progenitors, andtheyfurthersuggestthat,inthisregard, vivo aswellinvitro.Hence,ourdatahaveprovidedevidencefortheabilityof not todriveeithertypeofcellintodifferentiation. Furthermore,anon-cellautonomouseffect of rate inthesecells.Bycontrast,GATA2 overexpressionissufficient toforcesuchcellsandneuroblastomastopdividi Upregulation ofproneuronalgenes,suchas (Artavanis-Tsakonas etal.,1999;Roegiers andJan,2004). and arrestofproliferation:lateralinhibitionasymmetricdivision two majormechanismsinvolved inthechoicebetweenmaintenance downstream ofNotchreceptoractivation. Thispathway underlies pathway insustainingprogenitorproliferationismediated through different signallingpathways. Themost widelyimplicated coordination betweenproliferationanddifferentiation isachieved 2003; Galderisietal.,OhnumaandHarris,2003).Correct or, conversely, anexcess oflater-born celltypes(Cremisietal., of progenitorsbeforetheendhistogenesis(Ohnumaetal.,2002) nervous systemdevelopment inordertoavoid eitherthedepletion cycle exit anddifferentiation must be preciselycoordinatedduring events. Thisimpliesthatprogenitorcellproliferationversus cell and glialcelltypesinacharacteristicorderofdifferentiation neural progenitorsthatcanindividually give risetobothneuronal the neuroectodermandarecomposedofmultipotentproliferating organised inelaboratecircuits.Allneuronsarederived from The nervous systemisacomplex tissueincluding multipletypesof INTRODUCTION KEY WORDS:GATA2, Cellcycle,Neuronal progenitors, Posteriorneuraltube,Mouse,Chick proliferation capacityofmouseembryonicneuroepithelialcellsinculture.Indeed, silencing oftheNotchpathwaybutalsoappearstobeindependentproneuralfunction.Consistently, GATA2 alsocontrolsthe cyclin D1transcriptionandoftheexpressionp27/Kip1protein.Interestingly, thisfunctionalaspectisnotonlyassoci that proliferation ofneuralprogenitorsintheembryonicspinalcord.Usinggain-andloss-of-functionmanipulations,wehaveshown implication ofGATA2, atranscriptionfactorthatisinvolvedinseveralneuronalspecificationpathways,thecontrolof between mechanismsthatcontrolcellcycle,specificationanddifferentiation. Inthisrespect,wehavestudiedthepossible coordination ofproliferation,fatedetermination,differentiation andmorphogenesis.Thisprobablyreliesoncomplexinterplay Postmitotic neuronsareproducedfromapoolofcyclingprogenitorsinanorderlyfashionthatrequiresproperspatialandtemp Abeer ElWakil*, CédricFrancius* proliferation ofneuronalprogenitors The GATA2 transcriptionfactornegativelyregulatesthe Development 133,2155-2165(2006)doi:10.1242/dev.02377 Accepted 27March 2006 § † *These authorscontributedequallytothis work Marie Curie,Paris6,France. Salpêtrière, 105boulevard del’Hôpital,75013Paris,FranceandUniversitéPierre et UMR CNRS7000,CytosqueletteetDéveloppement,FacultédeMédecinePitié- 3boulevard del’Hôpital,75013Paris,France 83 prevent themfromdifferentiating; aphenomenonknown aslateral and Jag1),whichwillactivate Notch inneighbouringcellsand levels ofeithertwo typesofNotch ligand,deltaorjagged(Dll1 2002; Bladeretal.,1997).Newborn neuronstransientlyexpress high progenitors toaneuronalfate andcellcycle exit (Bertrandetal., Genome Informatics)orneurogenins,promotescommitment of Author forcorrespondence (e-mail:[email protected]) Present address: CHUPitié-Salpêrière, UMRCNRS7091LGN,BâtimentCERVI, Gata2 can driveneuralprogenitorsoutofthecycleand,tosomeextent,intodifferentiation. Thiscorrelateswiththecontrolof ,† , AnnieWolff, JocelynePleau-Varet Mash1 ( Ascl1 – Mouse Gata2 can operateindependentlyofneuronaldifferentiation. in suchcrosstalk. GATA2 was firstdescribedasaregulatory of GATA2, atranscriptionfactor that containstwo C4zincfingers, therefore tobeakey issue. , proneuralgenesandthe machinery ofthecellcycle appears and Cepko, 2001).Deciphering crosstalkbetweenspecification dependent onspecificationgenes (DyerandCepko, 2001;Livesey suggesting thattheexpression ofcellcycle regulators maybe differentially involved indistinctdifferentiation pathways, Mouse GenomeInformatics),have beenshown tobe (Cdkn1c –MouseGenomeInformatics)andp27/Kip1(Cdkn1b – retina, cyclin D1andcellcycle inhibitors,such asthep57/Kip2 al., 2004)andsympathetic(Tsarovina etal.,2004)neurons.Inthe cascades leadingtothedifferentiation ofserotoninergic (Pattyn et et al.,2003),whereas neuronal precursorsinboththebrainandspinalcord(Scardigli Neurog2 has beenshown toregulate theexpression ofneurogenin2( trans-activation andthecellcycle machinery, arestilllimited.Pax6 specification, modulationofNotchsignalling,pro-neuralgene concerning thegeneticevents, whichmostprobablylink Edlund andJessell,1999;Tanabe etal.,1998).However, data been shown toinfluencethecellcycle (Dubreuiletal.,2000; 2004; Tsudaetal.,2002). regions, have beenreported(Francoetal.,1999;Hofmann Notch signallingandotherpathways, actinginspecific precursor state(EdlundandJessell,1999).Indeed,crosstalkbetween a multipotentneuralprogenitortowards apost-mitotic neuronal extrinsic andintrinsiccuesarebelieved togovern theprogressionof precursor (Petersenetal.,2002;Roegiers andJan,2004). Other whereas theotherdaughtercellkeeps dividing andremainsa components inhibitingNotchsignallingandasaresultdifferentiates, case ofasymmetricdivision, onlyonedaughtercellinherits inhibition ofneurogenesis(Artavanis-Tsakonas etal.,1999).Inthe Here, wereportseveral linesofdatasupportingtheinvolvement Regulatory proteinsinvolved inneuronspecificationhave also – MouseGenomeInformatics)inseveral populations of Gata2 Gata2 † and JeannetteNardelli to inhibittheproliferationofneural inactivation enhancestheproliferation Mash1 has beenplacedupstreamofgenetic Gata2 RESEARCH ARTICLE expression wasobservedin †,§ ng but ated with Ngn2 2155 oral ;

