8).SinceSox9isrequiredforthe maintenance ofSox9expression duringthegliogenicphaseof specification genes.Notchsignalingisrequiredforthe appears toinvolve aroleintheexpression ofatleastcertainglial- maintenance. gliogenesis invivo independentofitseffects onprogenitor indicate thatphysiologicalNotchsignalingisrequiredfornormal Moreover, thelossofSox9expression cannotcompletelyexplain The mechanismbywhichNotchsignalingpromotesgliogenesis Rbpsuh presence ofNrg(B), all glia-containingcolonies(G-containing).Inthe Wnt1-Cre colonies. ( types, includingmultilineageandglia-containing controls cellsformedsimilarnumbersofallcolony Wnt1-Cre experiments) of5nMNrg.Instandard medium(A), experiments) andpresence (B,fourindependent absence (A,standard medium;nineindependent 35 mmdish)underadherent conditionsinthe dissociated andplatedatclonaldensity(500cellsper (*, of persist inthesympatheticchainabsence Fig. 5.Undifferentiated neuralcrest progenitors ( culture inthepresence ofneuregulin (Nrg). in thepresence ofNrg(O-R). multilineage colonyformedby control cellsinthepresence ofNrg(K-N)anda conditions (G-J),amultilineagecolonyformedby formed by cells understandard conditions(C-F),anN+Mcolony from withinamultilineagecolonyformedbycontrol A directly orindirectlyregulates Sox9expression. , B Rbpsuh P <0.05) fewermultilineagecolonies(N+G+M)and E13.5 sympatheticgangliafrom control and ) + + C-R Rbpsuh Rbpsuh Rbpsuh and canundergogliogenesisin Scl ) Typical photosofasinglefieldview (Fig. 8)(Muroyama etal.,2005), -deficient cellsunderstandard fl/fl fl/fl Wnt1-Cre mouse embryoswere cells formedsignificantly Development 134(13) + Rbpsuh Rbpsuh Rbpsuh -deficient cells fl/fl deletion is cells and

DEVELOPMENT numbers ofp75 rescued byadditionofNrg to theculture(Fig.1H).Sincenormal we observed inotherregions ofthePNS,this deficit was not formed multilineageNCSCcoloniesinculture.Incontrasttowhat observed asignificant reductioninthefrequency ofgutcellsthat maintenance ofundifferentiated neuralprogenitorsinvivo. We glial-specification genes. required inmammalstomaintaintheexpression ofatleast certain some numb Germline deletionof premature neurogenesisandadepletionofneuroepithelialcells. possible thatearlierdeletionof neural stemcellsinotherregions ofthenervous system,itis gestation. Althoughwedidnotobserve aprematuredepletionof required intheguttomaintainnormalnumbersofNCSCsinto late E10.5 (Fig.1C),theseresultssuggestthatNotchsignaling is later inneuraldevelopment. Ourfailure toobserve adepletionof maintenance earlyinneuraldevelopment andpromotegliogenesis developmental time.Notchsignalingmightpromoteprogenitor maintenance ofneuralprogenitors islikely tovary over Zhong etal.,2000).However, theroleof Notchsignalinginthe Hatakeyama etal., 2004;Hitoshietal.,2002;Petersen a lossofneuroepithelialprogenitors (delaPompaetal.,1997; required fortheexpression oftheglial-specification gene Nonetheless, thesedatademonstratethatjustasNotchsignalingis mechanisms bywhichNotchsignalingregulates gliogenesis. expansion asweobserved. Theremustthereforebeadditional oligodendrocyte formation(Stoltetal.,2003),rather thanan Sox9 the gliogenicdefectsinabsenceof Physiological Notchsignalspromote gliogenesis These datadonotruleoutaroleforNotchsignalinginthe Drosophila and by itselfisassociatedwithatransientreductionin numbl + , allleadtoprematureneuronaldifferentiation and glia (Udolphetal.,2001),Notchsignalingisalso neural crestprogenitorsmigratedintothegutat Notch1 , or Rbpsuh Rbpsuh Rbpsuh , or would have ledto Hes1 , becauselossof and Hes5 gcm , or in progenitors from thesegangliatoformgliainculture inthepresence sensory andsympatheticganglia invivo, despitetheabilityof Notch receptors. if progenitorsindifferent domainsofthespinalcordexpress different deletion mighthave different effects than depending onpreciselywhenconditional