in mammaliancorticogenesis. progression andintheacquisitionofapoptoticorneuronalcellfate.Theresultsprovidenewevidenceforcomplexrole progenitor poolswithdistinctendogenous of either Kevin R.Jones et al.,1998).Thesecellshave beenshown toactaspluripotent progenitors, namelytheRC2-positive radial glial(RG)cells(Götz distinct amygdalarnuclei(Tole etal.,2005). the rostralVPandLPappearsessentialforspecification of Similarly, thehighexpression level of low whereas thecaudomedialcorticaldomains,whichnormallyshow Pax6 Sey/Sey play aroleinprogenitorregionalization. Thus,inhomozygous expression incorticalprogenitorsalongtheanterior-posterior axis 2001). Recentdataindicatethatthedifferent levels of 1996; Stoykova etal.,2000;Torresson etal.,2000;Yun etal., (Kroll andO’Leary, 2005;Muzioetal.,2002b;Stoykova etal., pallium (LP),andsubsequentlyextending dorsallyand medially molecular identitystartingattheventral pallium(VP) andlateral (Hill etal.,1991),thecorticalprogenitorsacquireventral 2000). Inthe E15.5 (Muzioetal.,2002a;Stoykova etal.,1997;Stoykova etal., mediolateral-low gradient,withexpression lostprogressively after cortex, pallial-subpallial boundary(Toresson etal.,2000).Indeveloping expression of regional specification ofthetelencephalon.As earlyasE8.5,the In thebrain, pivotal rolesinthemorphogenesisofeye, pancreasandbrain. Pax6 isanevolutionarily conserved transcriptionfactor with INTRODUCTION Accepted 17January 2007 4 3 2 1 KEY WORDS:Corticogenesis,Cre/ conditional activationoftwo glial progenitorsandendowsthemwithneurogenicproperties.UsingaCre/ During development, Joachim Berger Conditional activationof (2007)doi:10.1242/dev.02809 Development 134,1311-1322 *Author forcorrespondence (e-mail:[email protected]) repressive interactionsbetween 1991), outliningtheanlageoffuturecortex, while cross- dorsal partofthetelencephalicprimordium(Walther andGruss, transgenic micecausesprogenitorapoptosis University ofColorado,Boulder, Colorado,USA. Dulbecco Telethon InstituteatIRCCSFondazione SantaLucia,Rome,Italy. DFG, CenterofMolecularPhysiology theBrain(CMPB),Göttingen,Germany. Max PlanckInstituteforBiophysicalChemistry, D-37077Göttingen,Germany. The expression of Pax6 is normallyexpressed atthehighestlevel, arereduced, Pax6 embryos, therostrolateralregions ofthecortex, where Pax6 expression, areexpanded (Bishopetal.,2000). Pax6 Pax6 is expressed inaprominentrostrolateral-highto or Pax6 4 Pax6-5a acts asapatternformationgene,involved inthe , PeterGruss 1 loss-of-function (LOF)mouse , SilkeBerger is confined totheneuroepitheliumof Pax6 Pax6 inhibits progenitorproliferationinthedevelopingcortex.Uponactivationoftransgenic is expressedinarostrolateral-hightocaudomedial-lowgradientthemajorityofcorticalradial specifies themajorityofcortical Pax6 loxP 1 Pax6 and AnastassiaStoykova isoforms, 1 , Overexpression, , Tran CongTuoc Pax6 and Pax6 in theprogenitorsof Pax6 Gsh2 expression levelsatdifferent developmentalstagesshowdefectsincellcycle Small eye and establish the Pax6 Pax6-5a Pax6 1,2 , , Marcello D’Amelio Pax6-5a ( Pax6 Sey , onthecorticogenesisoftransgenicmice.We foundthatactivation ) 1,2, , Progenitor, Apoptosis,Mouse in thedevelopingcortexof generate resulting in * The codingsequenceof (Niwa etal.,1991)followed by a A Construction ofplasmidsandgeneration oftransgenicmice activation ofthetwo (GOF) expression intransgenicmiceandstudiedtheeffects ofthe developed aninvivo systemforconditional Retrovirus-mediated overexpression of proliferation isinfluencedbybothisoforms(Haubstetal.,2004). mediated exclusively bythePax6 isoform,whereasprogenitor 1997). Dorsoventral patterningandboundaryformationseemtobe of targets (Czerny et al., 1993;Epsteinet1994b;Kozmik etal., isoforms,Pax6 andPax6-5a, possiblyhaving different sets (Götz etal.,1998;Warren etal.,1999). (Hartfuss etal.,2001;Stoykova etal.,1996)anddifferentiation and Osumi,2004;Stoykova etal.,1997),boundaryformation adhesive properties(Caricetal.,1997;Hartfuss2001;Nomura cycle (Estivill-Torrus etal.,2002;Götz1998),migratory and et al.,2000),andtheRGprogenitorsshow defectsintheirmitotic respectively) (Schmahletal.,1993;Stoykova etal.,1996;Stoykova overgrown ventricular andsubventricular zones(V.Z. andS.V.Z., At birth,thecorticalplate(CP)of 2002; Malatestaetal.,2000;Miyata2001;Noctor2004). progenitors, generatingbothneuronalandglialcells(Heinsetal., of thetwo isoforms, corticogenesis. We found thatectopicactivation oroverexpression Pax6 vitro andcelllineageexperiments indicatedaneurogenic activity of cortical progenitors.Furthermore,activation oftransgenic MATERIALS ANDMETHODS Pax6 of thecorticalprogenitorscontainingdifferent levels ofendogenous which seemstodependonthedistinctspatiotemporalsensitivities , andmassive apoptosisindifferent progenitorpools, vivo causesmisregulation of themitoticcycle, premature loxP In vertebrates, alternative splicing generates two different Pax6 Using aCre/ . (Hack etal.,2004;Heins2002;Haubst2004). - egfp JoP6 loxP pJoP6 -polyA cassettewas clonedintothe -based approach,westudiedtheeffect of and 3 , FrancescoCecconi JoP6-5a and loxP Pax6 pJoP6-5a Pax6 -based recombinationapproachwehave Pax6 mice bypronuclear microinjection.Transgenic and isoforms indifferent progenitorsduring or , respectively. Theplasmids wereusedto Pax6-5a Pax6-5a loxP Sey/Sey -( RESEARCH ARTICLE Xho , inhibitstheproliferationof was inserted intothe 3 Pax6 I)- , JessicaA.Gorski mice ishypocellular, with IRES Pax6 Eco in RGprogenitors - lacZ Pax6 RI siteof gain-of-function -polyA cassette. , specific pCAGGS Xho Pax6 Pax6

Pax6 1311 4 I site , in