DEVELOPMENT MATERIALS ANDMETHODS components andbyshuttingoff theNotchpathway. to beexerted by negative controlontheproliferation ofneuronalprogenitorsappears embryonic undifferentiated neuroepithelialcellsinculture.Such further insightintothispossiblenew aspectof specification genes,participateinitswithdrawal. Inordertogain Gata2 activation isturnedoninearlyprecursorsraisedthepossibilitythat Zhou etal.,2000).Thefact that,ineachoftheselineages, Pattyn etal.,2004)andV2interneurons(Karunaratne2002; 1999; Pata etal.,1999),serotoninergic neurons(Craven etal.,2004; andspinalcord,namelycranialnerves (Nardellietal., differentiation pathway ofdifferent typesofneuronsintheventral we show that to aquiescentandeventually differentiating stateinvivo. Inaddition, expression issufficient toforcecycling neuralprogenitorstoswitch is linked tothecellcycle. Ourdatahave establishedthat carried outanalysesaimedatabetterunderstandingofhow (Giudicelli etal.,2001)by entirely checked bysequencingandtransferredintopAdRSV-SP 1 hourbeforesacrifice. BrdU incorporation,pregnant femaleswereinjectedintraperitonally(2g/kg) Animals andembryosweregenotypedasdescribed(Tsaietal.,1994).For , identifiedbytheirbeatingheart,wereincludedinourstudies. of thehomozygousembryosstartstodecreasesignificantly. Onlysurviving be E0.5andembryoswerecollectedatE10.5,astagewhichthesurvival the pureC57Bl6background.Thedayofvaginal plugwas consideredto slightly improve thesurvival ofthe maintained inamixed C57Bl6/DBA2 background,whichwas foundto Mice heterozygousforthe Mouse embryos systems. Inthelattercase, including theuro-genital(Zhouetal.,1998)andnervous differentiation ofseveral other tissuesduringembryogenesis, al., 1994). and E11,owing tosevere defects inprimaryhematopoiesis(Tsaiet of functioninthemouseleadstodeathembryosbetweenE9.5 lineage specificationduringearlyhematopoiesis.Indeed that playsamajorroleintheproliferationofprogenitorsand 2156 gelatin andfrozen at–50°C.Serialsections(12 PBS 30%sucrose,embeddedin PBS containing15%sucroseand7% immunostaining andovernight forinsituhybridisation,wereequilibrated Embryos, fixed inparaformaldehyde (PFA) 4%for2-3hours Immunohistology andinsituhybridisation a encoding human sequence In ordertoobtainthePAdRSV-GATA2HA expression plasmid, theentire Electroporation ofchickembryosinovo the 3 collecting theembryos. was injectedintotheheartorumbilicalvein 30minutespriorto For BrdUincorporation,asingleinjectionof20mg/ml BrdU solution hours (occasionallyfor6-12hours)beforebeingfixed asdescribed below. 830 electroporator. Embryoswerefurtherincubatedforeither24or48 pulses of25Vfor50msecondseachwerethenappliedwithaBTXECM cord withanEppendorfFemtojetinjectoratstageHH12orHH15. Six DNA solution(2-3mg/ml)was injected inthehindbrainandspinal domesticus gallus Gilardi andPhilippeRavassard, respectively. Fertilisedeggs from pAdRSVGFP plasmidswerekindlyprovided by ChristoGoridis,Pascale cId2 (Dubreuiletal.,2000),pAdRSV-bGal (Giudicellietal.,2001)and LEICA CM3000 cryostat. Nco Ј I siteatthe5 end ofthecDNA. ThePCRproductwas clonedintoPGEM3, RESEARCH ARTICLE may influencethecellcycle andprobably, like other Gata2 Gata2 Gata2 Ј hens wereincubatedat38°Cinahumidifiedoven. The end, onecopy oftheHAepitopeandan also appearstobeindispensableforthe acts asapotentinhibitorofproliferationin by interferingwiththeregulation ofcellcycle Gata2 Gata2 Nco Gata2 I and was amplifiedbyPCRsoastointroduce -null mutation(Tsaietal.,1994)were Gata2 Eco is known toparticipateinthe –/– RV digestions.ThepCAGGS- embryos incomparisonwith ␮ m) werethencut with a Gata2 function, we Eco Gata2 RV siteat Gata2 Gata2 Gata2 Gallus loss in thesamegenetic backgroundandmaintainedas described (Nardelliet cells. was appliedtocalculatethepercentageofphospho-Histone3-positive Counting was carriedoutusingMetamorphsoftware. Thesameapproach and thecontrolsidesonatleastfoursectionsinthreedifferent embryos. nuclei andtheBrdU-positive nucleiwerecountedontheelectroporated E10.5 embryos.Inchickembryosmisexpressing GATA2-HA, allthe positive cells,distributed into10spinalcordsectionsfromtwo different embryos,BrdU-positive cellswerecountedamong850 mouse percentageof the 546. ThenucleiwerestainedwithDAPI beforemounting.To assess except thatthesecondaryantibodywas coupledtoAlexa Fluor488or coloured inPhotoshop(Adobe). pictures wereobtainedwithaTCSLeicaconfocalmicroscopeandfalse- mounted inFluorescentmountingmedium(DAKO). Blackandwhite solution, was addedfor30minutesatroomtemperature.Slideswerethen in PBS/0.1%Triton, thesecondaryantibody, dilutedinthesaturation with theprimaryantibodydilutedinsamesolution.Afterseveral washes foetal calfserum(FCS)(saturationsolution)for30minutesandincubated (Adobe). Cool Snapcamera(RopperScientific)andfalse-coloured inPhotoshop epifluorescence microscope,andpicturesweretaken withablackandwhite processed asdescribedearlier. Stainingwas analysedwithaBX60Olympus et al.(Ravassard etal.,1997).For furtherimmunostaining,sectionswere following thesupplier’s instructions. neural tubeexplants ofE9.5wild-typeand Mouse embryonicneuroepithelial cell cultures(ENC)wereinitiatedfrom Cell culture CCTCCCCTCC-3 GCGGT-3 pCAGGS-cId2, usingthefollowing primers:5 Id2 AGCACCGGGAAACGTGGTCCAGA-3 CTGCAAGAGGCGG-3 CCTCTGGAATGTGCACCAGTGTCC-3 al., 1996); Hunter, 1994);700 bp and cyclin D3(Wianny etal.,1998);full-length The mouseprobeswere: In situprobes Probes) werediluted1/2000. other speciesandcoupledeithertoAlexa Fluor488or546(Molecular antibodies, goatanti-mouse,-rabbitand-ratIgG,allhighlyabsorbedagainst 1/1000) andanti-(Abcam,1/1000).Thefluorescentsecondary Bioscience, 1/2000),anti-GATA2 (Santa-Cruz, 1/400),anti-GFP(Abcam, anti-phosho-Histone3 (UpstateBiotechnology, 1/1000),anti-p57/Kip2(BD 1/1000), ratmonoclonalanti-HAepitope(Roche,1/2000),rabbitpolyclonal neurofilament-associated protein(1/500;DSHB),anti-NeuN(Chemicon, anti-BrdU (DAKO, 1/1000),anti-Isl1(DSHB;1/20),3A10anti- p27/Kip1 (BDBioscience,1/2000),anti- The following primaryantibodies wereused:mousemonoclonalanti- Primary andsecondaryantibodies described (Lobjoisetal.,2004); fragments amplifiedbyPCR.Primersforcyclin D1amplification wereas antisense probesdevoid ofbacterial sequence weresynthesisedfromcDNA checked bysequencing.To monitortheactivation ofcyclin D1and primers: 5 cloned byRT-PCR fromE6.5chickretinatotalRNA, usingthefollowing cyclin D1(Lobjoisetal.,2004).Thefullchick laboratory); Mash1 cDNA for TUNEL stainingswereperformedwiththeApodetect BrdU stainingwas performedasdescribed(Ravassard etal.,1997), For immunostaining,sectionsweresaturatedwithPBS/0.1%Triton/10% In situhybridisationonsectionswereperformedaccordingtoRavassard probe was synthesisedbyPCRamplificationofthecDNA includedin and Ј Gata2 Ј Dll1 -GCGGCTCGAGAGCCAGCTTCGTGC-3 Ngn2 (F) and5 Ngn2 (Campos etal.,2001);and ; (Gradwohl etal.,1996).Thechickprobes were:full-length Sox2 Ј (Novitch etal.,2001); Gata2 (R). Ј Acc , -GTTAATACGACTCACTATAGAGCGTGGATT- Dll1 Gata2 Ј I/ -expressing cellsbeinginSphaseE10.5 (forward) and5 Xba and (Nardelli etal.,1999);cyclin D1,cyclin D2 I fragmentfor Jag1 Cash1 (obtained fromNicoleLeDouarin’s Cash1 ␤ Ј Ј III-Tubulin (Babco,1/10,000), primers were5 Jag1 Ј (reverse). Likewise, the chick Gata2 -GTAATACGACTCACTAT- Ј (reverse). ThecDNA was Sox2 -AAGCTTTCAGCCCCGT- p27 Hes5 , full-length (Groves etal.,1995);and Development 133(11) ; rat –/– cDNA (Toyoshima and -coding sequencewas embryos, generated Notch1 Ј (forward) and5 plus Ј kit (Qbiogen) -TGATGCG- Hes5 (Lindsell et Gata2 cDNA, Cash1 Ј - - ,

