regulates stem cells through Wnt/β-catenin signalling

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1 LETTERS O regultes stem ells through Wnt/β-tenin signlling Jolly Mzumdr,,7, W. Timothy O Brien, Rndll S. Johnson, Joseph C. LMnn, Jun C. Chvez 5, Peter S. Klein nd M. Celeste Simon,, Stem ells reside in speilized miroenvironments or nihes tht regulte their funtion. In vitro studies using hypoxi ulture onditions (< 5% O ) hve reveled strong regultory links etween O vilility nd funtions of stem nd preursor ells. Although some stem ells re perivsulr, others my oupy hypoxi nihes nd e regulted y O grdients. owever, the underlying mehnisms remin unler. ere, we show tht hypoxi induile ftor α (IF α), prinipl meditor of hypoxi dpttions, modultes Wnt/β tenin signlling in hypoxi emryoni stem () ells y enhning β tenin tivtion nd expression of the downstrem effetors LEF nd TCF. This regultion extends to primry ells, inluding isolted neurl stem ells (SCs), nd is not oserved in differentited ells. In vivo, Wnt/β tenin tivity is losely ssoited with low O regions in the sugrnulr zone of the hippompus, key SC nihe 7. if α deletion impirs hippompl Wnt dependent proesses, inluding SC prolifertion, differentition nd neuronl mturtion. This deline orreltes with redued Wnt/ β tenin signlling in the sugrnulr zone. O vilility, therefore, my hve diret role in stem ell regultion through IF α modultion of Wnt/β tenin signlling. Ativtion of the Wnt/β-tenin pthwy is hrterized y ) stiliztion of ytoplsmi β-tenin fter reeptor enggement y Wnt lignds; ) β-tenin nuler trnslotion, ) β-tenin intertion with lymphoid enhner-inding ftor-/t-ell ftor- (LEF/TCF) trnsription ftors; nd ) stimultion of trget genes,9. To determine whether this pthwy is modulted y O vilility, we trnsiently trnsfeted murine ells nd P9 emryonl rinom (EC) ells with luiferse-sed TOP-Flsh (TCF optiml promoter) Wnt reporter plsmid, nd ompred reporter tivity under normoxi (% O ) nd hypoxi (.5% O ) onditions. Exposure to hypoxi signifintly enhned reporter tivity (-fold) in oth emryoni ell lines (Fig. ; Supplementry Informtion, Fig. S, ). ypoxi exposure lso inresed levels of nuler β-tenin, LEF- nd TCF- proteins (Fig. ). Anlysis of hypoxi ells y quntittive rel-time PCR (qrt PCR) showed inresed expression ( -fold) of Wnt trget genes, suh s Axin nd Dkk (Dikkopf-), nd β-tenin tivtors, Lef nd Tf (Fig. ). This is onsistent with our previous dt showing inresed Lef expression in hypoxi mouse ells. IF-α protein stiliztion nd upregultion of the IF-α trget Pgk onfirmed the indution of hypoxi response (Fig., ). ypoxi exerted similr effet in stimulted ells. Wnt pthwy stimultors, inluding -romoindiruin-ʹ oxime (BIO) nd lithium hloride (LiCl), or Wnt--onditioned medium (Wnt- CM), enhned reporter tivity y out -fold, wheres exposure to hypoxi inresed TOP-Flsh tivity 5 -fold in stimulted ells, ompred with untreted ontrol ells (Fig. d; Supplementry Informtion, Fig. S, ). Exposure to hypoxi lso further inresed expression of Wnt trget genes Lef nd Dkk in stimulted ells (Fig. e). TOP-Flsh ssys in RAi-medited β-tenin-depleted ells onfirmed the involvement of β-tenin in hypoxi-indued luiferse tivity (Fig. f). We exluded the possile involvement of other signlling pthwys thought to promote β-tenin stiliztion (for exmple, Akt/PIK) y showing tht fter inhiition of glyogen synthse kinse-β (GSK-β), GSK-β phosphoryltion levels remined unhnged under hypoxi onditions (Supplementry Informtion, Fig. S). Colletively, these dt indite tht nd P9 EC ells mintin onstitutively tive Wnt signlling tht is β-tenin-dependent, nd mrkedly enhned y hypoxi. ypoxi indution of Wnt signlling ws lso evident in ell prolifertion ssys. The numer of hypoxi ells ws inresed when ompred with normoxi ells (Fig. g). Cell survivl ws lso inresed, s hypoxi exposure hd modest effets on ell yle, ut signifintly redued poptoti ell deth (Supplementry Informtion, Fig. S, d). Addition of Wnt- CM, whih stimultes ell expnsion nd self-renewl, inresed the numers of oth normoxi nd hypoxi ells, ompred with untreted ontrols. By ontrst, tretment with Dikkopf- (DKK-), n extrellulr Wnt pthwy inhiitor, exlusively deresed the numer of hypoxi ells (Fig. g). Of note, DKK- tretment downregulted TOP-Flsh tivity in oth normoxi nd hypoxi ells (Fig. h), suggesting tht hypoxi sensitizes ells to the growth effets of Wnt/β-tenin signlling. Armson Fmily Cner Reserh Institute, Division of emtology-onology, University of Pennsylvni Shool of Mediine, Phildelphi, PA 9, USA. Division of Biologil Sienes, U C Sn Diego, L Joll, CA 99, USA. Deprtment of Physiology nd Biophysis, Cse Western Reserve University, Clevelnd, Ohio, USA. 5 Trnsltionl Mediine, Wyeth Reserh, Collegeville, Pennsylvni, PA 9, USA. owrd ughes Medil Institute, University of Pennsylvni, Phildelphi, Pennsylvni 9, USA. 7 Current ddress: Clinil Biomrkers, Onology R&D, GlxoSmithKline, Collegeville, Pennsylvni 9, USA. Correspondene should e ddressed to M.C.S. (e-mil: eleste@mil.med.upenn.edu) Reeived July ; epted 5 August ; pulished online 9 Septemer ; DOI:./n nture ell iology VOLUME UMBER OCTOBER 7 Mmilln Pulishers Limited. All rights reserved.