DEVELOPMENT of BrdU BrdU labelingindex was estimated asthepercentageoftotalcellnumber TUNEL labeling,theApopTag kitwas used(Intergen, Purchase,NY).The labeling). SecondaryantibodieswerefromMolecularProbes(1:500).For of theprimersJoP6F andJoP6R. simultaneous expression oftransgenic stop cassetteplacedinfront ofthe Fig. 1.Theconstructfortransgenic activationof (Cell SignalingTechnology) on8 antisera againstpHH3(Biomol), neuron-specific classIII (BAbCO), 5-bromo-2-deoxyuridine(BrdU;Roche),nestin(Chemicon)and (Moorman etal.,2001).We usedmousemonoclonalantibodiesagainstPax6 2000) andinsituhybridizationanalysiswas accordingtoMoormanetal. Immunohistochemistry was carriedout asdescribed(Ashery-Padan etal., Immunohistochemistry with theprimersJoP6F, JoP6RandJoP65aR(Table 1). mice wereidentified byGFPfluorescence.GenomicPCRwas performed Primersequence(5 Pax6 Name Target Table 1.Primersusedinthisstudy 1312 Pax6-5a N-CAM (Promega) (Epsteinetal.,1994a). generated bycloningfourPD-andfive HD-consensussequencesinto Itga5 -ahrnFCTATGGTGCGGCTGCTGGTA Pxn F R-cadherin Tnc 8 F Itgb3 18S RESEARCH ARTICLE + cells onDAPI-stained sectionsinequallysizedareasmedial o6a GTCGCCACTCTTGGCTTACTCCCTCCG JoP65aR o6 CACAACCGTTGGATACCTGCAGAATTCGG JoP6R o6 CCTGGGCAACGTGCTGGTTATTGTGCTG JoP6F CCGCTTCAGCTGAAGTCGCA R GCACCTGGACTTTTGCATCTG R TCCTCCATGTCTTTGCCCTTG R GTACTCCAGAATCCGGGTGAA R GGAGTTCTCGGGAACGTTGAT R GGAACTCTCCAGTTCATCCAAGA R CTCCCGAATGGTGGTGGATGT R R R CCTGGTTGGTATCCCGGGA F CACAGCGGAGTGAATCAGCTT F ATTGTCATCTTCGTCCTGCTCC F GGATTCTGGAGTCCTCACTGTA F CCGGCTGTTACTGGAGCTGAA F GCAACCAAGGACAATGTGTGTCA F F QT00128849 QT01036875 ␤ tbln(u1 ac) andrabbitpolyclonal -tubulin (Tuj1; Babco), ␤ -actin (Abcam),andactivated caspase3 ␮ Pax6 m cryostatsections(5 Pax6 and Ј to 3 and lacZ Ј ) orQiagencatalognumber lacZ coding sequences.AfterCre-mediated recombination, the via anIRESsequence from theresulting construct pPD Pax6 and ␮ . The construct m forBrdU pHD pGL3 were pJoP6 of the expression ofthe actin/CMV fusionpromoter(Niwa etal.,1991),driving ubiquitous that containsafloxed For conditionalactivation of vivo The systemforconditionalactivationof RESULTS listed inTable 1. normalized tointernal18SRNA (dataasmeans±s.d.).Primersequencesare SYBR GreenPCRKit(Qiagen).Assayswereperformedintriplicateand for q-PCRwiththeQuantiTect Rev. KitandtheQuantiTect Total RNA isolatedfromcortex was quantified byopticaldensityandused q-PCR analysis performed usingLipofectamine2000(Invitrogen) and3 Transient transfectionofSAOS2 (ATC# HTB-85)andHeLacellswere Luciferase reporter assay pHH3 pallium (MP)anddorsal(DP)fromcortex ofbothgenotypes. shown asmeans±s.d. sized unitsofcontrolanddouble-transgenicapicalVZsurface. Dataare transgenic cortex (Fig.2A).Sectionsof was detectedusingDNA isolatedfrom successful recombinationanadditional261bpDNA fragment JoP6 respectively. GenomicPCRwithDNA isolatedfromthe cortex of Fig. 1),whichbind5 monitored byPCRwiththeprimersJoP6FandJoP6R(arrows in 2002). Recombinationinthe recombination inmostofthepallialprogenitors(Gorskietal., 5a et al.,1995), plasmids: JoP6 JoP6 transgenic mouselinegeneratedwiththisconstructwas named exhibited widespreadGFPfluorescenceintheentirebrain, which activities (datashown asmeans±s.d.). triplicate andluciferaseactivities werenormalizedtointernalcontrol Assay System(Promega) after48hours.Theassaywas performedin by Pax6++ inFig.7).CellsweresubjectedtotheDual-LuciferaseReporter . For invitrooverexpression of In ordertotesttherecombinationofintegrated construct, Pax6 gfp contains the + ae werecrossedwithfemale males brains resultedina1831bpDNA fragment, whereasafter -stop cassetteisexcised, leadingtosimultaneousexpression cells werecountedandcomparedover thewholecortex inequally and asecondreporter, pCMV pmRb-pJ3Omega115ROX-p and ␤ gfp -actin/CMV fusionpromotor anda pPax6 P reporter gene(Fig.1).UponCrerecombination values werederived usingStudent’s Ј gfp pJoP6rec and 3 (Maulbecker andGruss,1993), -stop cassetteundercontrolofthe Pax6 Pax6 gfp Ј lacZ . Arrows indicate theannealingsites of thefloxed -stop cassetteiseliminatedallowing we generatedaconstruct( , 16 JoP6 (Bernards etal.,1989),and , viaanIRESsequence.The ␮ Emx1 g of JoP6 ; Emx1 pPax6 Development 134(7) JoP6 IREScre ; Emx1 IREScre gfp was used(indicated ␮ loxP g ofthefollowing -stop cassette, control cortex mice directing IREScre p53Luc t Pax6 -flanked -test. mice was double- pJoP6 pJoP6- (Stuart in JoP6 gfp ␤ - - ) .

DEVELOPMENT JoP6 increase inluciferaseactivity, indicatingfunctionalityoftransgenic Pax6.Error barsindicates.d. choroid plexus, whereas the olfactory bulb(ob)andscattered cellsinthediencephalon(di)ofE15.5 co-transfected withthe pJoP6 E18.5 gal). The isolated brainsafterwhole-mount stainingfor 2002). Thefunctionofthesecondreporter, cells mightcorrespondtoGABAergic interneurons(Gorskietal., of the magnifications oftheframedarea inI as compared withthe metencephalon (mt),aswellthe inset) progenitors. ( the cellsinVZandSVZof unrecombined DNAtemplatesandfrom fluorescence inthethinCPandstriatum(str).( of thelevel JoP6 was switchedoff inmostofthecorticalcellsVZandCP Activation of ( pJoP6 ofrecombination in Fig. 2.Efficiency gal of the activation oftransgenic smaller thaninthecontrol,was intensively stained,indicating from the progenitors aswellpostmitoticcellsoftheCPand( L ) SAOS2cellswere transientlyco-transfectedwithaluciferasereporter constructcontainingeithertheHD(blue)orPD(r + ; aggregates aswellsomeindividual cellsweredetected in Emx1 ; JoP6 Emx1 or plasmid andcorticesfrom wild-type(negativecontrol), lacZ gfp pJoP6rec JoP6 IREScre -stop cassette(Fig.2B,B JoP6 (I) and IREScre reporter throughoutbraindevelopment. AtE12.5, ; Pax6 Emx1 Pax6 , and brain showed only non-specific stainingofthe , whichexpress JoP6 F transcripts inthe transgenic mice,indicatingsuccessfulexcision IREScre ) AtE15.5, during corticogenesis JoP6 JoP6 ; Emx1 pJoP6 cortex. ( ; Pax6 Nex-Cre cortex. ( IREScre control plasmid.Co-transfectionof ␤ . We furtherfollowed theexpression JoP6 -gal B JoP6 Pax6 (I , normalized to18SRNAindicateupregulation of JoP6 E B Ј ) cortex,showingectopicPax6expression inpostmitoticcellstheIZandlowerpartofCP. The ) E12.5sectionof + JoP6 Ј Ј ) AbundantGFPfluorescence isdetectedoncoronal sectionsofE14.5 ; . Luciferaseactivitiesare shownasrelative valuescompared withtheactivitymeasured inlysatesfrom cells cells are withinLP/VP, IZandthelowerpartofCP. ( ; demonstrate HoechstandPax6co-labeling.( Emx1 Emx1 Ј JoP6 pJoP6 control brain(top),are unstained.( ). RemainingGFP-positive ; Emx1 IREScre IREScre mice. plasmid and IREScre lacZ cortex are GFP-negative.Arrows indicatepreserved GFP C ␤ ) Whole-mountstainingfor -galactosidase ( ( A , was testedwith cortex, whereas thelevelof cortex, whichis H ) GenomicPCRwasperformedwiththeprimersJoP6FandJoP6RonDNAisolatedfrom the JoP6 ) atP28throughout thewholecortex.