DEVELOPMENT GCCGCTCTAGGCACCAA-3 (reverse); cyclin D3,5 BrdU incorporation. 10% serumandprocessedasENCforimmunostaining,transfection NB2a neuroblastomacellswereculturedinDMEMsupplementedwith Total RNA fromwildtypeor Semi-quantitative andquantitativePCRreactions Twenty-four hourslater, BrdU(10 confluency, thecellswereplaced inmediumcontaining0.5%FCS. cells wereseededonglasscover-slips in24-well plates.At50% exceeded 12.To comparetheproliferationratebyBrdUincorporation, cells wereperformedatmatchingnumbersofpassages,whichnever al., 2003).Allcomparative experiments betweenwild-typeand GATA2 andcellcyclearrest ofneuralprogenitors (reverse). GGGACA-3 ACGCACGATTTC-3 5 and CTCACG-3 CTTCTTGGGCGT-3 5 (forward) and5 CGT3 (reverse); cyclin E1,5 CTCTGTGCCACAG-3 up to95°Cgeneratedenaturationcurves. for 36cycles; then95°Cfor1minuteanda1°Cincrement/minute from55°C for 10minutes;30secondsat95°C,1minute65°Cand 72°C apparatus (Stratagene).Theprogramwas asfollows: aninitialstepat95°C product. camera andsoft-ware (BioRad)tocalculatetherelative amountofeach were separatedinBEt-agarosegelsandthenanalysedwithGel-Doc (30cycles). ThePCRproducts,allsizedbetween300and350bp, E1 Notch1(23cycles), cyclin D3and and and 30secondsat72°Cforactincyclin D2(20cycles), cyclin D1 step of95°Cfor4minutes,then45secondsat95°C,65°C polymerase (Roche).ThePCRprogramincludedaninitialdenaturation 0.5 with III(Invitrogen). Semi-quantitative PCRwas performed Superscript Quantitative RT-PCR kit. Quantitative RT-PCR was performedusingaSIGMASYBR Green Experiments wererepeatedtwiceonthreedifferent RNA preparations. treated withDNAse, thenchecked andquantifiedonAgilentchips. extraction. RNA was furtherpurifiedonRNeasycolumns(QIAGEN), tissue-dishandfollowing themanufacturer’s instructionsfor the prepared byaddingTRIZOL(Invitrogen) directlyontothecellsin 24 hourson conditioned mediumwas dilutedtwice withfreshmediumandappliedfor transfection, replacedbyfreshmediumandcollected24hourslater. This pAdRSV- hybridisation (Nardellietal.,2003).Controlcellsweretransfectedwith GATA2HA todetecttransfectedcellsbyX-Galcolorationbeforeinsitu immunostaining, pAdRSV- described (Nardellietal.,2003).Asthisprotocoldoesnotpermitfurther hours. Insituhybridisationontransfectedcellswas performedas control cellswas started30hoursaftertransfectionandallowed for15 transient expression experiments, BrdUincorporationintransfectedand were performedasdescribed(Nardellietal.,2003).DuringGATA2-HA on different coverslips. Transfection experiments andimmunostaining carried outonatleastfive fields,includingmorethan200cellsselected Nuclei werethencountedusingMetamorphsoftware. Countingwas carried outinduplicate,andallexperiments wererepeatedthreetimes. Fluorescence MountingMedium.Experimentsforeachtimepointwere stained withDAPI beforetheslidesweremountedwithDako treated aspreviously describedfortissuesections.Thenucleiwere with 10%FCS.Thecellswerethenfixed atdifferent timeintervals and Ј -TGGAGAGGGGCAGCTTGCCC-3 The primersequenceswereasfollows: cyclin D1,5 The quantitative PCRreactions(20 Reverse-transcription was performedfrom5 Ј -GCTTCCCTGAGGCTCTCCCTG-3 ␤ ␮ Gal alone.Theculturemediumwas changed 6hoursafter Ј l ofreverse-transcription reaction,usingFastStart Taq Gata2 Ј (forward) and5 (forward) and5 Ј -CTGTGGGCTCTGCATCCCACA-3 –/– Ј cells, platedonglasscover-slips in24-wellplates. Ј (reverse); cyclin D2,5 Ј Ј -CCTGGCCTTGATTCTGCACCG-3 (reverse); Notch1,5 (forward) and5 Gata2 Ј ␤ -TCAGGGCATCACACGTGAGTGTGT-3 Gal plasmidwas addedtopAdRSV- Ј Ј -TCAGGAGCAGCAGGGGTCTGC-3 (forward) and5 Ј –/– ␮ -TCGTCCGTGTGAGCACCCAC-3 g/ml ofculturemedium)was added embryonic neuroepithelialcellswas ␮ Ј l) wereperformedinaMX4000 (forward) and5 p27 Ј -TCTCGCACGTCGGTGGG- Ј Ј (27 cycles), andforcyclin -TGGCCGCAGTCAGGG- (reverse); actin,5 Ј -CAGTCGTCCGACTG- Ј -CTCTTTGATGTC- ␮ Ј Ј -AGGCCGGG- g RNA, with (reverse); Ј -TGTGGCC- Ј (forward) Ј -GTGG- Gata2 p27 –/– Ј Ј Ј , In thehindbrainandspinalcord, neuronalwith newborn precursors Gata2 RESULTS downregulation of In addition,weobserved thatGATA2-HA causedamarked limited coincidencewithGATA2-overexpressing cells (Fig.1J-L,N). on theelectroporatedside(28%comparedto47%)anda very embryos, demonstratedasignificantdecreaseinBrdU-positive cells incorporation, performedforhalfanhourbeforecollectingthe the Mphase(Fig.1G-I,M).Likewise, short pulsesofBrdU versus 11%)incellscontainingphospho-Histone3,apan-marker of ,GATA2-HA expression causedamarked decrease(5% HA epitope.Comparedwiththenon-electroporatedsideof ubiquitous expression ofthehumanGATA2 proteintaggedwiththe electroporation ofpAdRSV-GATA2HA, aplasmidthatdrives misexpression intheneuraltubeofchickembryos by progenitors fromthecellcycle, wefirstinduced To assessthepossibleroleof neuronal progenitors invivo Gata2 could participateincoordinatingcommitmentandcellcycle exit. of several differentiation pathways, raisedthehypothesisthat observations, andthefact that in alltheothersitesof (Fig. 1C,F).Furthermore,weobserved asimilarlyrestricted overlap of theGATA2-positive cellsinthespinalcordappeared tobecycling hybridisation weconfirmedthat,atE10.5,only9%(75among850) galactosidase expression inthespinalcord.Using insitu proliferating cells,asevidenced byBrdUincorporation,and (Zhou etal.,2000)reportedavery restrictedoverlap between expression was driven by differentiation pathway. Usingtransgenicmiceinwhich proliferating ornewly withdrawn fromdivision andengagedinthe medial andintermediatepositions,correspondingtocellseitherstill 1A,B,D, theonsetof both thecaudalhindbrainandspinalcord.Asshown inFig. relative tothespinalcord,althoughouranalyseswerecarriedoutin 2004). For thesake ofhomogeneity, weprincipallypresentdata neurons (Craven etal.,2004;Nardelli1999;Pattyn etal., (Fig. 1A)(Briscoeetal.,2000)andgeneratingserotoninergic plate correspondingtothep3domaindescribedinspinalcord further expression isobserved inathindomainadjacenttothefloor- 2000; Karunaratneetal.,2002;Zhou2000).Inthehindbrain, dorsal limitofthemotoneurondomain(Fig.1A,B)(Briscoeetal., the presumptive domainofV2interneuronprecursorsthatabut the cyclins, namely cyclin D1,D2and D3(CunninghamandRoussel, CDK4 andCDK6,whichisdependent oninteractionwithD-type principally