2 LETTERS β-tenin LEF- TCF- IF-α ART Fold hnge in mra (normlized to s) 7 5 CREB P9 P9 e Lef-D g umer of ells x kk- + Wnt + DKK- + W + W f Dys in ulture Lef- Tf- Dkk- + Wnt + DKK- P9 Axin Fzd7 Dys in ulture Pgk + tsi + tsi + Si + Si h P d 7 5 UT Si tsi β-t Atin β-t Atin + DKK- + DKK- + B + B Figure ypoxi tivtes Wnt/β-tenin signlling in mouse emryoni ells. () nd P9 EC ells trnsiently trnsfeted with TOP-Flsh nd prl-sv plsmids were grown under normoxi (, % O ) or hypoxi (,.5% O for h) onditions (n = 9). Dt indite luiferse ssys, performed using dul luiferse protool. () Western lot nlysis of β-tenin nd LEF-/TCF- in nuler extrts from ells under O onditions indited. CREB served s loding ontrol. () qrt PCR nlysis of Wnt trget gene levels in hypoxi ells reltive to S rra levels, normlized to normoxi ells (n = 9). (d) TOP-Flsh tivity in nd P9 EC ells treted with nm romoindiruin-ʹ oxime (B) nd ultured either in hypoxi or normoxi onditions (n = 9). (e) qrt PCR nlysis of Wnt- CM-treted ells (W) show inresed indution of Wnt trget genes under hypoxi, ompred with normoxi onditions (n = ). (f) Mrked redution in reporter tivity in nd P9 EC ells trnsfeted with pools of sira ginst β tenin t the indited O levels (UT: untrnsfeted, tsi: trget sira, Si: ontrol sira) (n = ; left pnel). Western lot nlysis of silened β tenin h fter trnsfetion (right pnel). (g) Growth rte of ells treted with Wnt- CM, or the Wnt inhiitor DKK- ( ng ml ) under the indited O onditions (n = ). (h) TOP-Flsh tivity in ells treted with DKK- ( ng ml ; n =). P <.5, P <.5., Student s t-test (error rs represent s.d.). Unropped imges of lots re shown in Supplementry Informtion, Fig. S7. One of the primry meditors of hypoxi responses is IF-, heterodimeri trnsription ftor ontining n O -sensitive α suunit (IF-α) nd onstitutively expressed β suunit (IF-β, lso known s ART). To determine whether hypoxi tivtes Wnt signlling through IF-, we nlysed TOP-Flsh tivity in if α / ells. In ontrst to report showing IF-α-medited repression of Wnt signlling in olon ner ells, if α deletion signifintly downregulted TOP-Flsh tivity in hypoxi ells, ut hd miniml effet on sl tivity (Fig. ). Comined tretment of Wnt- CM nd hypoxi did not superindue TOP-Flsh tivity in if α / ells (Fig. ), nd loss of if α hd no effet on normoxi responses to Wnt stimultion (Fig. ). owever, normoxi IF-α stiliztion in wild-type ells y the hypoxi-mimeti deferoxmine signifintly restored reporter tivity (>.5-fold; Fig. ). We oserved similr regultory link etween hypoxi ART tivity nd Wnt/β-tenin signlling (Fig. ). Deletion of if α nd Arnt lso diminished expression of Wnt trget genes, inluding Dkk, Lef- nd Tf, under hypoxi onditions (Fig. d, e; Supplementry Informtion, Fig. S). Thus, hypoxi indution of Wnt/ β-tenin signlling is medited y IF-α/ART omplexes. Moreover, s expeted from inresed levels of oth β-tenin nd LEF- in hypoxi ells (Fig. ), we deteted inresed ssoition of nuler β-tenin, extrted from hypoxi ells with immunopreipitted LEF- (Fig. f). Intriguingly, we noted redued levels of β-tenin in whole-ell extrts of hypoxi if α / ells, ompred with hypoxi if α +/+ (Fig. e). As hypoxi indution of Lef nd Tf mra nd orresponding proteins strongly orrelted with IF-α protein umultion, we exmined whether IF-α ontriutes diretly to inresed trnsription of Lef/Tf genes. Anlysis of murine Lef nd Tf gene sequenes reveled multiple puttive REs (hypoxi response elements) spnning exon nd the upstrem promoter nd enhner regions (+ p; Fig. g). In hromtin immunopreipittion (ChIP) ssys, IF-α ssoition t eh genomi region tested ws -fold greter in hypoxi ells thn in normoxi ells (Fig. g). IF-α, therefore, regultes LEF-/TCF- protein undne nd funtion in emryoni ells. In prolifertion ssys, neither if α / nor Arnt / ells showed growth dvntge under hypoxi onditions when ompred with the orresponding wild-type if α +/+ nd Arnt +/+ ells (Supplementry Informtion, Fig. S, ). Stem nd ommitted progenitor ells n respond differently to low O signls 5. We therefore exmined whether hypoxi effets on Wnt/βtenin signlling re funtion of differentition stge. Emryoni ells were stimulted with neuronl supplements (B7) to form neuronl preursors. Therefter, we sujeted nd P9 EC ell-derived nture ell iology VOLUME UMBER OCTOBER Mmilln Pulishers Limited. All rights reserved.