( ; JoP6 Emx1 JoP6 cortex, whereas anadditional261bpDNAbandisseenwiththesample IREScre pJoP6rec and ␤ ␤ - - JoP6 JoP6 cortex reveals weak with eithertheHD-orPD-containingreporter constructinducedan D ; ; massively populated by progenitors showed strong invading itslower part(Fig.2F).AtE18.5,thelatecortical migrating throughouttheintermediatezone(IZ)towards theCP, expression was apparentinbothVZprogenitorsandcells the VZandinthinCP(Fig.2E).Later, atE15.5, Emx1 (Fig. 2H),similartotherecombination patternintheadult whereas atP28thewholecortex was extensively stained for confined toVZprogenitors ofthe JoP6 Emx1 Emx1 ) WesternblotconfirmshigherlevelofPax6intheE15.5 In contrasttotheendogenous ␤ -gal indicatesspecificexpression of ; Pax6-5a IREScre IREScre Emx1 IREScre J Pax6 , K ) q-PCRswithRNAisolatedfrom cortexof mro.TePRapiisa13 pfamn from embryos. ThePCRamplifiesa1831bpfragment cortex (bottom),whereas themesencephalon(mes)and IREScre in thedouble-transgeniccortices(J)andspecificelevation brain (Gorskietal.,2002). is infactdiminished,ascompared withthecontrols (K). ␤ -gal stainingofindividualoraggregated (arrows and G ) Strong I cortex exhibited –similartothe , I Ј ) Pax6immunohistochemistryonsectionsof ␤ -gal stainingisseenatE18.5inlate + ␤ cells intheVZ.Notepreserved -gal JoP6 ␤ -gal stainingandtheCPwas + cortex, whereas themajorityof RESEARCH ARTICLE postmitotic cells(Fig.2G), Pax6 lacZ JoP6 in thetelencephalon(tel), expression, whichwas ed) domainandeither control cortex, the JoP6 ␤ -gal staining– JoP6 control, ; Nex-Cre ␤ 1313 lacZ -gal

DEVELOPMENT fewer mitoticcells attheapicalsurfaceofVZin appearance are scarce inthe thereafter, asindicatedbytheir dilutedcontentofBrdU. Nucleiofsuch were labeledattheendoftheirSphaseandunderwentmitosis JoP6 JoP6 nuclear migrationinthe point tocellaggregates). Note thesevere distortionoftheinterkinetic compared with mice (C cycle. Fig. 3.Activationoftransgenic 1314 (C,C pulse labelingofcorticalprogenitors atstageE11.0for90minutes JoP6 inhibition oftheproliferation rateintheMPandDPof minute BrdU pulse.EstimatedBrdU labelingindicesreveal asignificant ( 6-hour BrdU pulse,manynuclei are stillinthebasalVZ(arrows inD 0.5 mm.( significantly diminished,butcorticallayeringispreserved. Scalebars: labeling indexofthegenotypescompared. ( indicate theequallysizedareas usedfordeterminationoftheBrdU E , E Ј Ј ; ; ; ) ImmunolabelingofpHH3atstage E14.5reveals significantly Emx1 Emx1 Emx1 ) and6hours(D,D ( A Ј RESEARCH ARTICLE ,D , A B Ј IREScre IREScre IREScre , Ј ). Thearrows inCpointtonucleithecontrol cortex that B ) HEstainingofP23cross-sections of Ј ) Coronal 5 JoP6 cortex ascompared withthe cortex immunostainedforBrdU andDAPIaftera30- cortex (cx).Thesizeofthe mice. Ј ) in JoP6 ␮ m sectionsofE11.0 JoP6 JoP6 ; Emx1 control (C,D)and ; Pax6 Emx1 IREScre IREScre disturbs themitoticcell cortex, inwhich,evenaftera JoP6 C-D cortex (C JoP6 ; JoP6 Ј Emx1 ) Patterns ofBrdU) Patterns JoP6 control. Theframes JoP6 JoP6 control and IREScre Ј ; arrowheads control and ; ; Emx1 Emx1 cortex is IREScre IRES cre Ј ). as JoP6 apical surface ofthecontrolVZ(arrows inFig.3C).Inthe S phase,whentheBrdUpulsestarted)wereseenatornearto the nuclei withdilutedBrdUcontent(thatwereatthevery endoftheir predominantly withinthebasalregion oftheVZ(Fig.3C).Some intensively stained(S-phase)nuclei,which werestilllocated and determinetheBrdUlabelingindex (percentageof BrdU majority ofthecellsarestillproliferating.To labelS-phasenuclei Therefore, proliferationwas examined atstageE11.0,whenthevast proliferation and/orcellsurvival ofthecorticalprogenitors. incorporation, mostoftheprogenitorsincontrol into Mphase(Takahashi etal.,1993).After90minutesofBrdU and progressively moving totheapical VZsurface, wherethey enter nuclear migration,enteringintoSphaseatthebasalsurface ofVZ recombination was strong. for MPand of JoP6 42±6% fewer mitotic cellsattheapicalVZsurface of areas ofVZwithphospho-histone H3(pHH3)atE14.5revealed cell cycle arrestorextended Sphase.Quantitationofequallysized controls was demonstratedforthe suggests thatconditionalactivation oftransgenic JoP6 sections fromadultP23brainsrevealed thatthethicknessof Examination ofHematoxylinandEosin(HE)-stainedhistological cell cycleofearlycorticalprogenitors Activation oftransgenic sequences (Fig.2L). demonstrating thebindingactivity ofPax6 toitstarget consensus the transgenicPax6 was indicatedbyco-transfection experiments pallium (DP)of and 16±7%,was foundinthemedialpallium(MP)anddorsal BrdU was used.Significant reductioninproliferation,by14±6% only thelevel ofthe Ror not affected, asindicatedbylayer-specific markers [ was prominent hypocellularity, thecorrectpositioningoflayers compared withthecontrol( from thetotalnumberofDAPI were retainedatthebasalVZ(Fig. 3Dandarrows inD 3E,E the apicalVZ,whereasin the intensively stainedprogenitornucleiof thecontrolVZreached mutant VZ(arrowheads inFig.3C addition, faintly stainedaggregates ofcellswere visibleinthe with dilutedBrdUcontentwereseenattheapicalVZ(Fig.3C appeared tobedistributed throughouttheVZ,andonly afew cells the cellnuclei(Fig.2I Hoechst stainingconfirmed Pax6 immunoreactivity exclusively in indicating ectopicexpression. Co-labelingwithPax6 antibodyand ectopic Pax6 immunoreactivity intheIZandCP(Fig.2I,I that inthecortex ofthedouble-transgenic Interestingly, however, theresultsfromq-PCRassayrevealed of theendogenousPax6 locusoccurs(Pinsonetal.,2006). isoform, but alsooftheother, indicatingthatpositive autoregulation NIH3T3 cellsinvitroincreasesthecellularlevels ofnotonlythat expressing onlyoneisoform(Pax6 orPax6-5a) inNeuro2Aand 2J,D, arrow). Recentdataindicatethattheintroduction ofconstructs PCR andadditionallyfor During neurogenesis,proliferatingnucleifollow interkinetic Pax6-5a ␤ Ј ; ; ; Emx1 ). Taken together, theseresultsindicatethatin vivo activation Emx1 Emx1 and Cux2 IREScre IREScre IREScre a infact diminished(Fig.2K was n JoP6 =6 forDP;Fig.3B,B as comparedwith ( otxwssgiiatyrdcd(y4±% as cortex was significantly reduced(by43±3%) cortex, theS-phaselabeledprogenitornuclei Cutl2 Pax6 ; Ј Emx1 ). Thehigherlevel ofPax6 ascomparedwith ); notshown]. Thisphenotypestrongly transcript was elevated, whereasthelevel JoP6 P IREScre <0.001, + ; JoP6 Nex-Cre Pax6 cells), ashort,30-minutepulseof Ј mice, respectively ( ). After6hoursofBrdUlabeling, JoP6 JoP6 n ; Emx1 =12; Fig.3A,A misregulates the ; Emx1 mice ( by westernblotting(Fig. Ј ), two regions where JoP6 IREScre ). Development 134(7) The functionalityof IREScre P ; <0.001, Emx1 Pax6 VZ thesenuclei JoP6 Clim1a Ј cortices byq- P ). Despitethe Ј <0.001, might affect IREScre ) indicating cortex had n =14; Fig. ( Ldb2 + mice, Ј cells ). In n =5 Ј ), ),

