GSK-3 is a master regulator of neural progenitor homeostasis

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1 GSK-3 is mster regultor of neurl progenitor homeostsis Woo-Yng Kim 1, Xinshuo Wng 1, Yohong Wu 1, Brdley W Dole 2, Stish Ptel 3, Jmes R Woodgett 3 & Willim D Snider 1 The development of the rin requires the exquisite oordintion of progenitor prolifertion nd differentition to hieve omplex iruit ssemly. It hs een suggested tht glyogen synthse kinse 3 (GSK-3) ts s n integrting moleule for multiple prolifertion nd differentition signls euse of its essentil role in the RTK, Wnt nd Shh signling pthwys. We reted onditionl muttions tht deleted oth the nd forms of GSK-3 in mouse neurl progenitors. GSK-3 deletion resulted in mssive hyperprolifertion of neurl progenitors long the entire neurxis. Genertion of oth intermedite neurl progenitors nd postmitoti neurons ws mrkedly suppressed. These effets were ssoited with the dysregultion of -tenin, Soni Hedgehog, Noth nd firolst growth ftor signling. Our results indite tht GSK-3 signling is n essentil meditor of homeostti ontrols tht regulte neurl progenitors during mmmlin rin development. GSK-3 is serine-threonine kinse with estlished roles in reeptor tyrosine kinse nd Wnt/Frizzled signling. In mmmls, the GSK-3 fmily onsists of two memers, GSK-3α nd GSK-3β, whih hve 98% sequene identity in their kinse domins. Reent studies tht hve mostly used phrmologil pprohes hve suggested tht GSK-3 is importnt in n rry of settings inluding regultion of trnsription ftor levels in multiple signling pthwys, ell polrity nd neurotrnsmitter signling 1 3. Reently, phrmologil evidene hs implited GSK-3 signling in the regultion of emryoni stem ell self-renewl 4 6. Furthermore, it hs een suggested tht the Deleted in Shizophreni (DISC1), whih hs een implited in some fmilil ses of shizophreni nd depression, regultes neurl progenitor prolifertion nd my t in prt vi regultion of GSK-3 tivity 7. These oservtions rise the question of how GSK-3 funtions in reltion to neurl progenitor self-renewl nd neurogenesis during rin development. Development of the rin requires exquisite oordintion of progenitor prolifertion nd differentition to hieve optimized funtionl potentil. Reently, multiple signling pthwys, inluding Wnts, firolst growth ftors (FGFs), Soni Hedgehog (Shh) nd Noth, hve een identified s regulting neurl progenitors 8. However, the mnner in whih these signls re oordinted to hieve exquisite sptil nd temporl ontrol over neurogenesis is unknown. GSK-3s re uniquely well-positioned to oordinte multiple prolifertion nd differentition signls euse they potentilly regulte ritil nodes in eh of these pthwys. Inhiition of GSK-3 is ritil to nonil Wnt signling leding to inresed β-tenin levels in ll of the ell types studied to dte 1. Furthermore, GSK-3 is inhiited downstrem of FGF signling vi phosphtidylinositol-3 kinse (PI3K) 9. Severl reent studies in ell lines hve demonstrted GSK-3 regultion of My fmily proteins vi the PI3K pthwy In Drosophil, it is known tht the GSK-3 homolog Shggy regultes Hedgehog signling 14. Finlly, severl studies in ell lines hve demonstrted tht GSK-3 inhiition modultes the Noth pthwy To dte, however, the effets of deleting GSK-3 speifilly in neurl progenitors on signling vi these pthwys nd on progenitor self-renewl nd susequent rin development hve not een determined. We used onditionl knokout strtegy in Gsk3 null kground to speifilly trget Gsk3 in the developing nervous system. We found tht GSK-3 defiient progenitors were loked into the rdil progenitor phse, s genertion of oth intermedite neurl progenitors (INPs) nd postmitoti neurons ws mrkedly suppressed. Our findings suggest tht GSK-3 deletion removes homeostti ontrols on neurl progenitors, shifting the lne towrd self-renewl nd wy from neurogenesis. RESULTS GSK-3 deletion mrkedly enhnes progenitor prolifertion To investigte the funtions of GSK-3 in the developing mmmlin nervous system, we generted Gsk3 / (Gsk3 null) nd Gsk3 loxp/loxp mie nd rossed these with well-hrterized line tht drives expression of Cre reominse in CNS progenitors from out emryoni dy 1 (E1) 18,19. Gsk3 null mie tht hrored n exon 2 deletion nd Gsk3 loxp/loxp mie were generted y flnking exon 2 with loxp sites (Supplementry Fig. 1) 2,21. Neither Gsk3 null nor Gsk3 loxp/loxp ; mie hd mjor rin developmentl mlformtions (W.-Y.K. nd W.D.S., unpulished oservtions) 22. Western lots showed tht the α nd β forms of GSK-3 were eliminted in the developing rins of Gsk3 / ; GSK Gsk3 loxp/loxp ; mie (Supplementry Fig. 1). Compred with the ontrol 1 Neurosiene Center, University of North Crolin Shool of Mediine, Chpel Hill, North Crolin, USA. 2 MMster Stem Cell nd Cner Reserh Institute, MMster University, Hmilton, Ontrio, Cnd. 3 Smuel Lunenfeld Reserh Institute, Mount Sini Hospitl nd Deprtment of Medil Biophysis, University of Toronto, Toronto, Ontrio, Cnd. Correspondene should e ddressed to W.D.S. (wsnider@med.un.edu). Reeived 13 July; epted 1 Septemer; pulished online 4 Otoer 29; doi:1.138/nn.248 nture NEUROSCIENCE dvne online pulition

2 Brin Phospho-histone H3 BrdU DAPI Cortex Reltive hnge (versus ontrol, %) Spinl ord Sox2 Nestin DAPI Control littermtes (Gsk3 +/ ; Gsk3 loxp/+ ; ), Gsk3 / ; Gsk3 loxp/loxp ; mie hd igger heds (Supplementry Fig. 1) t every developmentl stge from E12.5 until they died t P. Brins from ontrol mie hd smooth nd stright orties in oronl setions t E13.5 (Supplementry Fig. 1), wheres the rins of Gsk3 / Gsk3 loxp/loxp ; mie were mrkedly onvoluted. Gsk3 / Gsk3 loxp/loxp ; rins lso showed sustntilly enlrged ventrl rin res, inluding the gnglioni eminene nd ventrl thlmus (Supplementry Fig. 1). We mesured the ventriulr pil surfe lengths nd found 2.7-fold inrese in GSK-3 deleted rins ompred with ontrol rins (Supplementry Fig. 1). To explore the sis of the onvoluted ortex, we ssessed neurl progenitors in the Gsk3 / ; Gsk3 loxp/loxp ; rin using n ntiody to Sox2, neurl progenitor mrker. In ontrol rins t E13.5, Sox2-positive progenitors were distintly distriuted surrounding the ventriles (Fig. 1). We lso oserved Sox2 expression in limited prt of the dorsl gnglioni eminene. In Gsk3 / ; Gsk3 loxp/loxp ; rins, Sox2-positive neurl