3 LETTERS if-α +/+ if-α / + Dfx if-α +/+ if-α / + W + W Reporter tivity (Fold) ART +/+ ART / ART Res d Fold hnge in mra (normlized to s) 7 5 Lef- if-α +/+ if-α +/+ if-α / if-α / Tf- e f P9 if-α +/+ IP: WB: β-tenin LEF- CRE B β-tenin LEF- CRE B if-α / ART +/+ ART / ART Res g Exon P P β-tenin Lef- % Input LEF- LEF- TCF- IF-α ART tin Tf- Fold hnge in IF-α IP (normlized to migg IP) RE P P RE,, RE RE P RE P RE,5, Exon RE 7 P P P I.C P P I.C Lef- Tf- ATG Figure IF- (IF-α/ART omplex) medites hypoxi-indued Wnt signlling in emryoni ells. () Compred with hypoxi if α +/+ ells (left pnel), if α / ells (middle pnel) show ttenuted TOP-Flsh tivity under hypoxi onditions. Reporter tivity ws inresed in normoxi ells treted with DFX for h (n = 9; right pnel). () TOP- Flsh tivity in if α +/+ nd if α / ells stimulted with Wnt- CM (W) nd ultured under the indited O onditions (n = 9). () TOP-Flsh tivity is ttenuted in hypoxi Arnt / ells, ompred with hypoxi Arnt +/+ ells (n = 9), nd resued in hypoxi Arnt Res ells (Arnt / ells with restored Arnt expression). (d) qrt PCR nlysis of if α / ell extrts for Wnt trget gene expression (n = ). (e) Western lot nlysis of β-tenin, LEF- nd TCF- proteins in whole-ell extrts of null ells (indited y neurons expressing the neuronl mrkers β-tuulin III (β-tuiii) nd douleortin (DCX) (Fig. ; Supplementry Informtion, Fig. S) to TOP-Flsh ssys. The trnsfetion effiieny ws monitored using GFP s reporter (dt not shown). Wheres hypoxi enhned TOP-Flsh tivity in undifferentited nd P9 ells (Fig. ), it did not hve ny effet on the emryoni ell- derived neurons. This oservtion suggests tht hypoxi seletively tivtes Wnt/β-tenin signlling in undifferentited ells. We lso oserved hypoxi indution of Lef nd Tf genes exlusively in undifferentited ells (Fig. ). Furthermore, neuronl differentition oinided with signifint loss of seline Lef /Tf levels ( 5-fold; Fig. ). We suggest tht the Lef nd Tf loi eome epigenetilly modified nd therefore inessile to IF- in differentited ells. the sene of speifi protein) or orresponding wild-type ells ultured either under normoxi or hypoxi onditions. Atin served s the loding ontrol. (f) Immunopreipittion with LEF- ntiody ws performed on nuler extrts of normoxi or hypoxi ells. CREB served s the loding ontrol. (g) ells were ultured under % or.5% O for h nd then ssyed y ChIP. After immunopreipittion with ntiodies ginst IF-α or isotype ontrol, DA extrts were ssessed y qrt PCR. The results of eh genomi region tested (upper pnel) re expressed s fold differene etween IF-α IP nd mouse IgG ontrol (lower pnel). Lef oding region (I.C) served s ontrol. Dt shown re men ± s.d. (n = ; P <.5, P <.5., Student s t-test nd one-wy AOVA ( d, g). Unropped imges of lots re shown in Supplementry Informtion, Fig. S7. It hs een shown tht LEF/TCF overexpression inreses β-tenin nuler trnslotion, nd tivtes trnsription in vrious emryoni ell types 5,. To investigte whether β-tenin umultion in hypoxi ells involved Lef indution, we engineered LEF--overexpressing ells (-Lef ). LEF- overexpression resulted in inresed levels of nuler β-tenin under normoxi onditions reltive to vetor ontrols (Fig. d). Also, -Lef ells showed enhned ell prolifertion, nd were sensitive to DKK- inhiition (Supplementry Informtion, Fig. S). This oservtion repitulted Wnt/β-tenin-dependent hypoxi effets on ell prolifertion (Fig. g). Moreover, we reprodued nuler β-tenin IF-α intertion in hypoxi ells (see Supplementry Informtion, Fig. S), similrly to previous oservtions in differentited neoplsti ells, further highlighting the nture ell iology VOLUME UMBER OCTOBER 9 Mmilln Pulishers Limited. All rights reserved.