deletionoccurs,and differentiation (Wang etal.,1998).Different resultsmightbeobtained that foundaninhibitoryeffect ofNotchsignalingonoligodendrocyte (Genoud etal.,2002).Ourresultsarealsoconsistentwithotherstudies during latefetaldevelopment afterconditional study thatalsoobserved increasedspinalcordoligodendrogliogenesis contrast withthoseofYang etal.,ourresultsareconsistentwithaprior development (Figs6,7).Althoughthese results would appearto oligodendrocyte differentiation atlater stages ofspinalcord material). Moreover, we observed asubstantialincreasein littermate controlsatE11.5(seeFig.S4inthesupplementary differentiation andadecreaseinthenumberofOlig2 relatively lateconditionaldeletionof neural progenitorsinmostlocationscouldhave beencausedbya Olig2 Yang etal.,we didnotobserve any difference inthefrequency of deletion, althoughthisoccurredlaterinourstudy. However, unlike observed alossofthecentralcanalinspinalcordafter development. AsYang etal.observed after oligodendrocyte differentiation atlaterstagesofspinalcord multilineage coloniesincultureorchangesastrocyte or test whether the E11.5neuraltube(Yang etal.,2006).However, thisstudydidnot spinal cordusing neurogenesis. The nearcompleteabsenceof gliogenesis in A recentstudyconditionallydeleted + cells orNgn2 Notch1 Cre the Olig2 co-label withBrdU (N,arrow), whereas atleast someof between arrowheads). We didnotobserveanydifference showed thatthecenterof per genotype).( section (three tosevensectionspermouse,three mice 50 of thesamesection.Scalebars:50 F,H,I are montagesofmultiplenon-overlappingimages contained mainlyBFABP progenitors (I),incontrasttocontrol micethat neural tubecontainedmainlyOlig2 Rbpsuh Rbpsuh of neurons intheE14.5spinalcord. oligodendrocytes withoutaffecting thenumbers of astrocytes andincreases thegenerationof Fig. 6.Deletionof and more Olig2 numbers ofOlig2 (activated caspase-3 controls inthefrequency ofcellsundergoingcelldeath per sectionandsignificantlymore Olig2 disorganized orabsentcentralcanalin center oftheneuraltube,whichwasmarkedbya in gliogenesiswere particularlypronounced inthe of neurogenesis appeared similar(E,F).Thedifferences than sectionsfrom control littermates,althoughlevels from thethoracicneuraltubeofE14.5 Nestin-Cre ␮ + + Rbpsuh m inH,I;20 deletion ledtoalossofcellsthatcouldform cells between fl/fl fl/fl + Nestin-Cre mice (compare AtoB,andHI).( mice containedfewerBFABP cells from fl/fl and observed anincreaseinneuronal mice hadsignificantlyfewerBFABP + H-O ␮ oligodendrocyte progenitors (C,D) + m inJ,KforJ-O. cells present incontrol micedidnot + + Rbpsuh ) Highermagnificationimages Nestin-Cre Nestin-Cre Rbpsuh RESEARCH ARTICLE cells, datanotshown).PanelsA- + Notch1 radial glia(H).Thesmall Rbpsuh Notch1 fl/fl Notch1 reduces thegeneration Nestin-Cre + mice andlittermate + Rbpsuh deletion, particularly , aftertheonsetof Rbpsuh + ␮ in thedeveloping Rbpsuh oligodendrocyte m inA,BforA-F; deletion, wealso Notch1 + + Nestin-Cre Nestin-Cre ( + A-F fl/fl radial glia(A,B) progenitors in + cells per Rbpsuh fl/fl mice did(O, ) Sections -deficient G mice and deletion Rbpsuh ) Rbpsuh Nestin- + + 2443 fl/fl + cells

DEVELOPMENT when Nrg isaddedtotheculturemedium.Onepossibilitythat required forgliogenesisinvivo inaway thatisnotrecapitulated failed toformgliainvivo. ThisindicatesthatNotchsignalingis sympathetic gangliainvivo (datanotshown) andyetthesecellsstill et al.,1997).However, Nrg was expressed around vivo (Dongetal.,1995;Meyer andBirchmeier, 1995;Riethmacher and Anderson,1997)isnecessaryforgliogenesisinthePNS NCSCs inculturetoacquireaglialfate (Morrisonetal.,1999;Shah but thatNrg canbypassthisrequirementinculture.Nrg instructs Notch signalingisrequiredforgliogenesisinthesegangliavivo with somepriorreports(Gabayetal.,2003).Ourdatasuggestthat from thoseemployed underphysiologicalconditions,consistent