progenitor Mitoti index Apoptosis index BrdU index Cell-yle re-entry index Figure 1 GSK-3 deletion leds to mssive inrese in neurl progenitor prolifertion. () Neurl progenitor pools re mssively expnded in Gsk3 / ; Gsk3 loxp/loxp ; () mie. Setions of developing forerin nd spinl ord t E13.5 were immunostined with n ntiody to Sox2. Left, neurl progenitor pools were hevily expnded throughout the ererl ortex nd ventrl re in the Gsk3 / ; Gsk3 loxp/loxp ; rin setions. Sle r represents 9 µm. Middle, highmgnifition imge of ererl ortex immunostined for Sox2. Sle r represents 1 µm. Right, the entrl nl ws losed s result of progenitor hyperprolifertion in Gsk3 / ; Gsk3 loxp/loxp ; spinl ord nd there ws extension of progenitors into the developing dorsl horn. Sle r represents 2 µm. () Enhned progenitor division in Gsk3 / ; Gsk3 loxp/loxp ; rins. The numers of phosphohistone H3 nd BrdU-positive dividing ells were inresed in the setions of Gsk3 / ; Gsk3 loxp/loxp ; ererl ortex t E13.5 ompred with ontrols. The phospho-histone H3 leled mitoti ells were mostly ner ventriles in ontrol setions. They were not limited in ventriulr region, ut rther spred throughout the developing ortil wll in Gsk3 / ; Gsk3 loxp/loxp ; setions. Sle rs represent 5 µm (left pnels) nd 1 µm (right pnels). () Quntifition of mitoti, BrdU leling, ell yle re-entry nd poptosis indies. n = 1 setions from eh of three mutnts nd ontrol emryos per eh ondition. P <.1. pools were mssively expnded (Fig. 1). At the level of gnglioni eminene, this expnsion ws lso prominent in ventrl forerin. We oserved this progenitor expnsion t every rostro-udl level tht we exmined, inluding spinl ord (Fig. 1). To further ssess this hyperprolifertion, we mesured the proportion of ells in vrious ell-yle phses t E13.5. Twie s mny ells were positive per oronl setion for the mitoti mrker phospho-histone H3 in Gsk3 / ; Gsk3 loxp/loxp ; ererl ortex ompred with ontrols (Fig. 1, nd Supplementry Fig. 2). Western lotting onfirmed tht phospho-histone H3 expression ws mrkedly inresed (Supplementry Fig. 2). We then ssessed the numers ells in S phse fter short-term BrdU pulse. The numer of BrdU-positive S phse ells ws lso inresed (Fig. 1, nd Supplementry Fig. 2). These results indite tht mny more ells were undergoing division in Gsk3 / ; Gsk3 loxp/loxp ; rins thn in ontrols t E13.5. To further identify the underlying uses of the expnsion of the progenitor pool, we investigted ell-yle length nd re-entry. The numer of Ki67 nd BrdU doule-positive ells fter short nd long BrdU pulses in Gsk3 / ; Gsk3 loxp/loxp ; ortex ws inresed (Fig. 1), inditing oth shortening Tr2 Figure 2 Conversion of rdil progenitors to INPs is suppressed y GSK- 3 deletion. () The numer of Tr2-positive INPs ws mrkedly redued in the Gsk3 / ; Gsk3 loxp/loxp ; rin setions. E13.5 ererl ortex smples from ontrols nd Gsk3 / ; Gsk3 loxp/loxp ; mie were immunostined with n ntiody to Tr2. Nulei were ounterstined with DAPI. Right pnels show higher mgnifition of smll oxes (white dotted) in left pnels. Sle rs represent 5 µm (left pnels) nd 2 µm (right pnels). () Western lotting showed tht the level of Tr2 ws mrkedly redued in the Gsk3 / ; Gsk3 loxp/loxp ; rin tissues. Tr2 DAPI dvne online pulition nture NEUROSCIENCE

3 Gsk3 +/ ; Gsk3 loxp/+ ; Gsk3 / ; Gsk3 loxp/loxp ; Gsk3 +/ ; Gsk3 loxp/+ ; Gsk3 / ; Gsk3 loxp/loxp ; Sox2 Tuj1 Figure 3 GSK-3 deletion in progenitors strongly inhiits neurogenesis. () Mrked thinning of ortil res filled with Tuj1-positive ells in Gsk3 / ; Gsk3 loxp/loxp ; rin setions t E13.5. Sle r represents 5 µm. () This ws lso oserved with Tr1, deeperlyer neuron mrker, stining. Sle r represents 5 µm. () NeuN-positive neurons were lso mrkedly less frequent in Gsk3 / ; Gsk3 loxp/loxp ; ortil setions. Sle r represents 1 µm. (d) Quntifition of Tuj1-, Sox2-, Tr1- nd NeuN-positive ells. The rtios of Tuj1-, Sox2- nd Tr1-positive ells to the DAPI-stined ells per field were mesured nd shown s reltive hnge versus ontrol. Reltive hnges of NeuN-positive res in Gsk3 / ; Gsk3 loxp/loxp ; tissue smples were sored y mesuring NeuN-positive res with ImgeJ softwre. n = 1 setions from eh of three mutnt nd ontrol emryos per eh ondition. P <.1. (e) Western lotting showed tht the neuronl mkers Tuj1, MAP2, SMI32 nd NeuN were downregulted in Gsk3 / ; Gsk3 loxp/loxp ; rin tissues. However, the progenitor mrkers Nestin nd Px6 were upregulted. Tissue lystes from three mutnt nd ontrol rins per eh ondition were used. (f) Quntifition of western lot dt. We used three independent lots from eh ondition for quntifition. e Tuj1 MAP2 SMI32 NeuN Px6 Nestin Tr1 DAPI Gsk3 +/ ; Gsk3 loxp/+ ; Gsk3 / ; Gsk3 loxp/loxp ; d f Reltive hnge (versus ontrol) Reltive nd intensity (versus ontrol) of the ell yle nd inresed ell-yle re-entry. Immunostining nd western lotting with n ntiody to tivted spse-3 reveled tht levels of poptosis were not signifintly different etween ontrol nd mutnt ortex t E13.5 (P <.1; Fig. 1 nd Supplementry Fig. 2). Tken together, these results indite tht the expnded progenitor pool in Gsk3 / ; Gsk3 loxp/loxp ; rin is the result of inreses in ell-yle rte nd re-entry, ut not of enhned progenitor survivl. GSK-3 deletion strongly Inhiits neurogenesis To ssess the progression of the rdil progenitors to INPs, we immunostined ererl ortex smples using n ntiody to Tr2, speifi mrker for INPs 23. To our surprise, we found tht the numer of Tr2-positive ells ws redued y 64% (±2.7%) in Gsk3 / ; Gsk3 loxp/loxp ; orties (Fig. 2). Western lotting showed tht the level of Tr2 in Gsk3 / ; Gsk3 loxp/loxp ; rin ws mrkedly deresed (Fig. 2). Our results indite tht GSK-3 tivity is required to regulte the onversion of Sox2-positive rdil progenitors into INPs during ortil development. We next exmined the numer of neurons in oth ontrol nd mutnt setions using immunostining with n ntiody to Tuj1 tht reognizes postmitoti neurons. The numers of Tuj1 positive neurons were deresed y out 6% in Gsk3 / ; Gsk3 loxp/loxp ; ortil setions (Fig. 3). We oserved similr dereses with Tr1 nd NeuN, two ommonly used neuronl mrkers (Fig. 3 d). These morphologil results were onfirmed y western Tuj1 Control Sox2 Tr1 Tuj1 MAP2 SMI32 NeuN DAPI Control NeuN NeuN PAX6 Nestin lotting. The