impairtheactivity ofcyclin-dependent kinases,suchas point, thusforcingthemtotake theG0branch.Theseinhibitors increased sufficiently toprevent themfromreaching therestriction cycle inG1,whenthe concentration ofcellcycle inhibitorshas between To confirmsucharole, weinvestigated possibleinteractions machinery Interaction between progenitors. actively participateintheswitchtopost-mitotic stageinneural GATA2 can behave asaninhibitorofthecellcycle andmaythus (Fig. 1O,P).Theseresultswereconsistentwiththehypothesis that expression ispredominantly associated is sufficient toarrest theproliferation of Gata2 and thecellcycle. Inmost cases,cellsleave thecell Sox2 Gata2 , apan-marker oftheneuralprogenitorstage Gata2 Gata2 Gata2 activation occursincellslocalised Gata2 Gata2 expression (datanotshown). Such cis-regulating elements,Zhouetal. RESEARCH ARTICLE Gata2 and thecellcycle in thewithdrawal ofneuronal has beenimplicatedupstream is ventrally expressed in Gata2 Gata2 2157 lacZ ␤ -

DEVELOPMENT motoneurons and moreventral precursors (Fig.2C).CyclinD3 dorsoventral axis, althoughtoalesserextent atthelevel of conspicuously detectedinthe marginal layeralongthe entire activation observed intheprogenitors,transcripts were showed two levels oftranscription.Incontrasttothegeneral low newborn precursors. Atthesamestage,cyclin D2,like cyclin D1, cells suggestedthatthishighlevel ofexpression was associated with V1. Furthermore,thesemi-lateralpositionofmajoritythese expressing V2interneuronsandmoredorsalinterneurons,suchas domain mostprobablyincludesthepresumptive domainof which abuts thedorsallimitofmotoneurons(Fig. 2A,E).This 2158 transcripts weredetectedatlow levels (cyclin D1 patterns ofcyclin D1andD2.Asshown in Fig.2A,B,cyclin D1 surprising distinguishingfeatureswereobserved intheexpression of theirrespective transcriptsintheventral neuraltube.AtE10.5, affect D-cyclin expression, wefirstcloselyexamined thedistribution 1998; Pagano etal.,1994).Speculatingthat and tosustainthemaintenanceofquiescence(Meyyappan etal., been reported,athighexpression levels, toexert theoppositeeffect the progressiontowards theG1/Stransition, D-cyclins have also 2001; Murray, 2004).However, inadditiontotheiractive roleduring a highlevel ofexpression (cyclin D1 maturing neuronprecursors.Strikingly, withintheventricular zone, ventricular zoneandnotatallinthemarginal layer, whichcontains RESEARCH ARTICLE high ) was observed inadomain Gata2 could probably low ) inthe Gata2 - antibody 6and 12 hoursafterelectroporation(data notshown). In activation was inducedincellsthatwerenotstainedbytheanti-HA degradation, weprovided evidence thatendogenouschick possibility thatthelackofGATA2-HA detection was duetoits also intheneighbouringnegative cells(Fig.2J-L).To exclude the endogenous cyclin D1,weobserved aclearactivation ofthe as inthecaseof , wechecked itsexpression inelectroporatedembryos.Indeed, this effect was relatedtotheactivation oftheendogenous neighbouring non-expressing cells(Fig.2H).Wondering whether occurred notonlyinGATA2-HA-expressing cellsbut alsoin activation ofcyclin D1(Fig.2F-H).Surprisingly, sucharegulation HA misexpression inthespinalcordofchickembryosdrove ectopic assessed in D1 innewly committedprecursors.Thishypothesiswas further suggested that distinguished bycyclin D1 observed adecreaseinthenumberofnewborn precursors seemed tobesignificantlyaffected (datanotshown). Bycontrast,we embryos, thetranscriptionpatternofneithercyclin D2nor cyclin D3 of thecellcycle, but alsotocellspecificity. InE10.5 E10.5 appearedtovary individually accordingnotonlytothephase cyclin Dgenesat summarise, thetranscriptionlevels ofthethree expression was restrictedtotheventricular zone(Fig.2D).To antibodies andhybridisedwithchick embryos misexpressing GATA2-HA, stainedwithanti-HA- bar: inA,70 sides (blue,N)are compared. ( BrdU-positive cellsinthecontrol (red, M)andthetransfected in O,50 express motoneurons. Thepresumptive domainofV2precursors, which stained withantibodiesagainstIsl1(red), apan-markerof , firsthybridisedwith and J,K(L).( analysed byconfocalmicroscopy. (I,L)SuperimpositionsofG,H(I) phospho-Histone3 (pH3)(H)oranti-BrdU antibodies(K),and stained withanti-HAantibodies(G,J)andeitheranti- electroporation withthepAdRSV-GATA2HA plasmid,double- sections ofthespinalcord ofchickembryos24hoursafter area includedinthewhiterectangle inC.( BrdU immunostaining(C).( Hybridisation with ofaE10.5mouseembryoinjectedwithBrdU. ( progenitors andissufficient toarrest proliferation. Fig. 1.GATA2 expressiononinnewborn isturned indicated bythewhitearrow. ( Gata2 motoneuron andV1interneuronprecursors; inthehindbrain, A Transverse sectionofthecaudalhindbrainaE10.5mouse ) Gata2 Gata2 is alsoactivatedtoalesserextentinthep3domain, Gata2 Gata2 ␮ m inO,P. M ␮ gain-of-function experiments gene notonlyinGATA2-HA-positive cells,but , islocatedbetweentherespective domainsofthe , m inA-C;D,20 N might participateintheupregulation ofcyclin ) Thepercentages ofphospho-Histone3-and Gata2 high antisense RNA(B)wasfollowedby D-F Gata2 O B transcription (Fig.2B).This ) Highermagnificationofthe , , C P ␮ ) Adjacentsectionsofchick ) Transverse sectionofthe m inD-F;G,20 anti-sense RNA(green), then Sox2 Development 133(11) anti-sense RNA.Scale G-L . ) Transverse Indeed, GATA2- ␮ m inG-L; Gata2 Gata2 Gata2 –/–

DEVELOPMENT GATA2 andcellcyclearrest ofneuralprogenitors p57/Kip2 was barelydetectedinmotorand V2neuronprecursors distributed intheneural tube(datanotshown). Bycontrast, precursors inthespinalcord, 2N,P), thusappearingtobe a generalmarker ofpost-mitotic protein was continuously expressed inthemarginal zone(Fig. Cepko, 2001;Livesey andCepko, 2001).Althoughp27/Kip1 stage inseveral neuronaldifferentiation processes(Dyer and (Cunningham andRoussel,2001)markers ofthepost-mitotic proteins, whichareinhibitorsofCDK-cyclin Dcomplexes control ofthecellcycle, wenext examined theexpression ofKip process. activation appearstobegenerated by anon-cellautonomous endogenous conclusion, GATA2 misexpression ledtoactivation ofthe To furtherunderstand theimpactofGATA2 expression onthe Gata2 and cyclin D1genes,andsomeaspectsofthis p27 transcripts wereequally appeared toprecedethatofp27(Fig.2O).In in 2I).Higherexpression was observed in more dorsalregions and (Fig. 2I,M;notetheexpression gapobserved between Isl1andp57 2T), whereasthedistribution of is normallyexpressed (compareFig.2Owith2Sand2P domain dorsallyabutting themotoneuronprecursors, where