4 LETTERS P S B7 + B7 + -tuiii DAPI Merge 5 µm d e..... Cytoplsm P P9 VC Lef- P9P CREB -tenin LEF- Fold hnge in nmra (normlized to s) -tenin 7 5? Lef- P Tf- P 5 µm uleus IF- Lef-/Tf- -tenin LEF-/TCF- Wnt trgets/proesses Figure O regultion of Wnt/β-tenin signlling is differentition-stge speifi. () nd P9 EC ells were treted with (+) or without ( ) B7 neuronl growth nd differentition supplements, nd monitored for the formtion of neuronl progenitors (P) through the expression of neuronl mrker β-tu III. () Compred with hypoxi undifferentited ontrols, nd P9 EC ell-derived Ps demonstrte ttenuted TOP-Flsh reporter tivity under hypoxi onditions (n = ) () Ps derived from ells were ultured under normoxi or hypoxi onditions for h nd ssessed for Lef/Tf gene expression y qrt PCR. Dt shown in nd re men ± s.d. (n = ; involvement of LEF- in differentition-stge-speifi responses of the Wnt/β-tenin pthwy to hypoxi. Tken together, we propose model where IF-α regultes Lef nd Tf in undifferentited ells, leding to inresed nuler β-tenin LEF/TCF intertion, nd tivtion of Wnt/β-tenin trgets (Fig. e). To determine the physiologil relevne of the model desried ove, we next investigted whether deresed O levels orrelte with Wnt/βtenin tivity in vivo. We sreened for res of Wnt/β-tenin tivity in Wnt reporter (BAT GAL) mouse strin, using β-gltosidse immunostining 7. We ompred its distriution with tht of low O regions deteted y immunostining with the hypoxi mrker pimonidzole hydrohloride. Pimonidzole detets O prtil pressures of less thn mm g (~.% O ). Ative Wnt/β-tenin signlling ws deteted in lose proximity to low O regions in oth emryoni nd dult rin (Fig., ; Supplementry Informtion, Fig. S5 d). One suh region positive for Wnt/β-tenin tivity ws the sugrnulr zone of the dentte gyrus (Fig. ; Supplementry Informtion, Fig. S5e g), primry neurogeni nihe in whih dult hippompl progenitors (APs) proliferte nd differentite into dentte grnule neurons 7. Pimonidzole lerly mrked the sugrnulr zone, s identified y its ololiztion with the SC mrker Sox (Fig., d). Importntly, enumertion of lood vessels mrked y lminin immunostining reveled muh higher rtio of nulei to lood vessels within the grnule ell lyer of the dentte gyrus, ompred with other res in the rin, inluding the djent hippompl Oriens lyer, nd the ereellr grnule ell lyer (Fig. e g). Other vsulr mrkers suh s CD nd Indi ink showed similr trend (Supplementry Informtion, Fig. S5h k). This P <.5., Student s t-test). (d) Western lot nlysis of nuler extrts of -Lef nd the orresponding ontrol virus-trnsdued ells (VC) ultured under normoxi onditions. CREB served s the loding ontrol. (e) Shemti representtion of model wherein hypoxi (vi IF-α) indues β-tenin trnsriptionl effetors, Lef nd Tf, resulting in inresed nuler trnslotion of β-tenin (? indites mehnism is unknown), susequent intertion etween β-tenin nd LEF-/TCF- nd tivtion of Wnt trget genes. Sle rs, 5 μm. Unropped imges of lots re shown in Supplementry Informtion, Fig. S7. suggests tht hypoxi within the dentte gyrus, s independently onfirmed y IF-α stiliztion nd the expression of hypoxi responsive genes roni nhydrse IX (CAIX) nd vsulr endothelil growth ftor (VEGF), (Supplementry Informtion, Fig. S5l q), is proly onsequene of O limittion, ttriutle to fewer lood vessels eing present. To further exmine the potentil link etween IF-α nd Wnt/βtenin signlling in vivo, we engineered BAT GAL reporter line in the kground of onditionl glol IF-α mutnts (gif α Δ/Δ, BAT GAL Tg ). Beuse glol if α deletion results in emryoni lethlity 9, we rossed mie homozygous for the loxp-flnked (floxed) if α llele (if α f/f ) with if α f/f mie expressing tmoxifen-induile Cre driven y uiquitin C (UC) promoter, suh tht tmoxifen dministrtion would result in glol postntl if α (gif α Δ/Δ ) deletion (Supplementry Informtion, Fig. S, ). if α deletion redued hippompl Wnt signlling, s shown y loss of BAT GAL reporter tivity in mie expressing the BAT GAL trnsgene (n = ; Fig. h, i; Supplementry Informtion, Fig. S, d). if α deletion lso deresed expression of Wnt trget genes suh s Dkk (undeteted), Lef nd Axin (.5-fold; n = 5) in gif α Δ/Δ nimls (Fig. j). We used the sugrnulr zone s model stem ell nihe, to evlute the role of IF-α in stem ell funtions, euse Wnt/β-tenin signlling hs een identified s positive regultor of neurogenesis in the sugrnulr zone of dult hippompus 7. On the sis of the sugrnulr zone IF-α -Wnt /β-tenin onnetion, we hypothesized tht loss of IF-α my result in errnt neurogenesis in vivo. We nlysed the dentte gyrus of young dult (7 weeks) gif α Δ/Δ mie or ontrol mie nture ell iology VOLUME UMBER OCTOBER Mmilln Pulishers Limited. All rights reserved.