levels of the neuronl mkers Tuj1, MAP2, SMI32 nd NeuN in Gsk3 / ; Gsk3 loxp/loxp ; rin tissue were ll redued y 5 7% (Fig. 3e,f). In ontrst, the progenitor mrkers Nestin nd Px6 were upregulted y 1 2%. Together, these results indite tht GSK-3 is required for proper regultion of neurogenesis in the developing mouse rin. To ssess the effets on neurogenesis over time, we exmined Gsk3 / ; Gsk3 loxp/loxp ; rins t multiple rostro-udl levels t E15.5 (Fig. 4). Notly, we oserved tht there ws mssive expnsion of the re filled with Sox2-positive ells throughout the telenephlon in Gsk3 / ; Gsk3 loxp/loxp ; ortil wll in omprison with ontrols, where Sox2-positive progenitors were restrited to regions surrounding the ventriles (Fig. 4 d). Neurogenesis in GSK-3 mutnt rins t this ge ws strongly suppressed, s mrked y Tuj1 stining (Fig. 4e,f). Thus, inhiition of neurogenesis is persistent in the sene of GSK-3 signling. One implition of our findings is tht GSK-3 tivity towrd sustrtes tht influene prolifertion my e upregulted s the progenitor prolifertion diminishes nd s neurogenesis proeeds. To ssess this ide in generl wy, we mesured the phosphoryltion levels of -My nd β-tenin, GSK-3 trgets, t three developmentl stges, E13, E15 nd E17. Both phospho -My nd phospho β-tenin levels in rin lystes were inresed with inresing emryoni ge (Supplementry Fig. 3), suggesting tht GSK-3 tivity towrd the prolifertion regultors -My nd β-tenin is suppressed during the period of tive progenitor prolifertion nd inresed t lter stges. Ativtion of -tenin, Shh nd Noth signling To determine whether Wnt signling is involved in the expnsion of the progenitor pool in Gsk3 / ; Gsk3 loxp/loxp ; rins, we first exmined the expression levels nd the distriution ptterns of β-tenin. In ontrols, β-tenin ws highly enrihed long the pil surfe (Fig. 5). However, in Gsk3 / ; Gsk3 loxp/loxp ; rins, β-tenin ws highly expressed throughout the thikness of the developing ortex (Fig. 5). We lso oserved similr loss of the norml sptil expression pttern nd n inrese in the levels of β-tenin in the spinl ord (Supplementry Fig. 4). At the single-ell level, we found n inresed level of β-tenin in oth the nuleus nd the ytosol in GSK-3 mutnt rins (Fig. 5), inditing umultion nd trnslotion of β-tenin from the ytosoli omprtment to the nuleus. Western lots showed tht totl levels of β-tenin nd its trnsriptionl trgets Axin2 nd -Jun were inresed in Gsk3 / ; nture NEUROSCIENCE dvne online pulition

4 d Figure 4 Neurogenesis is inhiited through E15.5. ( d) The neurl progenitor pool ws mssively expnded throughout the rostro-udl neurxis in Gsk3 / ; Gsk3 loxp/loxp ; mie. Seril rin setions in the rostro-udl diretion were prepred from E15.5 mie nd were immunostined for Sox2 (green) nd Tuj1 (red). Sle r represents 4 µm. Rostrl rin setions re shown in nd, nd udl rin setions re shown in nd d. (e,f) Higher mgnifition showed tht Sox2-positive progenitor pools in GSK-3 mutnt ortex were even more expnded t E15.5 thn t erlier ges. Genertion of Tuj1-positive neurons ws severely inhiited through this stge. Sle r represents 1 µm. Left, ontrol rin setions. Right, Gsk3 / ; Gsk3 loxp/loxp ; setions. e Sox2 Tuj1 DAPI Gsk3 loxp/loxp ; rins (Fig. 5). Together, these dt indite tht β-tenin is mrkedly dysregulted in Gsk3 / ; Gsk3 loxp/loxp ; neurl progenitors nd suggest tht β-tenin/tcf/lef1 signling is hypertive in these ells. To further investigte the mehnisms responsile for the expnded progenitor pool, we exmined Shh signling. We first exmined overll levels of Shh downstrem effetors, Gli1 nd Gli2 proteins in Gsk3 / ; Gsk3 loxp/loxp ; rins t E14.5. Western lots showed tht levels of Gli1 nd Gli2 were mrkedly inresed, wheres levels of Gli3 were unhnged (Fig. 5). Gli1 hs een demonstrted to e diret trnsriptionl trget of Gli2 in neurl nd non-neurl systems 24. Therefore, we determined the distriution ptterns of Gli1 mrnas in Gsk3 / ; Gsk3 loxp/loxp ; rins using in situ hyridiztion. Gli1 ws strongly expressed in the ventriulr/suventriulr zone of GSK-3 mutnt dorsl telenephlon, wheres there ws little expression in ontrol dorsl telenephlon (Fig. 5d). Furthermore, we found mssive expnsion of Gli1 expression in mutnt ventrl telenephlon (Fig. 5d). Pthed, trnsriptionl trget of Shh signling 24, ws lso mrkedly upregulted, prtiulrly in dorsl regions of the gnglioni eminene (Fig. 5d). The inresed levels of Shh omponents were not ssoited with Shh protein levels, s no hnges were found in Shh expression etween ontrol nd GSK-3 deleted rin smples (Supplementry Fig. 5). To test whether the inreses in the levels of Shh omponents re funtionlly relevnt, we knoked downed Gli1 in dissoited Gsk3 / ; Gsk3 loxp/loxp ; progenitors using smll interfering RNA to Gli1. The sirna deresed the progenitor prolifertion y out 35% ompred with ontrols (Supplementry Fig. 5). These dt indite tht Shh signling is onstitutively tivted in Gsk3 / ; Gsk3 loxp/loxp ; rins nd tht Shh signling is importnt in mediting GSK-3 deletion effets. Finlly, to determine whether there my e norml tivtion of Noth signling in Gsk3 / ; Gsk3 loxp/loxp ; rins, we f exmined the expression level nd distriution pttern of Hes1, Noth downstrem meditor. Hes1 expression is normlly restrited to the ventriulr nd suventriulr zones (Fig. 6). However, we found tht Hes1 ws distriuted throughout the width of the ererl ortil wll of GSK-3 mutnts. The overll levels of Hes1 nd Hes5 were lso sustntilly inresed in Gsk3 / ; Gsk3 loxp/loxp ; rin lystes (Fig. 6). The widespred distriution nd upregultion of Hes1 suggests tht the Noth pthwy is tivted in the developing ortex of Gsk3 / ; Gsk3 loxp/loxp ; emryos. To ddress this possiility, we mesured the mount of leved intrellulr domin of Noth (NICD, tivted form of Noth) tht ws present. We found tht, ompred with ontrol rins, the mount of NICD in Gsk3 / ; Gsk3 loxp/loxp ; rins ws mrkedly inresed (Fig. 6). To ssess the inrese in Noth signling is funtionlly relevnt, we treted GSK-3 deleted progenitors with the gmm seretse inhiitor N-[N-(3,5-difluorophenetyl-L-lnyl)]-Sphenylglyine t-utyl ester (DAPT). This tretment redued the hyperprolifertion nd inresed the numer of MAP2-positive neurons (Supplementry Fig. 6). We onlude tht intivtion of endogenous