DEVELOPMENT control and et al.,2005;Heins2002),westudiedtheeffect of Given theneurogenicactivity of properties neurogenesis andchangescelladhesive E13.5 at stagesE11.5(A,A magnifications counterstained withHoechst).( ephrin A5reveals thattheapoptoticcellaggregates ofthe downregulated. Error barsindicates.d. antibody (Tuj1) againstneuron-specific classIII neuronal differentiation invivo byimmunostainingwithm Conditional proliferation, andcorticalhypocellularity. of interkineticnuclearmigration,areductioninprogenitor of transgenic Activation of premature differentiated Tuj1 Fig. 5. Fig. 4.Enhancedneurogenesis andaltered adhesiveproperties afteractivationoftransgenic ( JoP6 C , C Ј ; ) Immunolabelingagainstactivated Casp3confirmsenhancedapoptosisinthe Emx1 JoP6 Pax6 IREScre ; Emx1 JoP6 GOF atearlycorticogenesiscauses apoptosis. Pax6 Pax6 cortex againstnestin(D)andactivated Casp3(D ; IREScre Emx1 Pax6 Ј in theearlycorticalprogenitorsleadstoadefect during corticogenesis with highermagnification)andE14.5 (B,B VZ issignificantlydiminishedascompared with IREScre activation enhances cortices normalizedto18SRNA. + cells are foundectopicallyintheVZof Pax6 for theRGprogenitors(Hack E ) AtstageE18.5, apoptosis isnolongerdetectedin ␤ -tubulin. Despite Pax6 JoP6 onoclonal N-CAM GOF on ; ( Ј Emx1 A-B Ј ) illustratesapoptosisof ) illustratesthatmanyoftheapoptotic cellsare nestin Ј ) TUNELassayonsectionsfrom and R-cadherinare upregulated, whereas JoP6 IREScre JoP6 ; Emx1 , butthesizeofTuj1 cortex are positiveforephrinA5(arrows). with JoP6 genotypes, suggestingenhancedneuronaldifferentiation in zone ofLPatE13.5showed nosignificant difference between 4C,C were detectedintheapicalVZregions oftheMPat E11.5 (Fig. the neuronalbHLHtranscriptionfactor gene premature neurogenesisinsubsetsofcorticalprogenitors.Inaddition, the 21±2%reductionin in theVPandLPofE11.5 Genome Informatics)showed astrongerinsituhybridizationsignal IREScre ; JoP6 Ј Emx1 ), aswellintheLPandVPatE14.5(Fig.4B,B JoP6 at E14.5( ( IREScre ; P JoP6 Emx1 <0.001, ; Emx1 IREScre mice (Fig.4A,A B , B n Ј + IREScre JoP6 ) andE11.5( cortex. ( =12) mice,thethicknessofTuj1 JoP6 JoP6 mantle zone(M)isunchanged.Also, Pax6 ; Emx1 cortical progenitors, more severe atE11.5. (A,B) and ; Emx1 D-D in developingcortex. JoP6 IREScre Pxn Ј Љ ). Furthermore,ectopicTuj1 C IREScre ) DoubleimmunostainingofE13.5 ; , RESEARCH ARTICLE Emx1 C , . ( JoP6 Ј Tnc ). ( F ) Insituhybridization with + cortical thicknesscompared D , ; progenitors (D IREScre Emx1 Itgb3 ) q-PCRoftotalRNAfrom Nex IREScre and cortex ascompared ( Neurod6 Itga5 (A ( A Ј Ј ), suggesting Љ , ,B A and Ј are Ј ) The ) embryos – Mouse + mantle + 1315 cells

DEVELOPMENT still detectableinprogenitorsofthe confined toprogenitors(Fig.5D-D colocalization inmany casessuggestingthatthecell deathis membrane, andcytoplasmic activated caspase3(Casp3)showed against nestin,amarker forVZprogenitorsassociatedwiththecell et al.,2005).Interestingly, bothaggregates andsingleTUNEL vivo causesprogenitorapoptosisintheembryoniccortex (Depaepe Recent evidence hasindicatedthatoverexpression ofephrinA5in however, TUNELreactionrevealed nofurtherapoptosis(Fig.4E). expression ofthe transgenic mediolateral patterningintheE11.5 progenitor apoptosis,wefoundthatneitherthedorsoventral northe patterning ofthecorticalprimordium,therebypossiblyinitiating (Fig. 4F). suggesting that tenascin C( involved incell-cellinteractionandsignalingsuchaspaxillin( as comparedwiththecontrols,whereasexpression ofgenes –MouseGenomeInformatics) Informatics) andR-cadherin(Cdh4 adhesionmoleculesN-CAM(Ncam1–MouseGenome cell showed anincreaseintheexpression ofgenesencodingthe performed withRNA extracted fromE12.5 involved incelladhesionandsignaling.q-PCRs transgenic undergoes prematuredifferentiation. subset ofcorticalprogenitors,mainlythosewithintheVPandLP, Together, theseresultssuggestthatupon with thecontrol(seeFig.S1D,D 1316 contrast tothe markers (e.g. affected, asindicatedbythenormalexpression ofcorresponding as causingapoptosis(Fig.4C,C against activated Casp3implicatedthecaspase-dependentpathway although atalower level (Fig.4B,B of progenitors. analyzed theeffect oftransgenic with distinctspatiotemporalactivation oftheCrerecombinase,we Ifaprc2 –MouseGenome Informatics).Byutilizingmouselines Hartfuss etal.,2001)(Ngn2isalso known asNeurog2,andRC2 in the In ordertostudywhetherthecorticalhypoplasiaseenin cortex causesapoptosis Activation oftransgenic experiments arenecessarytoelucidatetheirindividual roles. cells afteractivation oftransgenic strongly suggeststheirinvolvement intheaggregation ofcortical were reduced(Fig.4D).Thechangeintheexpression ofthesegenes detected intheproliferatingVZof TUNEL reactions.AtstageE11.5,massive apoptosiscouldbe in vivo mightalsoinvolve anincreaseincelldeath,weperformed into Ngn2 neurogenesis, E11)andRGcells (afterE13),whichcanbedivided vertebrate cortex: neuroepithelialcells(at the beginning of Several typesofprogenitorscontribute toneurogenesisinthe progenitor pools Pax6 material). When weexamined whether Because ofthecellaggregation detectedafteractivation of JoP6 GOF inducesapoptosisinspecificcortical RESEARCH ARTICLE + ; /Pax6 Pax6 Pax6 Emx1 Tnc Dlx1 JoP6 ,itgi ea3( ), integrin beta3 Pax6 – expression was stillmaintained,asjudgedbythe (Fig. 2E),wefurtheranalyzedtheexpression of IREScre , RC2 , lacZ Wnt3a control (Fig.5A,A might actinanephrin-A5-dependentmanner cortex show enhancedephrinA5expression, + reporter intheVZandCP(seeFig.2G); /Pax6 , Emx2 + Pax6 Ј and RC2 Pax6 ). AtstageE18.5,theactivation of Pax6 Itgb3 ; seeFig.S1inthesupplementary Ј Љ in thesupplementarymaterial). ). AtstageE14.5,apoptosiswas overexpression affects theearly JoP6 Ј activation indifferent subsets ) andintegrin alpha5( ). Labelingwithantibodies JoP6 Ј in thedeveloping ). Doubleimmunolabeling + Pax6 JoP6 Pax6 /Pax6 ; JoP6 Emx1 ; Emx1 . However, further ; Emx1 activation invivo, a – ; Emx1 (Guillemot, 2005; IREScre IREScre IREScre IREScre cortex was Pax6 cortex, in cortex, cortex Itga5 + Pxn GOF cells ), ) JoP6 still detectableatE18.5inpostmitoticcellsoftheCP of Although recombinationandthusactivation oftransgenic survival Pax6 the MP),undergo apoptosis. recombinase inthemajorityofRC2 cre specifically inPax6-positive RGprogenitors,weusedthe expression orenhancementofthe high sensitivity oftheearlyprogenitors MPtoectopic low level orarePax6-negative (Fig.6A,B).Theresultssuggesta progenitors thatareexpressing effect of 6D,D detected insingleprogenitorsaswellcellaggregates (Fig. indicated by After overexpression of progenitors asearlyE13.5(Götzetal.,1998;Heins2002). et al.,2000).Inthedouble-transgenic postmitotic neuronsaftertheirexit fromthemitoticcycle (Schwab Cre transgenic 5G). Inordertodirectlyassessthespecific effect oftheactivation of the Deregulation ofproliferation