5 LETTERS DG GCL β-gl Sox, DAPI d GCL P e Lminin DAPI Oriens lyer (Or) Oriens lyer (Or) µm Sox, P f µm µm g ulei: lood vessel rtio 5 Or CG DG gif-α f/f gif-α Δ/Δ h i BAT-GAL µm j Fold hnge in nmra (normlized to s) Lef- gif-α f/f gif-α Δ/Δ Axin Dkk- k DCX, DAPI l DCX, DAPI m BrdU, DAPI n DCX, DAPI gif-α f/f gif-α f/f nif-α f/f nif-α f/f gif-α Δ/Δ gif-α Δ/Δ nif-α Δ/Δ µm µm 7 µm µm o p q 5 umer of DCX + ells/field gif-α f/f gif-α Δ/Δ DCX + ells with neurites/field (perentge) gif-α f/f gif-α Δ/Δ umer of BrDU + ells/field 5 5 umer of DCX + ells/field nif-αf/f nif-α Δ/Δ + DMSO nif-α Δ/Δ +SB7 Figure Deletion of if α in vivo suppresses dult hippompl neurogenesis. () Wnt tivity in the dult dentte gyrus (DG) is mrked y β-gltosidse (β-gl) immunostining (red) in BAT GAL reporter mie. Arrowheds indite the sugrnulr zone (SGZ) in the DG. () ypoxi regions within the dult hippompus mrked y pimonidzole stining (P, green) inlude the dult DG. () The sugrnulr zone of the DG is mrked y the stem ell mrker Sox. (d) Cololiztion of pimonidzole with Sox + ells indites hypoxi sugrnulr zone. Arrowheds (, d) indite the orienttion of the sugrnulr zone of the grnule ell lyer (GCL). (e, f) Lminin stining mrks lood vessels in the DG (e), nd in the djent hippompl Oriens lyer (Or) (f). (g) Quntifition of the nulei: lood vessel rtio in the DG, Or nd ereellr grnule lyer (CG). Dt shown re men ± s.e.m. (n = ; 5 setions per niml, P <.5, Student s t-test). (h, i) X-gl stining of the DG of if α f/f, BAT GAL Tg (h) nd gif α Δ/Δ, BAT GAL Tg (i) nimls (n = ; 5 setions per niml). (j) qrt PCR nlysis of hippompl extrts for Wnt trget genes (Lef-, Axin nd Dkk; gif α f/f, n = nd gif α Δ/Δ, n = 5). (k, o) Signifint redution of DCX + ells (rrows) in gif α Δ/Δ sugrnulr zone nd GCL, ompred with ontrol if α f/f nimls (n = 7). (l, p) There were signifintly fewer DCX + ells with neurites in gif α Δ/Δ DG, s ompred with gif α f/f DG. DCX + ells with proesses greter thn μm were ounted s positive for neurites (n = 7). (m, n, q) Lk of neuronl if α in the sugrnulr zone redues SC prolifertion (m, q left pnel), s indited y BrdU + ells (rrows;, nd SC differentition (n, q right pnel), s indited y DCX + ells (rrows) indite DCX + ells with neuritis; rrowheds indite the diretion of sugrnulr zone (n = ). (q) Quntifition of BrdU + nd DCX + ells in nif α Δ/Δ DG treted with SB7 ( mg kg ) every other dy for weeks. Vehile-treted nif α Δ/Δ nd untreted nif α f/f mie served s ontrols. Dt shown re men ± s.e.m. (n = ; P <.5, P <.5., Student s t-test (j, o, p) nd one-wy AOVA (q). Sle rs:, μm; d, μm; f, μm; h, μm; k, μm, l, μm; m, 7 μm; n, μm. lking Cre (gif α f/f ) for ells expressing the erly neuronl mrker douleortin (DCX). gif α Δ/Δ dentte gyrus showed.-fold redution in DCX + ells, ompred with gif α f/f ontrol nimls (n = 7; Fig. k, o). Furthermore, the pity of DCX + neurons in the gif α Δ/Δ mie to form neurites ws mrkedly redued (-fold; n = 7; Fig. l, p), phenotype ritil for integrtion of the neworn neurons into the existing neuronl iruitry. To distinguish etween ell-intrinsi versus ell-extrinsi effets underlying the effet of IF-α on hippompl neurogenesis, we further nlysed dult neurogenesis in neuron-speifi if α deletion model. To seletively delete IF-α from mouse neurons, we rossed if α f/f mie with Rg no.5 (R) mie expressing Cre driven y the lium/lmodulin-dependent kinse (αcmkii) promoter,. Cre expression in the R nimls results in postntl neuronl if α nture ell iology VOLUME UMBER OCTOBER Mmilln Pulishers Limited. All rights reserved.