GSK-3 enhnes Noth signling, whih then inhiits neurogenesis in Gsk3 / ; Gsk3 loxp/loxp ; mie. Both onstitutive nd trgeted tivtion of β-tenin enhnes the prolifertion of neurl progenitors 25,26. These findings rise the questions of whether the effets of GSK-3 deletion tht we oserved re entirely medited y inresed levels of β-tenin. To ddress this issue, we inhiited β-tenin signling y trnsfeting dissoited Gsk3 / ; Gsk3 loxp/loxp ; ortil progenitors with dominntnegtive TCF (dntcf) onstrut tht lks the N-terminl sequenes required for β-tenin inding 27. Overexpression of dntcf only prtilly inhiited prolifertion of GSK-3 deleted progenitors, reduing prolifertion mrkers y out 5% (Fig. 6d,e). The perentge of proliferting ells ws still lmost twie s high s tht of ontrols (Supplementry Fig. 6). Thus, sustntil frtion of GSK-3 deleted progenitors ontinued to proliferte when β-tenin signling ws suppressed. Notly, inhiition of Noth signling y trnsfetion of dominnt-negtive Hes1 (dnhes1) lso resulted in redutions of progenitor prolifertion y round 5% (Fig. 6d,e). However, inhiition of oth β-tenin nd Noth signling redued prolifertion in GSK-3 deleted ells to levels elow 5% (Fig. 6d,e). These results re onsistent with the ide tht independent regultion of β-tenin nd Noth signling medite GSK-3 s effets. GSK-3 regultion of -My downstrem of FGF/PI3K My fmily proteins re ritil for the regultion of prolifertion in multiple settings nd GSK-3 hs een shown to diretly regulte the stility of oth -My nd the relted fmily memer N-My in ell lines 1. We stined Gsk3 / ; Gsk3 loxp/loxp ; progenitors with n ntiody to -My nd found tht there ws mssive dvne online pulition nture NEUROSCIENCE

5 Figure 5 Dysregultion of β-tenin nd Shh signling omponents s result of GSK-3 deletion. () Sptil expression of β-tenin ws disrupted in Gsk3 / ; Gsk3 loxp/loxp ; rin setions. Left, expression of β-tenin ws highly enrihed long the pil surfe of ontrol ortil setions (rrow heds). In mutnts, β-tenin ws highly expressed throughout the developing ererl ortil wll. Right, higher-mgnifition imges showing tht β-tenin in Gsk3 / ; Gsk3 loxp/loxp ; setions ws present in oth the nuleus nd ytosol. Sle rs represent 2 µm (left) nd 5 µm (right). () Western lots showed tht the expression of β-tenin nd its trget genes, inluding Axin2 nd -Jun, were inresed in the Gsk3 / ; Gsk3 loxp/loxp ; rins. () There were mrked inreses in Gli1 nd Gli2 protein levels in the Gsk3 / ; Gsk3 loxp/loxp ; rins. (d) Shh signling ws enhned in E14 Gsk3 / ; Gsk3 loxp/loxp ; rin setions. Left, Gli1 mrnas were expressed t low levels in E14 dorsl telenephlon ontrols. There ws signifint indution of Gli1 in the ventriulr nd suventriulr regions of Gsk3 / ; Gsk3 loxp/loxp ; smples (rrows). Sle r represents 1 µm. Middle, there ws mrked indution of Gli1 mrna levels in Gsk3 / ; Gsk3 loxp/loxp ; ventrl telenephlon (rrows). Sle r represents 2 µm. Right, Pthed mrna in ontrol tissues ws expressed in the dorsl prt of the ventrl telenephlon. Ventrl telenephli expression of Pthed mrnas in Gsk3 / ; Gsk3 loxp/loxp ; rins ws mrkedly inresed (rrows). Sle r represents 15 µm. SVZ, suventriulr zone; VZ, ventriulr zone. (e) Quntifition of western lot dt. Three independent lots per eh ondition were used for quntifition. P <.1. umultion of -My proteins in the nuleus (Fig. 7). Western lots onfirmed the mjor inrese of -My nd N-My in Gsk3 / ; Gsk3 loxp/loxp ; rin smples tht we oserved (Fig. 7). GSK-3 phosphoryltion of -My (nd N-My) t T58 is thought to promote -My degrdtion through uiquitin-protesome mhinery. We found tht T58 phosphoryltion ws lmost ompletely rogted in GSK-3 mutnt rin smples, s predited (Fig. 7). To test whether the inrese in -My levels is funtionlly relevnt, we inhiited -My funtion in dissoited Gsk3 / ; Gsk3 loxp/loxp ; progenitors with dominnt negtive -My (dn-my) onstrut in proedure similr to the one outlined ove for β-tenin. Overexpression of dn-my deresed progenitor prolifertion y 41 ± 5% ompred with green fluoresent protein (GFP) overexpression ontrol. GSK-3 regultion of My protein stility is thought to our downstrem of PI3K signling. A logil ndidte to regulte this signling would e the FGFs, whih re known to regulte progenitor prolifertion in the developing telenephlon vi reeptor tyrosine kinses 8. Therefore, we exmined the possiility tht GSK-3 medites FGF-PI3K signling in the regultion of neurl progenitor prolifertion. We ssessed progenitor prolifertion in ontrol nd GSK-3 defiient ortil ultures fter tretment with FGF2 nd/or LY2942. As expeted, ddition of FGF2 inresed progenitor prolifertion in ontrol ultures (dt not shown). Tretment with LY2942 deresed the numers of BrdU-positive Gli1 Gli2 Gli3 e 3 Reltive nd intensity (versus ontrol) 2 1 Gli1 β-tenin DAPI Gli2 Control Gli3 d Pi SVZ VZ Pi SVZ VZ Gli1 β-tenin Axin2 -Jun proliferting progenitors in FGF2-treted ontrol ultures y more thn 5% (Fig. 7,d). Notly, the inhiitory effets of LY2942 were ompletely suppressed in Gsk3 / ; Gsk3 loxp/loxp ; ortil ultures. We next ssessed -My levels in ortil ultures while inhiiting PI3K nd stimulting FGF. Tretment of ontrol ortil ultures with LY2942 sustntilly deresed -My levels (Supplementry Fig. 7). As expeted, phosphoryltion of -My t T58 nd GSK-3β t S9 were inresed nd deresed, respetively. These results suggest tht inhiition of PI3K dereses -My levels y enhning GSK-3 tivity. To ddress this possiility, we performed the sme experiments in Gsk3 / ; Gsk3 loxp/loxp ; ortil ultures. The derese in -My levels tht we oserved with LY2942 tretment ws olished in the lystes of GSK-3β deleted ultures (Fig. 7e). To ssess the potentil regultion of -My levels y FGF2, we treted ortil ultures with FGF2. Tretment with FGF2 mrkedly upregulted -My nd this ws inhiited y LY2942 (Fig. 7f). The inrese of -My in GSK-3 defiient ortil ells ws not ffeted y tretment with either FGF2 or FGF2 nd LY2942 (Fig. 7f). Tken together, these results strongly suggest tht GSK-3 medites FGF-PI3K regultion of -My. Multiple studies hve shown tht pproprite pil-sl polrity of progenitors is required for norml neurogenesis 28. To ddress progenitor polrity, we stined rin tissues with ntiodies ginst multiple ell polrity nd dhesion mrkers inluding PKCζ, Gli1 Pthed nture NEUROSCIENCE dvne online pulition