andapoptosisisassumedtoinvolve both transcriptional activation of Pax6 whereas thefate ofthepostmitoticneuronsisnotaffected. Pax6 P21 (Fig.6H,H Fig. 2I,J),noenhancementofapoptosiswas detectedatE14.5 and where thelevel of set ofprogenitorsthateitherexpress endogenous apoptosis detectedinthe situ hybridizationdetectionlimit(Muzioetal.,2002a),themassive Because theexpression of proceeds athighestlevel intheMPprogenitors(Lietal.,2003). resistant to Pax6-positive progenitorsofthe rostral VPappeartobemore inversely withtheirendogenous towards theelevation ofthe neurogenesis, earlycorticalprogenitorsshow differential sensitivity data notshown). Together, thesedataindicatethatattheonsetof transgenic cortex ascomparedwiththe seen atstageE11.5orE14.5inthe neither significantly increasedapoptosisnorcellaggregates were significant recombinationintheVP(asdetectedby crossed apoptosis isseenonlyrarely(Fig.6D Pax6 level alsoleadstocelldeath. midgestation corticalprogenitorsexpressing encoding transcriptionfactor recombinase isdirectedbytheE1enhancerelementofgene of prevents the inductionofapoptosisthroughtranscriptionalrepression To gaininsightintotheconsequencesofoverexpression of Recombination directedby Interestingly, intheVPof line (Zhuoetal.,2001).Thispromotesactivation ofCre p53 mouse line,whichinducesCrerecombinaseactivity in ; Ј Emx1 in vivo specifically inducescorticalprogenitorapoptosis, and itstarget -induced apoptosisdoes notinvolve ). (Ookawa etal.,1997).Previous evidence indicatedthatPax6 GOF inpostmitoticcellshasnoeffect oncell ( Therefore, weconcludethatoverexpression of JoP6 Trp53 Pax6 Pax6 IREScre Pax6 ␤ Ј mice withthe overexpression intheVPandLPprogenitors,we ) and -gal staining(Fig.6C),massive apoptosiswas expression innewly bornneurons,weused the and datanotshown). Thus,transgenicactivation of GOF, whereasthePax6-negative progenitors,or Pax6 mice, thesecellsdonotundergo apoptosis(Fig. Ngn2 pRb expression ishigherthaninthecontrols(see JoP6 Pax6 Pax6 ( Rb1 Pax6 are expressed atavery highlevels, JoP6 Emx1 Ngn2 ; Emx1 Pax6 in the )-dependent pathways, where pRb E1-Ngn2/Cre in MPatthisearlystageisthe Pax6 expression level, whichcorrelates Pax6 ; hGFAP-cre IREScre (Berger etal.,2004).Despite Ј IREScre at extremely low level (e.g.in ). To specifically examine the p53 JoP6 JoP6 JoP6 expression level: thehighly JoP6 expression level. starts atE9.5andinitially MP atE11.0involves a ; ; control (Fig.6E-F ; E1-Ngn2/Cre hGFAP-cre Nex-Cre Development 134(7) + /Pax6 , whereendogenous line, inwhichCre Pax6 Pax6 ␤ + at amoderate -gal staining), mice cortex, at extremely radial glial Pax6 cortex, as hGFAP- double- Pax6 in the Pax6 Ј Nex- and is

DEVELOPMENT of of amouse pPax6 P53 2H3, HeLaandHelaTAT (datanotshown). effect ofPax6. Similarresultswereobtained withthecelllinesNIH- activity decreased.pRBwas notabletosignificantly enhancethe endogenous promoter inahumanosteosarcomacellline(SAOS2) lacking this issue,westudiedtheeffect ofPax6 ontheactivity ofthe that theapoptosisinducedbytransgenic showed noeffect onthe whereas uponstrongoverexpression of In vivooverexpression of repression of activation of activation ofthecelldeathpathway throughadirecttranscriptional Pax6- 2004), suggestingtheexistence ofapossiblerelationship between al., 1995).Remarkably, Pax6 alsobindsdirectlytopRb(Cvekl etal., the effect on binds tothehuman Activation of JoP6-5a the invivo activation of leads toinhibitionofcellproliferation (Haubstetal.,2004).To study that retrovirus-mediated overexpression ofboth Previous GOFexperiments inprimarycorticalculturesindicated inhibits progenitor proliferation Pax6 promoter followed bytheluciferasegenewas co-transfectedwith , or pRb- barely influencedluciferaseactivation viathe based onthesame principlesas pPax6-5a pRb pRb and p53 p53 p53 Pax6 expression plasmid.AsillustratedinFig.7,expression and p53 , norbyabolishmentofthepRb-dependentactive gene activity hasnotbeenassessedsofar (Stuart et activity. P53 expression plasmidsintheabsenceandpresence during corticogenesis p53 -dependent apoptosis.Inanattempttoaddress P53 ( . Thereporterplasmid Pax6-5a TP53 promoter. Together, theseresultssuggest ) promoterwithlow affinity, although , wegeneratedthetransgenicline Pax6-5a Pax6 JoP6 Pax6 in vivo isdueneitherto moderately p53Luc Pax6-5a (see Materialsand the Pax6 P53 P53 containing the and expression promoter, promoter Pax6-5a P53 - either the developed a conditionalGOFapproachthatallows activation of pJoP6-5arec transgenic respectively; andPax6 immunostaining indicatedtheexpression of was monitoredbyGFPfluorescenceand To studytheinvivo functionof DISCUSSION isoforms invivo. results suggestadifference inthebiologicalactivity ofthetwo growth bycontrollingprogenitor proliferation,cellcycle levels ofPax6 playanessentialroleintheregulation ofcortical progenitors towards evidence foradifferential spatiotemporalsensitivity of cortical methods). Uponcrossingwiththe apoptotic patternof weaker comparedwiththe 5a transgenic Pax6-5a (Fig.8E).q-PCRshowed enhancementof 394 bpfragments);expression ofthetwo reportergenes JoP65aR detectedrecombinationofgenomicDNA (1955bpand of the proliferation ( a mildbut significant reduction(by 10±2%) inprogenitor of theBrdUindex intheE11.5DPafter30minuteslabelingrevealed aggregates wereformed( stages E11.5andE13.5revealed nodifference, andnocell JoP6 expression inthe ; Emx1 JoP6-5a Pax6 Pax6-5a IREScre with P or the <0.001, control (H). JoP6 not detectadditionalapoptosisinthecortexof in the in thedorsaltelencephalon.( ␤ JoP6 of E11.5 in apoptosisisdetectedviaTUNELassayonsections corticostriatal border). ( recombination intheLPandVP(arrow indicates ( endogenous progenitor apoptosisdependingonthe Fig. 6.Activationoftransgenic JoP6 expression intheMPand( demonstrates extremely faintendogenous situ hybridizationonE11.0wild-typesection same region (arrow). ( of anE11.5 JoP6 E14.5 indicatessuccessfulrecombination inthe G line was tested:PCRwithprimersJoP6Fand -gal indicatesrecombination inpostmitoticneurons ) E14.5sectionof cortex (Fig.8F).Statisticalanalysisofthe pHD (Fig. 8A-D ; ; ; JoP6-5a Nex-Cre Emx1 and hGFAP-cre JoP6-5a Pax6 Pax6 Pax6-5a n JoP6 n JoP6 and =5; Fig.8G,G =4; Fig.8D,D IREScre + GOF invivo. We foundthatdifferent JoP6 radial glialprogenitors. ( embryos (H ; ; (F) and Pax6 ; Emx1 hGFAP-cre pPD Emx1 cortex and( reveals massiveapoptosiswithinthe Ј ; isoform. Inthiswork, we provide E1-Ngn2/Cre ). Co-transfectionexperiments of Emx1 Pax6 expression level. Pax6 JoP6 RESEARCH ARTICLE IREScre indicated thefunctionalityof IREScre JoP6 C F ) , ␤ IREScre ; F Ј Ј Nex-Cre ) ascompared withthe shows extensiveapoptosis during corticogenesiswe ). Nevertheless, estimation -gal stainingofsectionat Ј ; B Ј E1-Ngn2/Cre ) Nosignificantdifference .Taken together, these ). cortex, whichwas much ) TUNELassayonE11.0 D enhancement inthe and controlcorticesat H , D , embryo indicates H line, thefunctionality Ј ) TUNELassayon Ј ) TUNELassaydid cortex stainedfor Pax6 ␤ E ) -gal staining, ( ␤ A leads to (F -gal staining gfp ) Ј Pax6 ) embryos. Pax6 and Pax6- in 1317 Pax6 lacZ