6 LETTERS Phse DAPI estin DAPI, estin 5 E-SC A-SC GFP 5 µm 5 µm DAPI Sox DAPI, Sox µm d.5.5 de-sc da-sc e 5 A-SC f DAPI β-tuiii DAPI, β-tuiii Fold hnge in nmra (normlized to s) 9 Lef- da-sc VC IF-αTM DAPI DCX DAPI, DCX µm Figure 5 euronl if α regultes neurogenesis through Wnt/β-tenin signlling. () E SCs (E-SC) or dult SCs (A-SC) isolted from the hippompus of mie ged weeks were ultured s neurospheres, nd used for TOP-Flsh ssy under normoxi or hypoxi onditions (n = ). () Trnsfetion effiieny for SC TOP-Flsh ssys ws monitored with GFP in -dy post-dissoition neurospheres (white rrows in phse). () eurospheres were ssyed for progenitor mrkers inluding estin (upper pnel) nd Sox (lower pnel). (d) TOP-Flsh ssy in differentited E (nif α Δ/Δ ) deletion in the forerin, inluding Sox + ells in the sugrnulr zone of the dentte gyrus (Supplementry Informtion, Fig. S5r t). Similrly to glol if α deletion, neuronl if α loss signifintly redued the numer of BrdU + (whih lels proliferting SCs nd progenitors) ell (.-fold) nd DCX + neworn neurons (-fold) in the sugrnulr zone of nif α Δ/Δ mie s ompred with nif α f/f ontrol nimls (n = ; Fig. m, n, q). Doule-immunostining with BrdU nd DCX onfirmed tht DCX + ells were derived from SCs nd APs (Supplementry Informtion, Fig. Se). Moreover, similrly to gif α Δ/Δ DCX + ells, the pity of DCX + ells from nif α Δ/Δ mie to form neurites ws mrkedly redued (-fold; n = ; Supplementry Informtion, Fig. Sf, g). Importntly, the neurogenesis defets were resued phrmologilly in nif α Δ/Δ mie treted with GSK- inhiitor SB 7 ( mg Kg ), ut not in vehile-treted nif α Δ/Δ mie (n = ; Fig. q). We lso mde similr oservtions in nif α Δ/Δ mie trnsdued with lentiviruses expressing onstitutively tive form of β-tenin (Δ-GSK β-tenin) (n = 5 per group; Supplementry Informtion, Fig. Sh, i). These models nnot exlude potentil role for IF-α in regulting trophi signls provided y the post-mitoti mture grnule neurons of the dentte gyrus in the survivl nd integrtion of SCs into hippompl iruitry. evertheless, IF-α lerly emryoni (de-sc), or dult SCs (da-sc) ultured under normoxi or hypoxi onditions (n = ). (e) qrt PCR nlysis of Lef levels in A-SCs or differentited A-SCs (da-sc) trnsfeted with IF-α TM plsmid or orresponding vetor ontrol (VC; n = ). (f) Differentition of neurospheres ws ssessed y the expression of differentition mrkers inluding β-tuiii (upper pnel) nd DCX (lower pnel). Dt shown re men ± s.d.; P <.5, P <.5., Student s t-test (, d, e). Sle rs:, 5 μm;, μm; f, μm. regultes dult neurogenesis y promoting SC/progenitor ell prolifertion nd differentition through Wnt/β-tenin signlling. We lso oserved tht hypoxi regultion of Wnt/β-tenin tivity shown y ells is shred y primry ells of the nervous system. Isolted emryoni nd dult neurl stem ells (E-SC nd A-SC) trnsfeted with TOP-Flsh plsmid showed signifintly inresed reporter tivity under hypoxi onditions (Fig. 5 ), nd this effet ws lost in differentited neurospheres (Fig. 5d, f). Of note, trnsient trnsfetion with normoxi-tive IF-α triple mutnt (IF-α TM[PA/P577A/ 5A]) plsmid inresed Lef levels y out -fold exlusively in the undifferentited neurospheres (Fig. 5e). Moreover, differentited neursopheres showed signifintly redued seline Lef levels s ompred with undifferentited ontrol neurospheres (Fig. 5e), reminisent of redued sl Lef expression oserved in ell-derived neurons (Fig. ). We onlude tht the regultion of Wnt/β-tenin y IF-α through Lef indution represents n importnt mehnism of stem ell homeostsis, nd my e pplile to stem ell miroenvironments eyond the hippompl neurogeni nihe. umerous dult stem ells hve een found losely ssoited with the vsulture 7. Interestingly, we now demonstrte tht low O levels my lso ontriute to stem ell miroenvironments. This suggests tht nture ell iology VOLUME UMBER OCTOBER Mmilln Pulishers Limited. All rights reserved.

7 LETTERS lthough some dult stem ells oupy well-oxygented perivsulr domins, others reside within hypoxi nihes nd re regulted y O deprivtion. Indution of Wnt/β-tenin signlling y IF-α represents moleulr mehnism underlying this regultion. These studies extend the positive effets of IFs previously shown for other stem ell pthwys, inluding Ot- nd oth- (refs, 9). The opposite result ws reported for CT olon ner ells, in whih IF-α inhiited Wnt/β-tenin tivity, suggesting tht the intertion etween IF-α ndwnt/β-tenin in undifferentited stem/ progenitor ells nd more differentited ell types (inluding neoplsti ells) is funtionlly distint. In summry, the present findings onfirm the emerging onept tht IFs not only t in metoli dpttions, ut n lso regulte ritil stem ell phenotypes. METODS Methods nd ny ssoited referenes re ville in the online version of the pper t ote: Supplementry Informtion is ville on the ture Cell Biology wesite. ACKOWLEDGEMETS We thnk K. Kinzler nd B. Vogelstein for the Wnt (TOP/FOP) reporter onstruts; P. Crmeliet