6 Figure 6 Chnges in Noth signling omponents nd ontriutions of β-tenin nd Noth signling to GSK-3 deletion effets. () Expnded zone of Hes1 expression in Gsk3 / ; Gsk3 loxp/loxp ; rins. Left, in ontrol rin setions t E14, Hes1 ws mostly expressed in the ventriulr nd suventriulr zone, where progenitors re normlly loted. However, Hes1 ws distriuted throughout the developing ortil wll in Gsk3 / ; Gsk3 loxp/loxp ; ererl ortex. Middle, higher mgnifition of smll oxes (red) from the left pnels. Right, Hes1-stined fields were merged with DAPI-stined imges. Sle r represents 15 µm. () Western lotting showed tht the level of Hes1 nd Hes5 were signifintly inresed in the Gsk3 / ; Gsk3 loxp/loxp ; rin lystes. () The level of NICD ws mrkedly inresed in Gsk3 / ; Gsk3 loxp/loxp ; rin lystes. (d) Inhiition of either β-tenin signling or Noth signling prtilly loked prolifertion of Gsk3 / ; Gsk3 loxp/loxp ; nestinre ortil progenitors in ulture. GSK-3 mutnt ells were trnsfeted with dntcf, dnhes or ontrol gfp. Cells were then ultured for 48 h nd immunostined with ntiody to Ki67. Overexpressing oth dntcf nd dnhes suppressed prolifertion of GSK-3 mutnt progenitors to higher degree thn overexpression of either dntcf or dnhes lone. Sle r represents 25 µm. Arrows indite trnsfeted ells tht were lso positive for Ki67. (e) The perentges of Ki67- nd GFP-positive ells reltive to totl trnsfeted ells (GFP positive) were ounted for quntifition. P <.1 (versus GFP), P <.1 (versus dntcf or dnhes). dherin, tin, APC nd EB1. The polrized pil onentrtions of the mrkers were signifintly redued in the ortex of Gsk3 / ; Gsk3 loxp/loxp ; nimls (Supplementry Fig. 8). These results demonstrte tht GSK-3 signling is essentil for pil-sl polrity in the developing ortex nd suggest tht GSK-3 my ontrol progenitor prolifertion in prt y orgnizing ellulr polrity. DISCUSSION GSK-3 is n importnt moleulr regultor of neurogenesis We found tht deletion of GSK-3 mssively expnded the rdil progenitor pool. The elimintion of either GSK-3 fmily memer lone or the elimintion of three Gsk3 lleles hd no ovious effet. This suggests tht mjor redution of GSK-3 signling is required to remove ritil GSK-3 medited homeostti ontrols during emryoni rin development. Determintion of the mitoti index with phospho-histone H3, BrdU nd Ki67 leling indited tht premture re-entry nd shortening of the ell yle ourred in progenitors in Gsk3 / ; Gsk3 loxp/loxp ; mie. Shortened ell yle hs previously een implited in redued responsiveness to differentition signls nd it hs een suggested tht ell-yle regultion is n importnt ontrol point for mehnisms tht ount for differenes in neuron numer etween rodents nd primtes 29. In ontrst with the expnsion of Sox2-positive rdil progenitors, genertion of neurons nd INPs ws mrkedly redued in the sene of GSK-3. Thus, neurl differentition ws rrested t the rdil progenitor stge. The effets of GSK-3 deletion on prolifertion nd differentition tht we oserved suggest tht temporl nd sptil regultion of GSK-3 tivity during nervous system development d gfp dntcf Hes1 DAPI dnhes dntcf, dnhes GFP Ki67 GFP + Ki67 + ells/ GFP + ells (%) is required to terminte prolifertion nd llow differentition t just the right moment to yield the orret numer of neurons. Our results rise the possiility tht regultion of the tivity of single kinse provides powerful nd simple mehnism for ontrolling neuron numer. GSK-3 deletion removes multiple homeostti ontrols The extent to whih the multiple mehnisms tht re known to regulte neurl progenitors operte independently or re oordintely regulted is unler. GSK-3 hs the, to the est of our knowledge, unique potentil to ontrol eh of these ritil homeostti mehnisms. Dysregultion of β-tenin levels is likely to e importnt for the hyperprolifertion of progenitors tht we oserved. Our findings re entirely onsistent with the notion tht pproprite regultion of β-tenin is ritil for the ontrol of progenitor prolifertion in multiple regions of the developing nervous system 7,25,26,3 32. Shh signling is lso ffeted y GSK-3 deletion. Our results suggest tht there is mmmlin ounterprt of the Drosophil GSK-3 homolog Shggy, whih regultes uitus interruptus ( homolog of mmmlin Gli proteins) in the Drosophil Hedgehog signling pthwy 14. The inresed prolifertion nd regultion of Shh signling omponents oserved in the ventrl telenephlon of GSK-3 deleted mutnts strongly suggest tht GSK-3 is ritil meditor of Shh signling in this region. GSK-3 is therefore ndidte for integrting Wnt/β-tenin nd Shh signling pthwys, oth of whih re known to ontrol neurogenesis in the ventrl telenephlon 32. Our dt lso indite tht Noth signling is inresed in GSK- 3 defiient rins. Noth signling is known to e importnt for the mintenne of stemness nd the identity of rdil glil ells during e Hes1 Atin Hes5 NICD Gsk3 +/ ; Gsk3 loxp/+ ; Gsk3 +/ ; Gsk3 loxp/+ ; Gsk3 / ; Gsk3 loxp/loxp ; Gsk3 / ; Gsk3 loxp/loxp ; gfp dntcf dnhes dntcf, dnhes dvne online pulition nture NEUROSCIENCE

7 Figure 7 GSK-3 medites FGF-PI3K signling to regulte -My in neurl progenitors. () Compred with ontrol progenitors, -My levels were mssively inresed in nulei (white dotted irle) of Gsk3 / ; Gsk3 loxp/loxp ; progenitors t E14. Sle r represents 5 µm. () Western lots showed tht levels of -My nd N-My were inresed in GSK-3 mutnt rin lystes. In ontrst, phospho -My ws rely detetle in GSK-3 mutnt rins. () FGF effets on progenitor prolifertion were medited in prt y PI3K nd GSK-3. Cortil progenitors from ontrol nd GSK-3 mutnt tissues were ultured with either FGF2 lone or FGF2 nd LY294 (LY) for 24 h. Then, the ultures were treted with BrdU for 6 h nd immunostined with n ntiody to BrdU. Tretment with FGF inresed progenitor prolifertion in ontrol ultures nd PI3K inhiited the FGF2 effets. GSK-3 mutnt progenitors showed no response to LY294. Sle r represents 7 µm. (d) The perentge of BrdU-positive ells ws ounted for quntifition. P <.1. (e) GSK-3 medited PI3K regultion of -My. Chnges in -My expression with LY2942 tretment were rogted in Gsk3 / ; Gsk3 loxp/loxp ; ortil ultures. The derese in phospho-akt nd phospho GSK-3β showed the effiieny of PI3K inhiition. (f) GSK-3 medited the effets of FGF