embryos, p27/Kip1proteinwas missingormarkedly reducedina engagement towards thepost-mitotic stage. positive control ofp27proteinexpression and,accordingly, inthe strengthened thehypothesisthat GATA2 canparticipateinthe obtained by with 2Q).Theectopicinduction oftheKip1/p27proteinwethen Fig. 2Owith2S)anddidnotseem tobeaffected (compareFig.2M not appeartocompensateforthe lackofp27/Kip1protein(compare altered (datanotshown). Interestingly, expression ofp57/Kip2did GATA2 -HA misexpression (Fig. 3A-C)further (I,M-P) or transverse spinalcord sectionsofwild-type immunofluorescent stainingperformedon ( (H,L) superimpositionofE-G(H)andJ-K(L). immunostained withanti-HAantibodies(F,J). Gata2 hybridised withachickcyclinD1(G)or cord ofelectroporated chickembryos, 70 (A,C,D,E) or of E10.5mouseembryos,wildtype ( loss- andgain-of-function. cycle regulators inthecontextofGATA2 Fig. 2.Analysisoftheexpression ofcell ( cyclin D1transcriptionisupregulated. inA,Bindicatethedomainwhere Brackets ventricular andthemarginalzones. delineate thepresumptive limitbetweenthe V2 andV1precursors. Broken linesinA,C-E the dorsallimitofmotoneurons andoverlaps concentration ofcyclinD1transcriptsabuts (green), showsthatthedomainofhigher performed withanti-islet1antibodies D3 (D)genes.InE,furtherstaining, cyclinD2(C)and for cyclinD1(A,B,E), was performedwithRNAanti-senseprobes expression in R,S,T, thatthesamedomainlacksp27 domain doesnotexpress p57/Kip2,andin arrows inIindicatethattheV2presumptive (MN) neurons are indicatedinIandP. White presumptive domainsofV1,V2andmotor coupled toAlexaFluor488.Therespective Alexa Fluor546andantimouseIgG2b visualised withanti-mouseIgG1coupledto and anti-Isl1antibodies,respectively (P,T) Doubleimmunostainingwithanti-p27 Superimpositions ofM,N(O)andQ,R(S). red) oranti-Kip1/p27(N,R).(O,S) green) were coupledwitheitheranti-Isl1(I, embryos. Anti-Kip2/p57antibodies(I,M,Q, I A-E F-H , M-T ␮ , transversesectionsofthespinalcord ) m inA-D;60 J-L ) Confocalanalysisofdouble (K) antisenseRNAprobe, then Transverse sectionsofthespinal ) p27 Gata2 RESEARCH ARTICLE Gata2 transcripts didnotseemtobe Gata2 –/– ␮ –/– (Q-T) E10.5mouse –/– m inE-L;80 (B); insituhybridisation embryos. Scalebar: Gata2 ␮ m inM-T. mutant Gata2 2159

DEVELOPMENT were notdividing (datanotshown) andwerenotexpressing GATA2 misexpressing cellshadnotmigratedtothemarginal zone, Fig. S1inthesupplementarymaterial).Bycontrast,restof of thesemarkers appearedtobeexclusive ofthatcyclin D1(see activated NeuNexpression (Fig. 4J-L).Atthisstage,theexpression Tubulin/Tuj1 (Fig.4D-F)andneurofilament4G-I),had and J,K(L).Scale bar: 80 immunostaining. (C,F,I,L) Superimpositions ofA,B(C),D,E(F),G,H(I) p27/Kip1 (B),Tuj1/ electroporation. HAimmunostaining (A,D,G,J,green) wascoupledwith sections ofachickembryomisexpressing GATA2-HA 24hoursafter These cellscontinuedtoexpress p27(Fig.4A-C), GATA2 misexpressing cellshadmigratedintothemarginal zone. carried out48hoursafterelectroporationshowed thatsomeofthe (see Fig.S1inthesupplementarymaterial).Furtheranalyses induction appearedtooverlap partiallywithcyclin D1activation expression was notdetected(Fig.3J-L). (Fig. 3D-F)andneurofilament3G-I),whereasNeuN early markers ofneuronaldifferentiation, suchas GATA2-HA misexpression was foundtoinducetheexpression of neuronal differentiation. Twenty-four hoursafterelectroporation, We next wanted toknow whethersucharolewas coupledto neuronal differentiation Gata2 2160 electroporation. postmitotic differentiation markers24hoursafter Fig. 3.GATA2-HA misexpression caninducetheexpression of RESEARCH ARTICLE control oncellcyclecanbeuncoupledfrom ( ␤ A-L IIItubulin (E),neurofilament (NF)(H)orNeuN(K) ) Confocalanalysisofadjacentspinal cord ␮ m. ␤ ␤ III-Tubulin/Tuj1 III-Tubulin/Tuj1 ␤ III- bar: 80 (C,F,I,L) SuperimpositionsofA,B(C),D,E (F),G,H(I)andJ,K(L).Scale Tuj1/ immunostaining (A,D,G,J,green) wascoupledwithp27/Kip1(B), cord ofchickembryos48hours afterelectroporation. HA immunofluorescent stainings performedonadjacentsectionsofspinal into thedifferentiation pathway. Fig. 4.Cellsmisexpressing GATA2-HA canfailtofurther progress impact of through inhibitionoftheNotchpathway. We thereforeanalysedthe GATA2 capacitytodrive cellsoutofthecellcycle mightbeachieved Gata2 therefore independentofthecellularcontext. restricted alongthedorsoventral axisoftheneuraltubeandwas neuronal differentiation. Notablythiseffect was notregionally sufficient tostopproliferationbut nottosystematicallyinduce pathway. zone andprobablyunabletoprogressalongthedifferentiation to differentiate normallyortobecomeblocked intheventricular 4J-L). GATA2-HA misexpressing precursorsthusappearedeither ␤ p27/Kip1 (Fig.4A-C)orneuronaldifferentiation markers suchas be informative asonlyslightdifferences couldbeobserved (datanot and effectors oftheNotchpathway suchas III-Tubulin (Fig.4D-F),neurofilament (Fig.4G-I)orNeuN In conclusion,gain-of-functionstudiesestablishedthat Ngn2 ␤ IIItubulin (E),neurofilament (NF)(H)orNeuN(K)immunostaining. ␮ counteracts Notchsignalling m inA-F;90 . Inthisregard, loss-of-functionstudiesdidnotturnoutto Gata2 loss- andgain-of-functionontheexpression of ␮ m inG-L. ( A-L ) Confocalanalysisofdouble Notch1 Development 133(11) , Dll1 , Hes5 Gata2 , Mash1 is

DEVELOPMENT Serrate1 and observed inthepresenceofId2alone(Fig.6G,J).Bycontrast, (Fig. 6E,F).ThiscanbeassignedtoId2functionasitwas also contrasting effect ofId2co-expression was theactivation of as assessedby GATA2-induced neuronaldifferentiation was notabolishedbyId2, activation andchick observed thatGATA2-HA was stillabletoinduce shown toinhibitthefunctionofproneuralproteins. We then misexpressed withId2,abHLHregulatory proteinthathasbeen Gata2 to analysetheconsequencesofinhibitingproneuralfunctionin proneural activity. To addressthisquestionspecifically, wedecided which raisedthepossibilitythatitcouldfunctioninconcertwith Thus, GATA2 appearedtobeastronginhibitorofNotchsignalling, to beactivated inresponsetoGATA2 misexpression (Fig.5D,E). that of Ngn2 GATA2 alone. the repressionof Mash1 required forGATA2 toextinguish Notchsignallingandtoinduce 6G-I). Theseresultsestablishedthat:(1)proneuralactivity isnot sections toA,D,G. Scalebar:70 or stainedwithanti-HAantibodies (A,D,G).