for if α / ells; nd E. Brown for U Cre ER T mie. We thnk X. Sun, Cse Western Reserve University, nd. Yu, University of Pennsylvni for tehnil ssistne with tissue hrvesting nd setioning. We thnk B. Keith nd the Simon l for disussion nd ritil review of the mnusript. This work ws supported y funds from the owrd ughes Medil Institute (MI), the Armson Fmily Cner Reserh Institute, nd the tionl Institutes of elth (Grnt o. M5 to P.S.K). M.C.S. is n investigtor of the MI. AUTOR COTRIBUTIOS J.M. nd W.T.O. designed, performed nd nlysed the experiments; P.S.K nd M.C.S ssisted in the interprettion of results; J.C.C. nd J.C.L. provided the αcmkii Cre if α f/f mie; R.S.J. provided the if α f/f mie; J.M. mde the figures nd wrote the pper; M.C.S. edited the pper. COMPETIG FIACIAL ITERTS The uthors delre tht they hve no ompeting finnil interests. Pulished online t nd permissions informtion is ville online t reprintsndpermissions/. Studer, L. et l. Enhned prolifertion, survivl, nd dopminergi differentition of CS preursors in lowered oxygen. J. eurosi., ().. Adelmn, D. M., Gertsenstein, M., gy, A., Simon, M. C. & Mltepe, E. Plentl ell ftes re regulted in vivo y IF-medited hypoxi responses. Genes Dev., 9 ().. Genev, O., Zhou, Y., Ludlow, J. W. & Fisher, S. J. Regultion of humn plentl development y oxygen tension. Siene 77, 9 7 (997).. Adelmn, D. M., Mltepe, E. & Simon, M. C. Multilinege emryoni hemtopoiesis requires hypoxi ART tivity. Genes Dev., 7 (999). 5. Dnet, G.., Pn, Y., Luongo, J. L., Bonnet, D. A. & Simon, M. C. Expnsion of humn SCIDrepopulting ells under hypoxi onditions. J. Clin. Invest., 5 ().. Morrison, S. J. et l. Culture in redued levels of oxygen promotes lonogeni sympthodrenl differentition y isolted neurl rest stem ells. J. eurosi., (). 7. Lie, D. C. et l. Wnt signlling regultes dult hippompl neurogenesis. ture 7, 7 (5).. Rey, T. & Clevers,. Wnt signlling in stem ells nd ner. ture, 5 (5). 9. vn de Wetering, M. et l. Armdillo otivtes trnsription driven y the produt of the Drosophil segment polrity gene dtcf. Cell, (997).. u, C. J. et l. Differentil regultion of the trnsriptionl tivities of hypoxi-induile ftor α (IF-α) nd IF-α in stem ells. Mol. Cell Biol., 5 5 ().. ito, A. T. et l. Phosphtidylinositol -kinse-akt pthwy plys ritil role in erly rdiomyogenesis y regulting nonil Wnt signling. Cir. Res. 97, 5 (5).. Rey, T. et l. A role for Wnt signlling in self-renewl of hemtopoieti stem ells. ture, 9 ().. Kidi, A., Willims, A. C. & Prskev, C. Intertion etween β-tenin nd IF- promotes ellulr dpttion to hypoxi. t. Cell Biol. 9, 7 (7).. Ying, Q. L., Stvridis, M., Griffiths, D., Li, M. & Smith, A. Conversion of emryoni stem ells into neuroetoderml preursors in dherent monoulture. t. Biotehnol., (). 5. uer, O. et l. uler loliztion of β-tenin y intertion with trnsription ftor LEF-. Meh. Dev. 59, (99).. Molenr, M. et l. XTf- trnsription ftor medites β-tenin-indued xis formtion in Xenopus emryos. Cell, 9 99 (99). 7. Mretto, S. et l. Mpping Wnt/β-tenin signling during mouse development nd in oloretl tumors. Pro. tl Ad. Si. USA, 99 ().. Rleigh, J. A. et l. ypoxi nd vsulr endothelil growth ftor expression in humn squmous ell rinoms using pimonidzole s hypoxi mrker. Cner Res. 5, 7 (99). 9. Ryn,. E., Lo, J. & Johnson, R. S. IF- α is required for solid tumor formtion nd emryoni vsulriztion. EMBo J. 7, 5 5 (99).. Drgtsis, I. & Zeitlin, S. CMKIIα Cre trnsgene expression nd reomintion ptterns in the mouse rin. Genesis, 5 ().. Brnov, O. et l. euron-speifi intivtion of the hypoxi induile ftor lph inreses rin injury in mouse model of trnsient fol ererl ishemi. J. eurosi. 7, (7).. Brth, A. I., Stewrt, D. B. & elson, W. J. T ell ftor-tivted trnsription is not suffiient to indue nhorge-independent growth of epithelil ells expressing mutnt β-tenin. Pro. tl Ad. Si. USA 9, (999).. u, C. J., Stur, A., Wng, L., Chen,. & Simon, M. C. The -terminl trnstivtion domin onfers trget gene speifiity of hypoxi-induile ftors IF-α nd IF-α. Mol. Biol. Cell, 5 5 (7).. Kiel, M. J., Yilmz, O.., Iwshit, T., Terhorst, C. & Morrison, S. J. SLAM fmily reeptors distinguish hemtopoieti stem nd progenitor ells nd revel endothelil nihes for stem ells. Cell, 9 (5). 5. Yoshid, S., Sukeno, M. & eshim, Y. A vsulture-ssoited nihe for undifferentited spermtogoni in the mouse testis. Siene 7, 7 7 (7).. Tvzoie, M. et l. A speilized vsulr nihe for dult neurl stem ells. Cell Stem Cell, 79 (). 7. Shen, Q. et l. Adult SVZ stem ells lie in vsulr nihe: quntittive nlysis of nihe ell ell intertions. Cell Stem Cell, 9 ().. Covello, K. L. et l. IF-α regultes Ot-: effets of hypoxi on stem ell funtion, emryoni development, nd tumor growth. Genes Dev., (). 9. Gustfsson, M. V. et l. ypoxi requires noth signling to mintin the undifferentited ell stte. Dev. Cell 9, 7 (5). nture ell iology VOLUME UMBER OCTOBER Mmilln Pulishers Limited. All rights reserved.