signling on -My regultion. Western lots showed tht the ddition of reominnt humn si FGF2 (1 ng ml 1 ) in ontrol ultures mrkedly indued upregultion of -My, whih ws suppressed y LY2942 tretment. However, neither the upregultion y FGF2 nor its suppression y LY2942 were oserved in Gsk3 / ; Gsk3 loxp/loxp ; ortil ultures. development Notly, Noth signling levels distinguish rdil progenitors from INPs tht hve ttenuted nonil Noth signling 34. Our results re onsistent with the ide tht high levels of Noth signling in Gsk3 / ; Gsk3 loxp/loxp ; ells suppress neurogenesis t the stge of rdil progenitors s they self-renew nd tht few ells progress to eome Tr2-positive INPs. Potentil intertions mong GSK-3 regulted pthwys An importnt issue is whether GSK-3 regultion leds to hierrhy of signling mehnisms tht regulte progenitor prolifertion or whether multiple GSK-3 regulted mehnisms eh ontriute independently to the finl outome. There is some evidene tht β-tenin n influene Noth signling nd n e regulted y the FGF pthwy 9. However, we dout tht stiliztion of β-tenin lone fully ounts for the effets tht we desrie here nd suggest tht severl other GSK- 3 regulted mehnisms exert independent effets. Elimintion of β- tenin in neurl progenitors redues, ut does not eliminte, progenitor prolifertion in vivo 3,32. Our dt indite tht overexpression of dntcf only prtilly inhiits prolifertion in Gsk3 / ; Gsk3 loxp/loxp ; neurl progenitors in vitro. These results suggest tht GSK-3 deletion my result in effets on Noth signling tht re independent of β-tenin. Severl studies hve suggested tht GSK-3 my diretly regulte Noth signling vi phosphoryltion of Noth1 or Noth2, or regulte the tions of the gmm seretse presenillin-1 (refs ). However, ll of this work hs een rried out in ell lines nd the importne of these findings in vivo is unler. e FGF FGF + LY -My Phospho-AKT GSK-3β -My DAPI BrdU DAPI WT LY LY f -My Finlly, our dt indite tht GSK-3 strongly regultes My fmily proteins nd tht FGF regultion of -My vi PI3K signling is disrupted y GSK-3 deletion. My fmily proteins re ritil for the regultion of prolifertion in multiple settings nd My fmily memer, N-My, is known from mouse geneti studies to e required for the mintenne of neurl progenitor prolifertion in ereellum nd ortex 1. Beuse -My is thought to e trnsriptionl trget of β-tenin 36,37, t lest some of the oserved inrese of -My in GSK-3 deleted progenitors my e ttriutle to β-tenin effets. However, we found nother mehnism of -My regultion, in whih FGF-PI3K ontrols -My levels vi GSK-3. Our findings re onsistent with work in ell lines demonstrting tht GSK-3 signling n diretly regulte the stility of My fmily proteins vi PI3K signling Indeed, we found tht GSK-3 phosphoryltion of -My t T58, whih is thought to e importnt for its stility, is regulted y PI3K. This FGF PI3K GSK-3 regultion of My protein stility in neurl progenitors is likely to e ritil for determining its effets, s the hlf-life of My protein in ell lines is extrordinrily rief, on the order of 2 min 38. Methods Methods nd ny ssoited referenes re ville in the online version of the pper t Note: Supplementry informtion is ville on the Nture Neurosiene wesite. -My DAPI WT d FGF BrdU + ells/totl ells (%) -My Phospho -My N-My Control LY LY FGF FGF FGF, LY FGF FGF, LY nture NEUROSCIENCE dvne online pulition

8 Aknowledgments We thnk F. Polleux, L. Pevny nd E. Anton for vlule dvie nd omments on the mnusript. We re lso grteful to L. Goins nd A. MKell for niml re nd M. Ait for tehnil support. This reserh ws supported y grnts from the US Ntionl Institutes of Helth (NS5968 to W.D.S. nd NS45892, whih supports the Confol nd Multiphoton Imging nd Expression Loliztion Cores of University of North Crolin Neurosiene Center) nd Cndin Institutes of Helth Reserh (MOP to J.R.W.). AUTHOR CONTRIBUTIONS W.-Y.K. designed nd onduted most of the experiments, nlyzed the dt, nd o-wrote the pper. X.W. ontriuted to the in vitro experiments nd ides. Y.W. onduted western lotting nd ontriuted tehnil ssistne. B.W.D. nd S.P. generted the GSK-3 mutnt lines. J.R.W. ontriuted to experimentl design, provided intelletul guidne nd o-wrote the pper. W.D.S. supervised the work, ontriuted to the experimentl design, provided intelletul guidne nd o-wrote the pper. Pulished online t Reprints nd permissions informtion is ville online t reprintsndpermissions/. 1. Dole, B.W. & Woodgett, J.R. GSK-3: triks of the trde for multi-tsking kinse. J. Cell Si. 116, (23). 2. Kokeritz, L., Dole, B., Ptel, S. & Woodgett, J.R. Glyogen synthse kinse-3: n overview of n over-hieving protein kinse. Curr. Drug Trgets 7, (26). 3. Beulieu, J.M., Ginetdinov, R.R. & Cron, M.G. The Akt GSK-3 signling sde in the tions of dopmine. Trends Phrmol. Si. 28, (27). 4. Sto, N., Meijer, L., Skltsounis, L., Greengrd, P. & Brivnlou, A.H. Mintenne of pluripoteny in humn nd mouse emryoni stem ells through tivtion of Wnt signling y phrmologil GSK-3-speifi inhiitor. Nt. Med. 1, (24). 5. Ying, Q.L. et l. The ground stte of emryoni stem ell self-renewl. Nture 453, (28). 6. Bone, H.K. et l. Involvement of GSK-3 in regultion of murine emryoni stem ell self-renewl reveled y series of isindolylmleimides. Chem. Biol. 16, (29). 7. Mo, Y. et l. Disrupted in shizophreni 1 regultes neuronl progenitor prolifertion vi modultion of GSK3et/et-tenin signling. Cell 136, (29). 8. Corin, J.G. et l. Regultion of neurl progenitor ell development in the nervous system. J. Neurohem. 16, (28). 9. Shimizu, T. et l. Stilized et-tenin funtions through TCF/LEF proteins nd the Noth/RBP-Jkpp omplex to promote prolifertion nd suppress differentition of neurl preursor ells. Mol. Cell. Biol. 28, (28). 1. Knoepfler, P.S. & Kenney, A.M. Neurl preursor yling t soni speed: N-My pedls, GSK-3 rkes. Cell Cyle 5, (26). 11. Behrd, M. & Dlton, S. Suellulr loliztion of glyogen synthse kinse 3et ontrols emryoni stem ell self-renewl. Mol. Cell. Biol. 29, (29). 12. Dole, B.W. & Woodgett, J.R. Exploring pluripoteny with hemil genetis. Cell Stem Cell 4, 98 1 (29). 13. Otto, T. et l. Stiliztion of N-My is ritil funtion of Auror A in humn neurolstom. Cner Cell 15, (29). 14. Ji, J. et l. Shggy/GSK3 ntgonizes Hedgehog signling y regulting Cuitus interruptus. Nture 416, (22). 