(B,C,E,F,H,I) Adjacent (C), hours afterelectroporation, hybridisedwithchick Gata2 cells atE10.5.Bycontrast,gain-of-functionstudiesshowed that shown), probablyowing tothelow distribution of GATA2 andcellcyclearrest ofneuralprogenitors misexpression. Fig. 5.TheNotchpathwayisshut off inthecontextof Cash1 Hes5 (Fig. 5D,F), misexpression causedaclearrepressionof Gata2 and neuronaldifferentiation; and(2)Id2appearstoalleviate gain-of-function context. To thisendGATA2-HA was / Jag1 did notappeartobeaffected byId2misexpression (Fig. (E), . Only Ngn2 Fg AC,teexpression ofwhichdoesnotoverlap (Fig. 5A,C),the ␤ ( A-I III-tubulin expression (datanotshown) Ngn2 Notch1 Spinalcord cross-sections ofchickembryos24 ) (F), Cash1 Notch1 Hes5 transcription observed inthepresenceof (Fig. 5G,H), , thechick ␮ repression (Fig.6C,D).Similarly, (H) and m. Hes5 Mash1 Hes5 (I) antisenseRNAprobes, (Fig. 5G,I)andeven of homologue, appeared Dll1 Cash1 Gata2 Dll1 (B), Serrate1 (Fig. 5A,B), -expressing (Fig. 6A,B) Gata2 . The sole Cash1 Ngn2 / Jag1 dissected eitherfromwild-typeor ENC culturesweregeneratedfromposteriorneuraltubeexplants inactivation ontheproliferationrateofENCinculture.To thisend, restriction ofproliferation,wedecidedtoassesstheimpact Speculating that agent suchasamutatedversion ofSV40T(Nardelliet al.,2003). a poorproliferationrateifthey donotexpress animmortalising Nardelli etal.(Nardellial.,2003)].Inaddition,thesecellsexhibit cord doexpress (ENC). Indeed,ENCisolatedfromeitherthehindbrainorspinal cultured cells,specificallytomouseembryonicneuroepithelialcells To furtherinvestigate thisaspect,weswitchedourinvestigations to that celldeathcouldaccountforsuchadifference (data notshown). embryos (datanotshown). TUNELstainingexcluded thepossibility mutant embryoswas infact morerestrictedthaninwild-type embryos. We foundthatthedistribution ofthetranscriptsin type andthemutated using allows detectionoftranscriptsgeneratedfrom boththewild- (data notshown). Second,asthe pattern ofphospho-Histone3and respectively, whereasno significant differences wereobserved inthe the distribution oftranscriptsinbothwild-typeand an increasein different attempts,itwas notpossibletoprovide evidence forsuch cycle exit invivo isincreasedproliferation.However, despite Another consequencethatcouldbeexpected fromdefective cell culture Gata2 higher inthewild-typethan PCR, wefoundthatthelevels of wild-type andmutantcells.By performingsemi-quantitative RT- This promptedustocomparethe statusofcyclin Dtranscriptionin implying ashorteningoftheG1 phaseintheabsenceofGATA2. necessary toreachtheG1/Sphase was shorterinmutantENC,thus cell cycle reactivation. Theseresultsestablishedthatthetime only 10%ofthewild-typecells,wereBrdUpositive 8hoursafter delayed inthewild-typeENC.Indeed,60%ofmutantcells, but entry intothecellcycle. Bycontrast,suchanincreasewas clearly BrdU-positive ENCincreasedsteadilyinthe mutantcellsuponre- compared attimeintervals. Asshown inFig.7G,thepercentageof the culturemedium,andrateofBrdUincorporation was re-enter thecellcycle byadditionoffoetalcalfserumandBrdUin quiescence byserumdeprivation for24 hours.ENCwereallowed to the wild-typeandofmutantENC.Bothculturesweredriven into BrdU incorporationwas performedintwo culturesrepresentative of division timeintheabsenceofGATA2. Inordertoconfirm this, wild-type andmutantENCwas probablyduetoashorteningofthe to differentiate. of thewild-typeandmutantcells,theircommonlow propensity (Fig. 7Fanddatanotshown) confirmedtheneuroepithelialcharacter (Fig. 7Danddatanotshown), andlackof more rounded.Widespread nestin(Fig.7B,C)andSox2expression flatten andspreadasmuchwild-typecells,remainingsmaller than aweekwhenseededat1:5dilution.Moreover, they didnot proliferation rate.Indeed,themutantcellsreachedconfluency inless in allfive casesthemutatedcellsmanifestedasignificantlyhigher out ofsixculturesoriginatingfrom dilution,every two tothreeweeks.Bycontrast,five passage, at1:4 cells derived fromtheseculturesdivided very slowly, allowing a wild-type explants survived andcould beexpanded inculture.The accordance withourprevious experiments, only11outofthe25 The marked difference intheproliferationrate between impedes theproliferation ofneuralcellsin Gata2 Gata2 Gata2 Gata2 knockout (KO) embryos.First,theexpression in culture,albeitatlow levels [Fig. 7Aand expression couldbeinvolved inthe allele (Tsaietal.,1994),wecouldassess Gata2 Gata2 Sox2 cyclin D1 Gata2 RESEARCH ARTICLE Gata2 –/– was foundtobeunaltered RNA probewehadbeen –/– ENC, three-andfourfold ␤ –/– III-Tubulin expression explants survived, and embryos atE9.5.In and cyclin D2 Gata2 Gata2 Gata2 Gata2 2161 were KO

DEVELOPMENT 2162 RESEARCH ARTICLE (BioRad). Scalebar:40 then analysedandquantifiedwithGelDoccamerasoftware quantitative RT-PCR. PCRproducts were separatedonBEtagarose gels, D1, cyclinD2,D3andE1, (blue) inB,C,E,F. (B)or Wild-type of serumandBrdU. ( phase atdifferent timeintervalsaftercellcyclerelease inthepresence anti-Tuj1/ with anti-GATA2 (A),anti-nestin(B,C,green), antiSox2(D,green) and (C-F) embryonicmouseneuroepithelial cells(ENC);immunostaining neuroepithelial cellsinculture. Fig. 7.GATA2 inhibitstheproliferation ofmouseembryonic than in transcripts inwildtypeENCcellswas foundtobe4.5-fold lower observations wemadeinvivo, theconcentrationof respectively, incomparisonwithwild-typecells.Consistentthe level oftranscriptsinmutantcellswas decreasedthree-andfivefold, performing quantitative RT-PCR experiments thatshowed thatthe concerning cyclin D1andcyclin D2werefurther confirmedby increased bythreefoldin approach demonstratedthatthelevel ofcyclin E1transcriptswas andof case ofcyclin D3 (D), butnoTuj1/ ( wild-type cells(notshown);D-Fcorrespond tothesamefield. (see Fig.S2in thesupplementarymaterial). These experiments transfected cellsculturedinmedium conditionedbytransfectedcells control cells(Fig.8B,D).Similar upregulation was obtainedinnon transcription (Fig.8A,C),which was detectedatvery low levels in appeared tomarkedly and widelyupregulate cyclin D1andD2 by transienttransfectionwiththe pAdRSV-GATA2HA construct, G Comparisonofthepercentage ofserum-starvedcellsreaching theS ) Consistently, Gata2 ␤ III-tubulin (F, red) antibodies.Nucleiwere stainedwithDAPI -deficient ENC. ␤ Gata2 III-Tubulin (F),canbedetectedin H ) Comparisonofthetranscriptionlevelcyclin ␮ m inA-C;20 complementation inmutantENC, achieved Gata2 Fig. 6. probes for performed withchickRNAantisense antibodies. Insituhybridisationwas antibodies and(G)withanti-GFP (A,C,E) Immunostainingwithanti-HA pACGGScId2 andpAdRSV-GFP (G-J). GATA2HA andpACGGScId2(A-F)orwith after electroporation eitherwithpAdRSV- cord sectionsofchickembryos24hours require proneural activity. sections. Scalebar:70 Ngn2 in G-J. p27 Gata2 ( mutant ENC(Fig.7H).Theresults (Fig. 7H).Inaddition,thesame A-F (F,J). (A,B;C,D;E,F;G-J)Adjacent Gata2 ␮ p27 ) Wild-type (A,B)and ) Wild-type –/– m inD-F. Cash1 (C) ENCsexpress Nestin.Sox2 and function doesnot Development 133(11) Notch1 (B,H), Gata2 ␮ Hes5 m inA-F;60 genes bysemi- –/– ( (D,I) and cells, asin A-J Gata2 ) Spinal Notch1 –/– ␮ m