of Nrg, suggeststhatgliogenicmechanismsinculturecandiffer 2444 RESEARCH ARTICLE Rbpsuh -deficient P shown]. We alsoobserved onlyasmall(but statisticallysignificant; Krox20 (Egr2–MouseGenomeInformatics)staining;datanot mice [thesenerves appearednormalbyelectronmicroscopy and defects inperipheralnerve gliogenesisin (Morrison etal.,2000).Inthecurrentstudy, wedidnotobserve any performed onNCSCsisolatedfromthedeveloping sciaticnerve NCSCs toacquireaglialfate inculturecamefromexperiments 1999). of ongoingbonemorphogenicproteinsignaling(Morrisonetal., neurogenesis togliogenesisbyovercoming theneurogenicinfluence Notch signalingisrequiredinvivo topromotethetransitionfrom <0.05 byapaired The originalevidence indicatingthatNotchligands caninstruct type ofcolony. number ofcellsundergoingcelldeath(activatedcaspase3 genotype). There wasnosignificantdifference inthe of reduction inthenumberofBFABP restricted progenitors. Scalebars:50 in multilineagecolonies,notanincrease inneuronal- increase inneuron-containing coloniesreflected theincrease likely toformastrocyte-only colonies(A). containing colonies(O-containing),butsignificantlyless multilineage colonies(N+A+O)andoligodendrocyte- significantly more likelythancontrol cellstoform in control and ( ( astrocytes (Gfap cultured atclonaldensitythenstainedforneurons (TuJ1+), Gfap littermate controls. We alsoobservedmore Sox10 Fig. 7. (*, expression. ( culture atlowdensityfor6hoursthenstainedGfap dissociated cellsfrom E19.5spinalcord andplatedthemin sections basedonfilamentousGfapstaining,weacutely mice pergenotype).Becausegliaare difficult tocountin spinal cord (three tosevensectionspermouse,three tofive oligodendrocyte lineagecellsinthe ( non-overlapping imagesofthesamesection. spinal cord (E,F, arrows). C,Drepresent montagesofmultiple oligodendrocyte lineagecellsinthe Nestin-Cre Representative multilineagecoloniesfrom control (I-L)and increased numbersofMbp Nestin-Cre increases inthenumbersofOlig2 including decreased numbersofGfap observed atE14.5persistedintheE19.5spinalcord, multilineage coloniesinculture. without depletingprogenitors thatcouldform oligodendrocytes intheE19.5mousespinalcord astrocytes andincreases thegenerationof Q I-P G Quantification revealed asignificant(*, ) ) Thepercentage ofspinalcord cellsthatformedeach Nestin-Cre P ) DissociatedE19.5thoracicspinalcord cellswere <0.05; 1000cellscountedpermouse,three miceper + cells andstronger Gfapstainingincontrol colonies. Rbpsuh + + Rbpsuh Rbpsuh t H -test) reductioninthefrequency atwhich + ) We observedasignificantlylowerpercentage Nestin-Cre Rbpsuh Nestin-Cre deletion reduces thegenerationof + ) andoligodendrocytes (O4+). fl/fl fl/fl fl/fl spinal cord ascompared with (M-P) spinalcord cellsshowmore cells inculture thatwere Gfap + + Rbpsuh + Rbpsuh oligodendrocytes (C,D)inthe + fl/fl + , Sox10 fl/fl astrocytes andsignificant Nestin-Cre Nestin-Cre ( Development 134(13) A-F Wnt1-Cre mice (datanotshown). ␮ + spinal cord cellswere m inA-F. astrocytes (A,B)and ) Thedifferences P + <0.05. The P and Mbp <0.05) + + Rbpsuh Rbpsuh + Rbpsuh + Pdgfr + + fl/fl fl/fl ␣ + fl/fl + )

DEVELOPMENT ganglia intheabsenceof almost completelossofgliogenesisinsensoryandsympathetic differentiation versus progenitormaintenance. We observed an distinguish betweeneffects ongliallineagedetermination/ loss ofcanonicalNotchsignalingongliogenesisinvivo andto gliogenesis. signaling playsaphysiologicalroleinperipheralnerve development. Asaresult,itremainsuncertainwhetherNotch from neuralcrestprogenitorsthatparticipatedinsciaticnerve This raisesthepossibilitythat Rbpsuh multilineage NCSCcoloniesformedinculturefrom Physiological Notchsignalspromote gliogenesis 2000; Scheeret al.,2001;Tanigaki et al., 2001;Yoon etal.,2004). in theCNS(Furukawa etal.,2000;GaianoHojo with theobservation