15. Espinos, L., Ingles-Esteve, J., Aguiler, C. & Bigs, A. Phosphoryltion y glyogen synthse kinse 3 et downregultes Noth tivity, link for Noth nd Wnt pthwys. J. Biol. Chem. 278, (23). 16. Uemur, K. et l. GSK3et tivity modifies the loliztion nd funtion of presenilin 1. J. Biol. Chem. 282, (27). 17. Jin, Y.H., Kim, H., Oh, M., Ki, H. & Kim, K. Regultion of Noth1/NICD nd Hes1 expressions y GSK-3lph/et. Mol. Cells 27, (29). 18. Tronhe, F. et l. Disruption of the gluoortioid reeptor gene in the nervous system results in redued nxiety. Nt. Genet. 23, (1999). 19. Yokot, Y. et l. The denomtous polyposis oli protein is n essentil regultor of rdil glil polrity nd onstrution of the ererl ortex. Neuron 61, (29). 2. MAuly, K. et l. Glyogen synthse kinse 3lph speifi regultion of murine hepti glyogen metolism. Cell Met. 6, (27). 21. Ptel, S. et l. Tissue-speifi role of glyogen synthse kinse 3et in gluose homeostsis nd insulin tion. Mol. Cell. Biol. 28, (28). 22. Kim, W.Y. et l. Essentil roles for GSK-3s nd GSK-3 primed sustrtes in neurotrophin-indued nd hippompl xon growth. Neuron 52, (26). 23. Sess, A., Mo, C.A., Hdjntonkis, A.K., Klein, W.H. & Brooli, V. Tr2 direts onversion of rdil gli into sl preursors nd guides neuronl mplifition y indiret neurogenesis in the developing neoortex. Neuron 6, (28). 24. Ding, Q. et l. Diminished Soni hedgehog signling nd lk of floor plte differentition in Gli2 mutnt mie. Development 125, (1998). 25. Chenn, A. & Wlsh, C.A. Regultion of ererl ortil size y ontrol of ell yle exit in neurl preursors. Siene 297, (22). 26. Woodhed, G.J., Muth, C.A., Olson, E.C. & Chenn, A. Cell-utonomous et-tenin signling regultes ortil preursor prolifertion. J. Neurosi. 26, (26). 27. Chen, S. et l. Wnt-1 signling inhiits poptosis y tivting et-tenin/t ell ftor medited trnsription. J. Cell Biol. 152, (21). 28. Götz, M. & Huttner, W.B. The ell iology of neurogenesis. Nt. Rev. Mol. Cell Biol. 6, (25). 29. Dehy, C. & Kennedy, H. Cell-yle ontrol nd ortil development. Nt. Rev. Neurosi. 8, (27). 3. Zehner, D. et l. Bet-tenin signls regulte ell growth nd the lne etween progenitor ell expnsion nd differentition in the nervous system. Dev. Biol. 258, (23). 31. Mhon, O. et l. A dynmi grdient of Wnt signling ontrols initition of neurogenesis in the mmmlin ortex nd ellulr speifition in the hippompus. Dev. Biol. 311, (27). 32. Gulsi, A.A. & Anderson, S.A. Bet-tenin medited Wnt signling regultes neurogenesis in the ventrl telenephlon. Nt. Neurosi. 11, (28). 33. Yoon, K. & Gino, N. Noth signling in the mmmlin entrl nervous system: insights from mouse mutnts. Nt. Neurosi. 8, (25). 34. Mizutni, K., Yoon, K., Dng, L., Tokung, A. & Gino, N. Differentil Noth signling distinguishes neurl stem ells from intermedite progenitors. Nture 449, (27). 35. Shimojo, H., Ohtsuk, T. & Kgeym, R. Osilltions in noth signling regulte mintenne of neurl progenitors. Neuron 58, (28). 36. He, T.C. et l. Identifition of -MYC s trget of the APC pthwy. Siene 281, (1998). 37. Clvisi, D.F., Ldu, S., Ftor, V.M. & Thorgeirsson, S.S. Ativtion of et-tenin provides prolifertive nd invsive dvntges in -my/tgf-lph heptorinogenesis promoted y phenoritl. Crinogenesis 25, (24). 38. Eilers, M. & Eisenmn, R.N. My s rod reh. Genes Dev. 22, (28). dvne online pulition nture NEUROSCIENCE

9 ONLINE METHODS Mterils. DAPT (Cliohem), reominnt humn si FGF2 (Sigm), reominnt humn epiderml growth ftor (EGF) (R&D system), B27 supplements (Invitrogen), N2 supplement (Invitrogen), BrdU (Sigm) nd LY2942 (Cell Signling) were purhsed from the ompnies indited. Plsmids enoding wild-type nd onstitutively tive β-tenin were generously provided y B. Vogelstein (Johns Hopkins University). Dominnt-negtive TCF, Hes1 nd -My were generous gifts from A. Chenn (Northwestern University), A. Strom (Krolinsk Institute) nd H. Sugimur (Hmmtsu University), respetively. Gli1 sirnas were otined from Invitrogen. Mie. Mie were red for ording to niml protools pproved y the Institutionl Animl Cre nd Use Committees of the University of North Crolin nd University of Toronto. Gsk3 / mie were engineered to hror n exon 2 deletion 2. Gsk3 loxp/loxp mie were generted so tht exon 2 is flnked y loxp sites 21. Nervous system speifi onditionl GSK-3 doule-knokout mie (Gsk3 / ; Gsk3 loxp/loxp ; ) were generted y mting Gsk3 /, Gsk3 loxp/loxp nd mie 18. Littermte Gsk3 +/ ; Gsk3 loxp/+ ; mie served s ontrols. Immunohistohemistry. Immunohistohemil leling of emryoni rin setions or dissoited neurl ells ws performed s previously desried 22. For primry ntiodies, we used rit ntiody to Sox2 (Chemion), mouse ntiody to Nestin (BD iosienes), rit ntiody to Tr1 (Chemion), rit ntiody to Tr2 (Am), rit ntiody to phospho-histone H3 (Upstte Bioteh), mouse ntiody to BrdU (Sigm), rit ntiody to Ki67 (Covne), rit ntiody to spse 3 (Cell Signling), rit ntiody to β-tenin (Cell Signling), mouse ntiody to Tuj1 (Sigm), mouse ntiody to tuulin (Sigm), mouse ntiody to NeuN (Chemion), rit ntiody to PKCζ (Cell signling), rit ntiody to dherin (Cell Signling), mouse ntiody to tin (Sigm), rit ntiody to APC (gift from K. Neufeld, University of Knss), mouse ntiody to EB1 (Sigm), mouse ntiody to γ-tuulin (Sigm), rit ntiody to Hes1 (Snt Cruz), rit ntiody to -My (Cell Signling) or mouse ntiody to GSK-3β (BD Biosienes). Approprite Cy2-, Cy3- or Alex dye onjugted seondry ntiodies (Jkson ImmunoReserh, Moleulr Proes) were used to detet primry ntiody inding. DAPI (Sigm) or Drq5 (Moleulr proes) ws used s nuler ounterstins. Morphometry. For the quntifition of rin size nd ventriulr pil surfe length, we imged ten different resyl violet stined rin setions t periodi distnes long the rostro-udl xis. The re of eh setion ws defined using the Freehnd seletion tool in ImgeJ (US Ntionl Institutes of Helth). The lulted ImgeJ vlues were verged nd the result ws relulted s reltive hnges versus ontrol. The sme rin setions were used to mesure ventriulr pil surfe lengths of ontrol nd Gsk3 / ; Gsk3 loxp/loxp ; rins. Ventriulr pil surfe length ws defined using the Freehnd line seletion tool in ImgeJ. For ell ounts, imges were tken with Zeiss LSM51 onfol mirosope nd Nikon Elipse epifluoresene mirosope. For imge nlysis, the Zeiss LSM51 imge rowser nd Metmorph