DEVELOPMENT expression inthesecells(datanotshown). with neuronaldifferentiation, asdeducedbythelackof was notassociatedwiththe inductionofp27/Kip1expression or data notshown). However, this clearinhibitionofcellproliferation not incorporateBrdUorexpress phospho-Histone3(Fig.8H-Jand cultures didnotgrow aswell,andGATA2-HA expressing cellsdid (Fig. 8E-G;seeFig.S3inthesupplementary material),transfected continued togrow normallyandmostofthemwereBrdUpositive neuroblastoma cells.Indeed,inbothcases,whereasthecontrol cells material) andnotablyofhighlyproliferatingNB2amouse GATA2 andcellcyclearrest ofneuralprogenitors proliferation of GATA2 transient expression alsocausedthearrestof the that thisnon-cell-autonomouseffect ismediatedbyasecretedfactor. observed forcyclin D1invivo. Furthermore,they stronglysupport transcription inENCcellsanon-cell-autonomousmanner, as provided evidence thatGATA2 inducescyclin D1andD2 incorporated BrdU (J).Scalebar:45 density and,especiallyGATA2-HA expressing cells,havenot whereas intransfectedcultures (H-J),cellshavenotreached thesame culture (E-G),cellshavenotstoppedgrowing andare allBrdU positive; mounting. (G,J)SuperimpositionsofE,F(G)andH,I(J).Inthecontrol immunostained (I,red); nucleiwere stainedwithDAPI(E,H)before 15 hourslater, processed forBrdU staining(F,I) andfurtherHA- in theculture medium30hoursaftertransfection,cellswere recovered transiently transfectedwithpAdRSV-GATA2HA (H-J).BrdU wasadded either withpAdRSV-GATA2HA andpAdRSV- probes. ( hybridisation withcyclinD1(A,B)orD2(C,D)antisenseRNA Fig. 8. pAdRSV- mouse neuroblastoma cells. Gata2 much more activecyclinD1andD2transcription,while Gata2 transient overexpression blockstheproliferation of E-J ␤ Gal alone,thentreated forX-Galcolourationandinsitu ) NB2amouseneuroblastoma cells,eithercontrol (E-G)or dfiin N complementedwith -deficient ENC Gata2 –/– ENC (seeFig.S3inthesupplementary ( A-D ␮ ) Gata2 m inA-D;15 –/– ␤ Gal plasmids(A,C)orwith ENC were transfected ␮ m inE-J. Gata2 ␤ exhibit III-Tubulin features ofD-cyclins intheearlyneural plate(Lobjoisetal.,2004; al., 2004)have beenshown tolargely accountfordistinctexpression with thefact thatgrowth factors andsignallingpathways (Lobjoiset implicated intheregulation ofthiscyclin gene,whichisconsistent upregulation indicatesthatenvironmental cuesare cyclin D1 neuronal differentiation. Bycontrast,theregion-specific aspectof denote amorespecificrole,which remainstobeunderstood,during consolidation ofthepostmitoticstage.Inaddition,itcaneventually maturation phaseofneurons,whichcouldthusberelated to transcription patternisconsistentwithageneralfunctionduring the (Meyyappan etal.,1998;Pagano etal.,1994). CyclinD2 cycle progressionandsustainthemaintenanceofquiescence transcription orfurtherstabilisationofthetranscripts,impede cell D-cyclins, whichcanresultfromeithermoreactive gene functions. Indeed,previous reportshave shown thathighlevels of distinct upregulation featuresappeartobeinvolved inother them toparticipateinthemaintenanceofcelldivision, whereas a low level ofactivation oftheD-cyclins maybesufficient toenable conversely, theG1/Stransition.Accordingtoourexpression studies, phase incommittingthecellstowards either theG0phaseor, Kip proteins,whichareknown toplayacrucialroleduringtheG1 in cellcycle withdrawal, wefocusedourstudiesonD-cyclins and To gainfurtherinsightintohow GATA2 caneventually participate components ofthecellcyclemachinery Gata2 conditions. require furthermolecularevents obviously absentinourculture irremediably progressalongthedifferentiation pathway, whichmay sufficient toconsolidatethepost-mitoticstageandenablecells culture. Consistently, wesuggestthatGATA2 maynotbealways electroporation. Furthermore,nodifferentiation was observed in and becomenegative forsuch markers 48hoursafter that partofthesecellsarenotabletokeep differentiating properly primarily observed inGATA2-misexpressing cellsinvivo, itappears vivo. However, ifoutsetsofearlyneuronaldifferentiation are further associatedwithcellcycle exit andneuronaldifferentiation in tubulin, neurofilamentandNeuNexpression, thiseffect canbe described forotherventral specificationgenes,suchas to engageinterminaldifferentiation. Thishas previously been to differentiation, andthisinstructscellstoexit thecellcycle inorder the possibilitythat as inculture,ENCorNB2acells.Asvisualisedbyp27, sufficient toimpedetheproliferationofneuralprogenitors,invivo As estimatedbyBrdUincorporation,GATA2 expression canbe neuronal differentiation may require furtherinstructiontoinduce Gata2 neuronal differentiation. effect was observed bothinconcertwithandindependentlyof in vivo andrestrictstheproliferationrateinvitro.Interestingly, this the division ofneuronalprogenitors,whichcanlead tocellcycle exit studies, wehave establishedthat (Dubreuil etal.,2000).Usingbothloss-andgain-of-function during motoneuronspecification(Tanabe etal.,1998)or a minorityofcellsexpressing et al.,2002;Pattyn etal.,2004;Zhou2000).Thefact thatonly neuronal differentiation pathways (Craven etal.,2004;Karunaratne molecular cascadesthattake placeduringtheearlystepsofseveral Gata2 DISCUSSION has beendemonstratedtobeadeterminingfactor in interferes withthecontrol ofexpression of is sufficient toinhibitcellproliferation but Gata2 is activated duringfinalcelldivision prior Gata2 Gata2 RESEARCH ARTICLE were foundtobemitoticraised exerts negative controlon MNR2 Phox2b 2163 / ␤ HB9 III-