DEVELOPMENT progenitors in of transgenic 16±7% fortheMPandDP, respectively. Despitethemuchlower level the BrdUlabelingindex ascomparedwiththecontrols:by 14±6%and progenitor proliferationatE11.0revealed asignificant reductionof layering anddorsoventral patterning.Subsequentanalysisof 43±3%) aftertransgenic We foundthattheadultcortex issignificantly reducedinsize(by cell cycleprogression Pax6 of theanti-tumorgene our assays,isunlikely tobedueadirecttranscriptionalregulation transcript. We show thattheinducedprogenitorcelldeathevident in proliferation ofcorticalprogenitorswas foundforthe induction ofneurogenesis.Asimilarroleincontrollingthe progression, theacquisitionofprogenitorapoptoticfate and Pax6 protein (3 Fig. 7.Effect ofPax6protein onactivityofthe 1318 results fromour laboratory(Götzetal.,1998)and fromothergroups of theVZ,aprocesstermedinterkinetic nuclearmigration.Previous nuclei oftheprogenitorsmigrate fromthebasaltoapicalsurface (Haydar etal.,2000).Duringthe transitionfromStoMphase,the express in vivo (this study). proliferation ofcorticalprogenitorsinvitro(Haubstetal.,2004) and proliferation inthedeveloping eye (Singhetal.,2002),itinhibits as invivo (thiswork). Similarly, although primary cellcultures(Haubstetal.,2004;Heins2002)as well epithelial cells(Ouyangetal.,2006),andcorticalprogenitors in human glioblastomacells(Zhouetal.,2005),cultivated corneal and proliferation intheneuralretinaofvertebrates (Marquardtetal.,2001) different controlfunctionsonprogenitorproliferation. in different environmental contexts, thetwo Pax6 isoformsexert Pax6-5a et al.,1998),thesefindings confirm thepivotal roleofboth Sey/Sey enhanced progenitor-proliferation ratefoundin the (Hack etal.,2004;Heins2002;Cartier2006).Given the Pax6 10±2%). Theseresultsareinlinewithvitroexperiments inwhich overexpression (16 human Note thatpRBslightlyincreases thePax6inhibitoryeffect on ‘empty’ vector pRb isindicated;error barsindicates.d.Theluciferaseactivityofthe Normalized luciferaseactivityinthepresence orabsenceoffunctional promoter activity. Bycontrast,Pax6-5ashowsnosignificanteffect. The majorityoftheS-phaseand many oftheM-phaseprogenitors transduction leadstotheinhibitionofprogenitorproliferation controls corticalprogenitor proliferation and P53 RESEARCH ARTICLE cortex atE10.5(Warren etal.,1999)andE14.5-E16.5(Götz Pax6 in controllingcorticalprogenitorproliferation.Remarkably, promoter intransientlytransfectedSAOS2cells,butupon , suggestingthat Pax6-5a pCMV ␮ JoP6-5a (Dominguez etal.,2004),but reducesproliferationof g of ␮ g without pRbwasgivenanarbitraryvalueof100. pPax6 pPax6 p53 expression, theproliferationrateofcortical ; Emx1 Pax6 ) doesnotpositivelyregulate activityofthe , indicatedby++)doesinhibitthe by Pax6. IREScre activation, having apreserved cortical Pax6 regulates cellcycle progression iewsihbtdaswell mice was inhibited Pax6-5a P53 seems topromote promoter. Pax6 Pax6 P53 Pax6 enhances Pax6-5a P53 . LOF and ( by JoP6 differentiation. Owingtothemassive apoptosisinthe in HeLacells(Cartieretal.,2006)have revealed prematureneuronal adult neurospheres(Hacketal.,2004;Heins2002)aswell (Ouyang etal.,2006). inhibition ofcellproliferationandretardationthecycle in cornealepithelialcelllinesandprimaryculturecauses cycle arrest(Cartieretal.,2006).Similarly, overexpression of reduces thenumberofcellsinSandG2–Mphases,indicatingcell analysis demonstratethatactivation of support ofthisconclusion,recentresultsfromquantitative FACS significantly smallerproportionofprogenitorsundergo mitosis.In many cellsseemtobestuckorprolongedinSphaseanda of Torrus etal.,2002).Hereweprovide evidence thatoverexpression cells foundintheSphaseasaresultofshortercellcycle (Estivill- nuclear movement ofcorticalprogenitorsisimpaired,withmore expression ofR-cadherin(Stoykova etal.,1997).After isolated corticalprogenitorsfrom Previously, wereportedonchangesintheadhesive propertiesof the corticalprogenitorsofE11.5 against activated Casp3andnestinrevealed abundant apoptosisin TUNEL assaysanddoubleimmunohistochemistry withantibodies of corticalprogenitors Pax6 a reliabletoolforsuchstudiesinvivo. system forconditionalactivation of dissect thespecific roleof these proteinsparticipate,furtherdetailedanalysisisrequired to involved incell-cellinteraction.Given thecomplex pathways inwhich expression levels ofintegrin beta3,paxillinandtenascinC,molecules its promoter(Duncanetal.,2000),aswelladecreasein the expression ofintegrin alpha5,whichcontains Pax6-binding sitesin of thesetwo genes.We alsofoundPax6-mediated inhibitionofthe (Andrews andMastick,2003),possiblybydirectgeneticinteraction R-cadherin mediating expression ofR-cadherin.Thelatterresultfurthersupportstheidea Pax6 (Holstetal.,1998;Yamaoka etal.,2000),aswellincreased which encodesacell-celladhesionmoleculepositively regulated by vivo wefoundstrongenhancementoftheexpression ofN-CAM, transgenic cortical progenitors(Araietal.,2005). maintenance ofproliferationversus neuronaldifferentiation in downstream target also beofinteresttotesttheexpression ofthepotential severe hypocellularityoftheadult cortical progenitorpoolearlyindevelopment andcontribute tothe from themitoticcycle anddifferentiate, whichwould diminishthe control), thethicknessofTuj1 E13.5 issignificantly reduced(by21±2%,ascomparedwiththe progenitors ofthe Pax6 (Warren etal.,1999)indicatedthatin unchanged. Furthermore,theectopicallylocatedTuj1 we foundthatalthoughthe estimation ofpossibleprematureneurogenesisisdifficult. However, CP of VZ, andtheenhanced GOF was directed intopostmitoticcellsby the Experiments involving Pax6 In additiontomisregulation ofthemitoticcycle, wefoundthat ; Emx1 JoP6 GOF conditioninvivo. Thus,subpopulationsofearly GOF invivoinducesapoptosis inspecificsets in vivo leadstodefectsofmitoticcycle progressionsuchthat Pax6 ; IREScre Emx1 activation causesaggregation ofcorticalprogenitors. IREScre JoP6 mice duringearlycorticogenesis,aquantitative Fabp7 Nex ; Pax6 Emx1 mice, suggestenhancedneurogenesisinthe Pax6 , recentlyreportedtobeinvolved in in situhybridizationsignaltheE11.5 Pax6 JoP6 -dependent functionincelladhesion IREScre transduction inRGcellculturesand JoP6 ; in theseprocesses.However, the JoP6 Emx1 Pax6 cortex seemtoexit prematurely ; Sey/Sey Emx1 ; Pax6 Emx1 Pax6 + IREScre described hereseemstobe mantle layerappeared IREScre in HeLacellsstrongly Development 134(7) IREScre LOF, theinterkinetic cortex andreduced cortical thicknessat Nex-Cre mice. When cortex. Itwould Pax6 + cells inthe mouse line GOF in Pax6 Pax6 Pax6