softwre (Moleulr Devies) were used. The numers of neurl progenitors nd neurons positive for BrdU, phosphohistone H3, Ki67, Sox2, MAP2, Tuj1, Tr1 or NeuN were otined s previously desried 25,39. Western lotting. Extrts from E13.5 telenephlon ws prepred using RIPA uffer nd the protein ontent ws determined y Bio-Rd Protein Assy system. Proteins were seprted on 3 8% or 4 12% SDS-PAGE grdient gels nd trnsferred onto nitroellulose memrne. The memrne ws inuted with mouse ntiody to GSK-3 (Invitrogen), rit ntiody to phospho GSK-3β (Cell signling), mouse ntiody to tin (Sigm), rit ntiody to phosphohistone H3 (Upstte), rit ntiody to tivted spse 3 (Cell Signling), rit ntiody to Tr2 (Am), mouse ntiody to Tuj1 (Sigm), mouse ntiody to MAP2 (Chemion), mouse ntiody to NeuN (Chemion), mouse ntiody to Px6 (Iow Hyridom Bnk), mouse ntiody to Nestin (BD Biosienes), rit ntiody to β-tenin (Cell Signling), SMI32 (Sternerger Immunoytohemils), rit ntiody to -My (Cell Signling), mouse ntiody to N-My (Snt Cruz), rit ntiody to -Jun (Cell Signling), rit ntiody to Hes1 (Snt Cruz), rit ntiody to NICD (Cell Signling), rit ntiody to Gli1 (Cell Signling), rit ntiody to Gli2 (Am), rit ntiody to Gli3 (Snt Cruz), rit ntiody to phospho-akt (Cell Signling), rit ntiody to phospho-mek (Cell Signling), rit ntiody to phospho- ERK (Cell Signling), rit ntiody to GluR (Cell Signling) or rit ntiody to (Cell Signling) t 4 C overnight. As seondry ntiodies, we used donkey ntiody to mouse or rit IgG onjugted to horserdish peroxidse (Amershm). ECL regents (Amershm) were used for immunodetetion. For quntifition of nd intensity, lots from three independent experiments for eh moleule of interest were used. Signls were mesured using ImgeJ softwre nd represented y reltive intensity versus tht of ontrol. ws used s n internl ontrol to normlize nd intensity. Progenitor ultures. Cererl ortex from E11 14 mie dorsl telenephlon ws isolted nd dissoited with triturtion fter trypsin/edta tretment. Then, the ells were plted onto poly-d-lysine/lminin oted overslips nd ultured for 2 d in the medium ontining neurosl medium, B27 nd N2 supplements, FGF2 (1 ng ml 1 ), nd EGF (1 ng ml 1 ). For differentition of neurl progenitors, the medium ontining FGF2 nd EGF ws removed nd repled y neurosl medium with 5% serum (vol/vol), B27 nd N2 supplements. To ssess the numers of proliferting progenitors nd differentited neurons, we rried out immunostining nd morphometry using ntiodies to Ki67, phosphohistone H3 nd MAP2, s desried ove. More thn 2 rndom mirosopi fields (1 nd 2 ) were nlyzed for eh ondition. For pturing imges of the ultures, we used Metmorph softwre nd Hmmtsu Or ER CCD mer tthed to Nikon Elipse mirosope. All imge nlysis for the mesurement of proliferting ells ws done with IPL softwre (Snlytis) or Metmorph softwre. Eletroportion. Mouse ortil progenitors were trnsfeted with vrious plsmids s desried previously 22. First, emryoni orties were dissoited nd suspended in 1 µl of Amx eletroportion uffer with 2 1 µg of plsmid DNA. Then, suspended ells were trnsferred to n Amx eletroportion uvette nd eletroported with n Amx Nuleofetor pprtus set on the G-13 progrm. After eletroportion, ells were plted onto oted overslips nd the medium ws hnged 4 h lter to remove the remnnt trnsfetion uffer. dntcf nd dn-my lk the β-tenin inding domin nd trnstivtion domin, respetively. dnhes1 hs mutted DNA-inding domin. A rtio of the numer of Ki67 nd GFP doule-positive ells to the numer of GFP-positive trnsfeted ells ws determined using the smpling methods outlined ove. BrdU dministrtion nd quntifition of mitosis. For prolifertion ssys, intrperitonel injetion ws performed into pregnnt mie t E BrdU (2 mg per kg of ody weight, dissolved in.9% sline, wt/vol) ws dministered either 24 h or 3 min efore the mouse ws killed for trnsient ssys. The mitoti index is rtio of the numer of phospho-histone H3 positive ells to the numer of DAPI-stined ells per field. To nlyze the BrdU leling index nd ell-yle re-entry, we exposed E13 ontrol nd Gsk3 / ; Gsk3 loxp/loxp ; nestinre mie to BrdU. Then rin slies were immunostined with ntiodies to BrdU nd Ki67. The BrdU leling index is the frtion of progenitor ells (Ki67 nd BrdU doule positive) to the numer of BrdU-positive ells fter 3-min BrdU pulse lel. For the nlysis of ell-yle re-entry, mie were exposed to single-pulse BrdU lel for 24 h. The frtion of ells leled with BrdU nd Ki67 reltive to the totl numer of BrdU-positive ells fter 24-h pulse lel ws determined. In situ hyridiztion. For rioproe synthesis, we used the pbsii plsmids ontining Gli1 nd Pthed (A. Joyner, Memoril Slon-Kettering Cner Institute). A 1,6-p EoRI frgment ontining zin-finger domin ws used to generte ntisense Gli1 rioproe. Pthed rioproe ws synthesized from n 841-p EoRI frgment. Digoxigenin-leled rioproes were synthesized using in vitro trnsription. Prformldehyde-fixed (4%, wt/vol) frozen rins from E12 14 emryos were setioned t 2 µm, treted with proteinse K (.5 µg ml 1 ), etylted, dehydrted ording to stndrd protools nd hyridized with digoxigenin-leled rioproes (1 µg ml 1 ) for 18 h t 65 C. Then, the tissue setions were rinsed nd inuted with 1:2, dilution of lkline phosphtse onjugted sheep ntiody to digoxigenin (Rohe) in loking uffer for 3 h t 2 25 C nd developed using nitro-lue tetrzolium hloride nd 5-romo-4-hloro-3 -indolyphosphte p-toluidine slt ording to the doi:1.138/nn.248 nture NEUROSCIENCE

10 mnufturer s instrutions. After developing, lkline phosphtse tivity ws quenhed y fixtion in 4% prformldehyde nd slides ontining tissue setions were mounted in Aqutex (EM Siene Hrleo). Sttistil nlysis. For exmining tissue setions, we used six E12 16 mie for eh experiment (ontrol mie, n = 3; Gsk3 / ; Gsk3 loxp/loxp ; mie, n = 3). Rndom fields hosen from more thn ten tissue setions from eh emryo were exmined. For ssessing ultured progenitors nd neurons, more thn 2 rndom fields from three mie were nlyzed for eh ondition. Sttistil signifine ws determined using n independent t test nd one-wy ANOVA followed y Tukey s post ho test. Dt re presented s mens ± s.e.m. 39. Cppello, S. et l. The Rho-GTPse d42 regultes neurl progenitor fte t the pil surfe. Nt. Neurosi. 9, (26). nture NEUROSCIENCE doi:1.138/nn.248