DEVELOPMENT of thiscyclin didnotseemtobealteredinvivo in D2, thiswas indiscrepancy withthefact thattheexpression pattern transcription hasbeenestablishedinENCcells.Inthecaseofcyclin V2 precursors.ThesameinfluenceofGATA2 oncyclin D1andD2 of cyclin D1invivo andmaythusparticipateinthisupregulation in we have shown thatGATA2 isabletocausetransientupregulation during thespecificationofneuralprogenitors.With regard tothis, necessary togainmoreinsightintotheeventual roleofthiscyclin regulation oftranscriptionactivation. Furtheranalysiswillbe cells exhibiting thehigherlevel of the distribution ofcellsexpressing GATA2 atE10.5andofventral embryos. Thiscouldpossiblybeexplained inanumberofways: (1) specification genessuchas Wianny etal.,1998).Downstream ofsignallingpathways, 2164 and tobemediated byasecretedfactor not yet identified.Thisisalso mediate thiseffect, whichappearstofunctioninabsenceofGATA2 effect, suggestthat endogenousfunctional ENC, whichconfirmedtheexistence ofthisnon-cellautonomous function analysesstronglysupportthat the controlofp27/Kip1proteinexpression. Ourgain- and loss-of- (Tsvetkov etal.,1999;Zhang2005)events largely accountfor Indeed, translational(Kullmann etal.,2002)andpost-translational synthesis inthemarginal layerreliesonpost-transcriptionalcontrols. distributed intheneuraltubesupportsideathatprotein Furthermore, thefact that progenitors withrespecttotheirpositionalongthedorsoventral axis. mechanisms underlyingcellcycle withdrawal amongneural yet beenelucidated,they denotethepotentialexistence ofdifferent functional consequencesofsuchdistinctregional features have not barely detectedinthemotoneuronandV2domains.Although continuously delineatesthemarginal zone,whereasp57/Kip2is the expression ofp27/Kip1and/orp57/Kip2proteins. In fact, p27 events mayberequiredtoconsolidatethepost-mitoticstage,suchas (Meyyappan etal.,1998;Pagano etal.,1994). towards theSphase,inparticularupregulation ofcyclin E This canresultintheinhibitionofevents underlyingtheprogression inactivate CDK2bydisruptingthecomplex itformswithcyclin E. of cyclin D1andD2.Indeed,athighconcentrations,D-cyclins can observed inENCcouldbeaconsequenceofthehigherexpression and takingplaceonlyinculture.Thedownregulation ofcyclin E (3) theexistence ofadistinctregulatory mechanisminvolving vivo, asdescribedinKO miceforD-cyclins (Kozar etal.,2004);and absence ofGATA2; (2)compensatorymechanismstakingplacein might betoolow toprovide clearevidence ofalteredexpression in autonomous manner. Complementationexperiments in Gata2 exogenous GATA2 expression inducedactivation oftheendogenous The latterhypothesisisstronglysupportedbythefact that and (2)thelackofauto-activation potentially inducedbyGATA2. in cellfate andthesubsequentdownregulation ofthe apossibleswitch for bytwo non-mutuallyexclusive hypotheses:(1) could notexplain it,thispronouncedrestrictioncouldbeaccounted restricted in in theventral spinalcordatE10.5,whichwas foundtobeeven more failure mightbeduetothelow distribution ofcellsactivating manifested byENCinculturebut was notobserved invivo. This situation couldbeincreasedproliferation.Thiswas overtly exit intheseprecursors.Theexpected consequence ofsucha by p57/Kip2,andthisraisestheissueofalterationcellcycle GATA2-deficient precursorsdoesnotappeartobecompensatedfor control invivo. Furthermore,thelackofp27proteinoccurringin Once cellshave beencommittedtoleave thecellcycle, further and cyclin D1genesinboth acell-autonomousandnon-cell- RESEARCH ARTICLE Gata2 mutant embryos.Consideringthatcelldeath p27 transcripts werefoundtobewidely Gata2 cyclin D2 may beimplicatedinthe Gata2 Gata2 can contribute tothis transcription which is notnecessaryto Gata2 Gata2 Gata2 mutant Gata2 Gata2 ; –/– that non-cell-autonomous mechanismiscontrolledinthespinalcordso gain furtherinsightintothistranscriptionalcontrolandhow this D2 maynotbedirectlyregulated byGATA2. Itwillbeinterestingto consistent withthehypothesisthattranscriptionofcyclin D1and Artavanis-Tsakonas, S.,Rand,M.D.andLake,R.J. References http://dev.biologists.org/cgi/content/full/133/11/2155/DC1 Supplementary materialforthisarticleisavailableat Supplementary material Pierre etMarieCurie,theAFM andtheARC(grants59463376). Maunoury forplasmids.ThisworkwassupportedbyCNRS,UniversityParis VI Fabienne Pituello,PhilippeRavassard, Pierre Savatierand SylvieSchneider- Guillemot, Tony Hunter, FrançoiseLapointe, RobinLovell-Badge,JaneJohnson, Brunet, DouglasEngel,JohanEricson,PascaleGilardi, ChristoGoridis,François quantitative RT-PCR experiments.We thankDavidAnderson,Jean-François of neuroepithelial cellcultures, andtoAbibaDoukani forherassistancein manuscript, toAnneDesmazières forherinitialparticipationinthegeneration Françoise Helmbacher, PaulaMurphyandSueOrsoniforcriticalreading ofthe insightful discussion,toJamesBriscoe,GillianButler-Browne, RacquelCooper, grateful toJacquesMalletforhisvaluablesupport,FabiennePituello We are indebtedtoStuartOrkinforproviding the Bertrand, N.,Castro, D.S. and Guillemot,F. The Notchsilencingcausedby Gata2 Campos, L.S.,Duarte,A.J.,Branco, T. andHenrique,D. Craven, S.E.,Lim,K.C.,Ye, W., Engel,J.D.,deSauvage,F. andRosenthal, Briscoe, J.,Pierani,A.,Jessell,T. M.andEricson,J. Blader, P., Fischer, N.,Gradwohl,G.,Guillemot,F. andStrahle,U. intervene duringneuronalfate determination. a possiblecrosstalkbetweenproneuralandspecificationgenesthat proliferation ofneuralprogenitors.They provide furtherevidence of allow ustodeterminetheway inwhich (Oberg etal.,2001;Schweisguth,1999).Althoughourdatadonot it mayrelyondegradation processesthatcontrolNotchturnover has beenreported(Wai etal.,1999).Finally, wecannotruleoutthat interference betweenNotchsignallingandcellcycle components consequence ofnegative regulation ofthecellcycle by Tsakonas etal.,1999).Second,Notchsilencingmaysimplybea other components(Huppertetal.,1997)(forareview, seeArtavanis- expression ofany componentoftheloopwillalterexpression of involves anauto-regulatory loop,whichmeansthatalterationofthe pathway mayoccurindifferent ways. First,Notchsignalling induction ofneuronaldifferentiation. Thiseffect ontheNotch manipulations isconsistentwiththearrestofproliferationand function of 2004; Pattyn etal.,2004)andnotwithproneuralactivity. associated withafunctioninneuronalspecification(Craven etal., et al.,2004),andwethinkthat described tobemechanisticallydistinct(Parras etal.,2002; Pattyn in -typespecification.Thetwo functionalaspectshave been Indeed, inadditiontoitsproneuralactivity, functional aspectof signalling, despite cell fatecontrol andsignalintegration indevelopment. specification ofneuralcelltypes. early cortex. mDll3 expression inthedevelopingmousebrain:role intheestablishmentof neural tube. protein codespecifiesprogenitor cellidentityandneuronal fateintheventral Development activity ofneurogenin1 iscontrolled bylocalcuesinthezebrafishembryo. hedgehog. A. In conclusion,ourstudieshave revealed new aspectsofthe Gata2 (2004). Gata2specifiesserotonergic neurons downstream ofsonic interferes withtheNotchpathway activation isrestrictedtoV2precursors. Development Gata2 J. 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