thatNotchactivation canpromote gliogenesis instruct NCSCstoacquireaglial fate (Morrisonetal.,1999)and consistent withourpriordemonstration thatNotchactivation can differentiation in the developing spinalcord.Theseresults are Rbpsuh similarly necessaryfortheregulation ofgliogenesisintheCNS,as its effects onprogenitormaintenance.Notchsignalingwas crucial roleinthepromotionofgliogenesisvivo, independentof This demonstratesthatphysiologicalNotchsignalingplays a progenitors thatcouldformgliainculturessupplementedwith Nrg. neurogenesis, anddespitethepersistenceofnormalfrequencies of excision ofboth deleted intheseNCSCs:only7±10%ofneurospheresshowed littermate cells(1.1±0.7%).However, This isthefirst studytoexamine theconsequencesofacomplete fl/fl deletion reducedSox9expression andastrocyte nerve cells(0.6±0.5%),ascomparedwithcontrol Rbpsuh Rbpsuh alleles ( Rbpsuh , despitenormalornearly n =6 independentexperiments). Rbpsuh was notefficiently deleted was notefficiently Wnt1-Cre + Artavanis-Tsakonas, S.,Rand,M.D.andLake,R.J. Anthony, T. E.,Mason,H.A.,Gridley, T., Fishell,G.andHeintz,N. References providing the Disorders andStroke (F32NS046202).We thankTasuku Honjoforgenerously National Research ServiceAward from theNationalInstituteofNeurological National InstitutesofHealth(R01NS40750).M.K.T. was supportedbya This workwassupportedbytheHoward HughesMedicalInstituteandthe later promotinggliogenesis. promoting thegenerationormaintenanceofneuralprogenitorsand signaling playsreiteratedrolesinneuraldevelopment, initially Together, theresultsfromthisandpriorstudiesindicatethatNotch providing Bixby, S.,Kruger, G.M.,Mosher, J.T., Joseph,N.M. andMorrison,S.J. Bigas, A.,Martin,D.I.andMilner, L.A. http://dev.biologists.org/cgi/content/full/134/13/2435/DC1 Supplementary materialforthisarticleisavailableat Supplementary material against BFABP; andAlexanderGowforthe Anderson forantibodiesagainstSox10andNgn2;ThomasMullerantibody manuscript; Tom JessellforantibodiesagainstHB9andChx10;David Bennett NovitchfortheantibodyagainstOlig2andvaluablecommentson the Leventhalfortechnicalsupport; (obtained viatheJacksonLaboratory);Tina Carlesso, N.,Aster, J.C.,Sklar, J.andScadden,D.T. Genes Dev. Brain lipid-bindingprotein is adirect targetofNotchsignalinginradialglialcells. cell fatecontrol andsignalintegrationindevelopment. 35 peripheral nervoussystemregulate thegenerationofneuraldiversity. (2002). Cell-intrinsicdifferences betweenstemcellsfrom different regions ofthe 2324-2333. myeloid differentiation inresponse todifferent cytokines. , 643-656. Wnt1-Cre 19 Rbpsuh , 1028-1033. mice; RudigerKleinforproviding fl mice usedintheseexperiments;AndyMcMahonfor Nestin-Cre at E13.5andE14.5,butSox9 cord byE19.5.( gradually dispursedthroughout thespinal ventricular zoneearlyindevelopmentbut Cre increased celldeathatE19.5.( control embryos,Sox9 In overlapping imagesofthesamesection. represent montagesofmultiplenon- E11.5 toE19.5were stainedforSox9.A-E embryos, Sox9 Fig. 8. Sox9 genotype andtimepoint).( embryos afterE12.5(three tosixmiceper increasingly depletedrelative tocontrol zone from E11.5toE13.5,butbecame by qRT-PCR atE14.5. expression of Nestin-Cre sections ofthespinalcord from control and E12.5 inthespinalcord. maintenance ofSox9expression after + + Rbpsuh cells appeared toundergocelldeath Rbpsuh (1998). Notch1andNotch2inhibit + + RESEARCH ARTICLE Mbp Rbpsuh Rbpsuh fl/fl Rbpsuh + embryos exhibitedreduced F is necessaryforthe cells were intheventricular cDNA probe. In ) fl/fl fl/fl (1999). Notchsignaling: Nestin-Cre (1999). Notch1-induced Nestin-Cre and Science embryos exhibited mouse embryosfrom + cells were inthe Mol. Cell.Biol. Sox9 ( G A-E ) Onlyrare 284 + H cells from + Transverse ) mice but not ) Rbpsuh , 770-776. Nestin- (2005). Neuron 18 2445 , Scl fl/fl

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