DEVELOPMENT immunohistochemistry aftera30-minuteBrdU pulseoncross-sections ofE11.0 sections ofbothgenotypesreveals nosignificantdifference inapoptosis(arrowheads pointtoendogenousapoptosis).( depth ofthecortex is populated by spreading intotheIZandCP, respectively, andatP28the entire 5a 394 bp)confirmsuccessfulrecombination inthe therefore, thata significant partofthe early(E9.5-E14.5)cortical inhibition oftheproliferation rateinthe the cell deathappearstooccur. Inparallel,alimitednumberof apparent atE11.5,itweakens byE14.5,andatE18.5noincreased whereas atE15.5andE18.5the cells, markingrecombinedsurvived progenitors,arefoundatE12.5, JoP6 1997), theapoptosisatbeginning ofneurogenesisin cleared outoftheratcortex in2hours20minutes(Thomaidouetal., apoptosis duringmammaliancorticogenesis. provide thefirst evidence that (Schwab etal.,2000),noapoptosiscouldbedetected.These results Activation of JoP6-5a transfected withaluciferasereporter constructcontainingeithertheHD(blue)orPD(red) domainandeither with RNAisolatedfrom E12.5cortexof the HD-orPD-containingreporter constructinducedanincrease inluciferaseactivity, indicatingfunctionalityoftransge express Fig. 8.Conditionaltransgenicactivationof shows ectopicantibodystainingindicatingCre-induced activationof recombination. ( Given thatapoptosisisavery fast process,withdeadcellbodies (A) ispartiallylostin Pax6-5a ; Emx1 ; Pax6-5a Emx1 IREScre expression levelinthe IREScre Pax6 uieaeatvte eenraie oc-rnfcin with the . Luciferaseactivitieswere normalizedtoco-transfections C ) Whole-mount mice ismassive. Althoughextensive apoptosisis eecpao n latr ub ( telencephalon andolfactorybulb. during corticogenesis JoP6-5a ; Emx1 ␤ JoP6-5a -gal stainingofE15.5 Pax6 ␤ IREScre -gal stainingisprogressively JoP6-5a JoP6-5a ; ␤ is involved inprogenitor (A Emx1 -gal Ј ). ( Pax6-5a ; control and JoP6-5a + Emx1 IREScre B ) Two products generatedbygenomicPCRwiththeprimersJoP6FandJoP65aR(1955bp cells. We assume, IREScre D telencephalon andolfactorybulb.Error barsindicates.d..( , ; via the D JoP6-5a Emx1 Ј with inset)ImmunostainingforPax6onE14.5 DP. JoP6 IREScre ␤ JoP6-5a ; Emx1 and -gal cortex, whereas the Pax6-5a JoP6-5a + IREScre mouse line. of theglutamatergic corticalprogenitors(Li etal.,2003).Therefore, 2002b). Thereafter, towards progenitors invivo have spatiotemporaldifferences intheirsensitivity regionalized recombination, weprovide evidence thatcortical monitored as Pax6 sections, whereasthelaterprogenitorsareaffected muchlessby accumulated enough apoptosis andareremoved fromthecortex beforethey have progenitors, wheretransgenic endogenous transgenic cortex, normalizedto18SRNA,indicateonlyaslightenhancementof in the ; Emx1 Subpopulations ofcorticalprogenitorssurvive JoP6-5a GOF. pJoP65a JoP6-5a IREScre Pax6 ( Pax6 A and JoP6 Pax6 brains showsstrong activationofthe , A control plasmid.Co-transfectionof ; GOF. Inthe ␤ Ј Emx1 JoP6-5a ) GFPfluorescence onsectionsofE14.5cortex is initiatedatE9.5predominantly intheMP, where -gal cortex andthe is onlybarelyexpressed, ifatall(Muzioetal., Pax6 IREScre + ␤ cells. Byusingdifferent mouselinesfor ; Emx1 -gal tobeconfidently registered onthin GOF progressively spreadstothemajority CP. ( JoP6 JoP6-5a IREScre E pJoP6-5a ) SAOS2cellswere transientlyco- Pax6 ; cortices reveals asignificant Emx1 G pJoP65a RESEARCH ARTICLE , and G Ј ) TUNELassayonE11.5brain is induced,undergo rapid IREScre plasmid showno H JoP6-5a , H or i a65.( nic Pax6-5a. Ј ) BrdU pJoP65arec cortex, activation of pJoP6rec ; lacZ Emx1 Pax6 reporter inthe IREScre GOF andare , which F with either ) q-PCRs cortices 1319 JoP6-

DEVELOPMENT Paae l,19)ad indeveloping (Plaza etal.,1999) and, pathways inthecontrolofcorticalprogenitorcelldeath. of geneticinterplaybetweenthe target gene inducing apoptosisaswell(Zhou etal.,2005).Inaddition,theCasp3 overexpression of 2001), extensive apoptosiswas detected,indicatingthatthe MP andPax6 Pax6-negative progenitorpool(neuroepithelialcellsatE9.5-E12in appears tobetheresultofeitherectopicexpression of the observed massive apoptosisintheMPof 1320 we testedtheeffect of mechanism oftheinducedapoptosisin (Hickman etal.,2002).Inanattempttostudythemolecular cycle arrestandapoptosis,inducingcelldeathupon activation Pax6 GOF condition,whichisprobablydependentupontheendogenous the corticalprogenitorshave different sensitivity towards the elevation of with thehighestlevel ofendogenousPax6 areresistanttofurther JoP6 2003). Nosignificant enhancementofapoptosiswas observed inthe by ahighdosageofPax6 (Marquardtetal.,2001;Scardigli recombination directedbythe the Pax6 overexpression intheearlyVPprogenitors,whereendogenous leads toapoptosisaswell.Inorderstudytheeffect of expressing Pax6 onlyfaintly. Also,inthe activation of proliferation andleadstoapoptosis (Yamaoka etal.,2000),and pancreatic 2006). Ectopicactivation of down cellcycle progressionandcausesapoptosis (Ouyangetal., Pax6 transgenic apoptosis. Evidencehasbeenpresentedthatahighcopy-number of cell cycle progression,but possiblyalsobytheinvolvement of in tissuegrowth, notonlybymodulatingprogenitorproliferationand where transgenic transgenic miceinvivo (Depaepeetal.,2005 recently beendiscovered inGOFexperiments forephrinA5in a similarapoptoticphenotypeofearlycorticalprogenitors has A5 expression inapoptoticcorticalprogenitors.Most intriguingly, overexpression of p53 pathway activation. Itisinterestingtonotethatafter indicate thatthisphenomenonisnotlikely tobeaconsequenceof the pRB. Therefore,althoughtheunderlyingmolecularmechanismof p53 is unabletotrigger p53 sequester pRbandabrogatepRb--dependentrepressionofthe Therefore, wealsotestedwhether, uponoverexpression, Pax6 could apoptotic gene the transcriptionofE2F-responsive promoters,includingthepro- allows theformationofpRb-E2Fcomplexes, whichactively repress binds tohypophosphorylatedpRb(Cvekl etal.,2004),whereas pRb contains Pax6 target sequences (Stuartetal.,1995).Pax6 protein the RC2 Accumulating evidence supportstheview that The tumorsuppressorgene Pax6 transcription uponstrongoverexpression andindependentlyof promoter, therebyinducingapoptoticfate. We foundthatPax6 E1-Ngn2/Cre ; expression level isatitshighest(Stoykova etal.,1997),weused expression level. E1-Ngn2/Cre overexpression incultivated cornealepithelialcellsslows RESEARCH ARTICLE + /Pax6 idcdaotssinvivo isstillunclear, ourresults -induced apoptosis Pax6 Parp Pax6 ␤ – Pax6 RG progenitors),oroverexpression of -cells oftransgenicmice inhibits progenitor + leads tomicrophtalmiainmice(Schedletal.,1996). radial glialcells(Malatestaetal.,2003;Zhuo p53 acts asaregulator of . Collectively, theseinvivo resultsdemonstratethat Pax6 Pax6 Pax6 suppresses tumorigenicityofglioblastoma cells cortex, suggestingthattheVPandLPprogenitors p53 line (Berger etal.,2004).Thislinedrives (Ookawa etal.,1997;Sellers1995). Pax6 wedetectedastrongenhancement ofephrin , in thePax6 becomes activated afterE13.5exclusively in activation invitro,but, bycontrast,inhibits expression onthe E1-Ngn2 Pax6 p53 + Pax6- is involved inthecontrol ofcell midgestation corticalprogenitors in undifferentiated andmature Pax6 Xenopus enhancer thatisactivated only and ephrin-A5 JoP6 expression inneuroretina JoP6 ), JoP6 raising thepossibility p53 expression of , ; ; hGFAP-cre Emx1 ; Emx1 Pax6 promoter, which Pax6 Pax6 IREScre - IREScre is involved in RGcells in theearly dependent cortex, cortex, Pax6 Pax6 Pax6 mice JoP6-5a GOF invivo indifferent cellularandexperimental contexts. to reveal thebiologicalsignificance oftheapoptosisinducedby al., 1995)(datanotshown). Therefore,furtherresearch isrequired enhanced apoptosisisdetectedinthedeveloping cortex (Grindley et attributed toabnormalapoptosis(Fukudaetal.,2000),but no the transitionofnasalectodermintoplacodehasbeen Pax6 the Pax6 isoformonly. However, inagreementwithresultsfrom progenitor apoptosisisaspecific featureoftheinvivo elevation of Therefore, presently, itcannot bestatedwhetherthedetected necessary toinduceapoptosiswas notachieved inthisassay. with thecontrols).ThepossibilityremainsthatPax6-5a level Ashery-Padan, R.,Marquardt, T., Zhou,X.andGruss,P. Arai, Y. A.,Funatsu,N.,Numayama-Tsuruta, K.,Nomura,T., Nakamura,S. and Gautier, 1998).Inthe at theneural-foldstageoverlaps withTUNEL-positive cells(Hensey Andrews, G.L.andMastick,S. References http://dev.biologists.org/cgi/content/full/134/7/1311/DC1 Supplementary materialforthisarticleisavailableat Supplementary material supported byEY014998. Scientist supportedbyTelethon andCompagniadiSan Paolo.K.R.J.was and F. Faustiforoutstandingtechnicalassistance.F.C. isanAssociateTelethon DeLaurenzi forSAOS2cells;A.Jacobyhelpfulcomments;andM.Daniel Frisen fortheephrinA5probe; M.Crescenzi forpRb; W. LucforHelaTaT; V. We thankK.-A.Nave,S.GoebbelsandM.Schwabforthe in contrast tothesubstantialelevation ofthelevel of controls), theincreaseinlevel of new invivo evidence foracomplex roleofthe apoptosis. Usingthe acquisition asthisinfluencescellproliferation,differentiation and modulation of presented inthisstudysupporttheview thatduringcorticogenesis,the proliferation inthedeveloping cortex. we find thatthetwo Pax6 isoformssuccessfullyrepressprogenitor Berger, J.,Eckert,S.,Scardigli, R.,Guillemot,F., Gruss,P. andStoykova,A. 5a Bishop, K.M.,Goudreau, G. andO’Leary, D. Bernards, R.,Shackleford, G.M.,Gerber, M.R.,Horowitz, J.M.,Friend,S. Caric, D.,Gooday, D.,Hill,R.E.,McConnell,S.K.andPrice,D. J. multiple phasesofmammaliancorticogenesis. Cartier, L.,Laforge,T., S.,Dubois-Dauphin, M.and Feki,A.,Arnaudeau, Cvekl, A.,Yang, Y., Chauhan,B.K.andCveklova, a singleretina intheeye. the lensprimordium isrequired forlensformationandcorrect placementof rat cortex. maintenance ofneuroepithelial cellsduringearlyembryonicdevelopmentofthe and Osumi,N. growth-promoting cueforpioneeraxons. the developingCNS. (2004). E1-Ngn2/Cre isanew lineforregional activationofCre recombinase in identity inthemammalianneocortexbyEmx2andPax6. transcription factorPax-6. Determination ofthemigratorycapacity ofembryoniccorticalcellslackingthe 6478. characterization ofitsencodedprotein. al. H., Schartl,M.,Bogenmann,E.,Rapaport,J.McGee,T., Dryja,T. P. et crystallins inlens. byPax6inocularcells:acaseoftissue-preferred expression of Neurobiol. expression ofneuronal (tubulin,postmitoticphenotype,andcellmigration. Krause, K.H. ; We wereunabletodetectapoptosisinthecorticalprogenitorsof Taken togetherwithalltheevidence available sofar, theresults JoP6 Emx1 (1989). Structure andexpression ofthemurineretinoblastoma geneand and ; ; IRESCre Emx1 Emx1 Pax6-5a J. Neurosci. 66 , 421-436. (2006). Pax6-inducedalterationofcellfate: Shapechanges, Pax6 IRESCre 20) Roleof (2005). IRESCre mice was muchlessevident (1.2-fold,ascompared Int. J.Dev. Biol. GOF experiments invitro(Haubstetal.,2004), Genesis expression levels iscrucialforprogenitor cellfate Pax6 25 mice (3.8-foldhigher, comparedwiththe , 9752-9761. mice. Itshouldbenoted,however, thatin Genes Dev. Development GOF approachdescribedhere,weprovide 40 Pax6 Fabp7 , 195-199. 48 (2003). R-cadherinisaPax6-regulated, , 829-844. 14 LOF mutant , adownstream geneofPax6,inthe Proc. Natl.Acad.Sci.USA , 2701-2711. 124 J. Neurosci. , 5087-5096. (2000). Regulationofarea Development 134(7) Sey 23 (2004). Regulationof Pax6-5a , 9873-9880. Science (2000). 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DEVELOPMENT Dominguez, M.,Ferres-Marco, D.,Gutierrez-Avino, F. J.,Speicher, S.A.and Depaepe, V., Suarez-Gonzalez, N.,Dufour, A.,Passante,L.,Gorski,J. Duncan, M.K.,Kozmik,Z.,Cveklova,Piatigorsky, J.andCvekl,A. Czerny, T., Schaffner, G.andBusslinger, M. Activation of Epstein, J.,Cai,Glaser, T., Jepeal,L.andMaas,R. Hickman, E.S.,Moroni, M. C.andHelin,K. Hensey, C.andGautier, J. Heins, N.,Malatesta,P., Cecconi,F., Nakafuku,M.,Tucker, K.L.,Hack,M.A., Epstein, J.A.,Glaser, T., Cai,J.,Jepeal,L.,Walton, D.S.and Maas,R.L. Hill, R.E.,Favor, J.,Hogan,B.L.,Ton, C.C.,Saunders,G.F., Hanson,I.M., Gorski, J.A.,Talley, T., Qiu,M.,Puelles,L.,Rubenstein,J.L.andJones,K.R. Fukuda, T., Kawano,H.,Osumi,N.,Eto,K.andKawamura, Estivill-Torrus, G.,Pearson,H.,vanHeyningen,V., Price, D. J.andRashbass, Holst, B.D.,Vanderklish, P. W., Krushel,L.A.,Zhou,W., Langdon,R.B., Guillemot, F. Grindley, J.C.,Davidson,D.R.andHill,E. Götz, M.,Stoykova,A.andGruss,P. Hartfuss, E.,Galli,R.,Heins,N.andGotz,M. 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