8 METODS DOI:./n METODS Cell ulture nd tretments. Arnt /, Arnt Res nd orresponding wild-type (Arnt +/+ ) ells hve een desried previously,. ells were ultured s desried previously. P9 EC ells (ATCC) were ultured s per the mnufturer s protool. ypoxi (.5%,.5% nd % O ) ws hieved using ERAell hypoxi worksttion with O ontrol. Deferoxmine (DFX; Cliohem) ws used s hypoxi-mimeti t finl onentrtion of μm for h. Wnt- onditioned medium (Wnt- CM) nd orresponding ontrol medium were olleted from L Wnt-A nd L ells, respetively (ATCC). LiCl ( mm, finl onentrtion) nd BIO ( nm, finl onentrtion) were purhsed from Sigm. Reporter ssy. Trnsient trnsfetions of ells with TOP-Flsh or FOP-Flsh plsmids were performed with Lipofetmine (Invitrogen) in -well pltes. Luiferse ssys were performed using the dul luiferse protool (Promeg) with the following modifition for sira: ells were first trnsfeted with sira ginst β tenin (O-TARGET plus SMART pool L---5, Dhrmon), nd fter h were o-trnsfeted with firefly ( ng) nd Renill luiferse ( ng) reporter gene expression plsmids. β-tenin knokdown ws verified 7 h fter trnsfetion y stndrd western lot proedures. Apoptosis nd ell prolifertion. For poptosis studies, undifferentited monolyers were grown on geltin-oted overslips, nd ultured under hypoxi onditions for the indited time period. TUEL + ells were visulized using n ApopTg kit (Millipore). TUEL + ells were enumerted s perentge of totl numer of ells in the field deteted y DAPI nuler stining. Three rndomly hosen fields per plte (n = ) were nlysed nd the men for eh ondition lulted. For prolifertion nlysis, ells were seeded on m pltes, nd two pltes per time point were ounted in hemoytometer over dys. Reominnt mouse DKK- (R&D systems) ws used t finl onentrtion of ng ml. For ell-yle nlysis, monolyers were ultured either under normoxi or hypoxi onditions, riefly pulsed with 5ʹ-romo-ʹ ʹ-deoxyuridine (BrdU; μm for min), stined with propidium iodide (PI) nd nlysed y flow ytometry. qrt-pcr nlyses. Totl RA ( μg of eh smple isolted using Resy; Qigen) ws reverse trnsried using high pity DA reverse trnsription kit (ABI) nd ssyed for gene expression y SYBR-GREE tehnology (ABI). The following primer sequenes were used: Lef-F, 5ʹ-TCCTGAAATCCCCACCTTCT-ʹ, Lef-R: 5ʹ-TGGGATAAACAGG CTGA CCT-ʹ; Tf-/7F, 5ʹ-CAGCTCCCCCATAC- TGTGAG-ʹ; Tf-/7R, 5ʹ-TGCTGT CTATATCCGCAGGAA-ʹ; Dkk-F, 5ʹ-ACGAAG AAATC ACA AAGC AGTAAG-ʹ; Dkk-R, 5ʹ-AAAA ATGGC GAG- CAC AGC-ʹ; AxinF, 5ʹ-GAG AGTGAGCGGCAGAGC-ʹ; AxinR, 5ʹ-CGG CTGA C TCG TTCTCCT-ʹ; Fzd7F, 5ʹ-GGGTA TCTCT GTGTA GCC CTGA-ʹ; Fzd7R, 5ʹ-AGA GGCAGGTGGATGTCTGT-ʹ; PgkF, 5ʹ-TACC TGCTG GCTGGA TGG-ʹ; PgkR, 5ʹ-CACAGCCTCGGCATATTTCT-ʹ; IF-α KOF, 5ʹ-GCA CTAGACAAAGTTCACCTGAGA-ʹ, IF-α KOR, 5ʹ CGCTAT- CCACATCAAAGCAA-ʹ. ChIP primers used were s follows: Lef- PF, 5ʹ-TTCCC AGCG CTCA- TCATCA-ʹ; Lef- PR, 5ʹ-CCTTTCGCTTCGGT TT TCCT-ʹ; Lef- PF, 5ʹ-AA AAC- A AAAC CCCAAATCACC-ʹ; Lef- PR, 5ʹ-TCACCGT GCAA AAC- CTCTC-ʹ; Lef- PF, 5ʹ-CGGCG TAGACGCTCTCAG-ʹ; Lef- PR, 5ʹ-CGCTTT CCCACTTAGAAGGAC-ʹ; Tf- PF, 5ʹ-ACACCGAAA CGTT- C TTG AGGC-ʹ; Tf- PR, 5ʹ-TCACCACGACCGATCACTGTT-ʹ; Tf- PF, 5ʹ-GGATGCAA CTTCCCAGACTGAG ʹ; Tf- PR, 5ʹ-GCTT AGAA- CCTGCTGTCCAGGA-ʹ. Immunopreipittion ssy. For hromtin immunopreipittion (ChIP) experiments, the sonited nuler extrts of hypoxi ells were immunopreipitted with nti-if-α monolonl ntiody (ovus Biologils), reverse ross-linked nd nlysed y qrt PCR. For immunopreipittion experiments, 5 μg of hypoxi nuler protein extrts were lered with either μl of nti-if-α polylonl ntiody (ovus Biologils) pre-oupled to protein-gsephrose (Rohe), or 5 μl of nti-lef- polylonl ntiody (Cell Signling) pre-oupled to protein-a sephrose (Rohe). Genertion nd nlysis of mie. Genertion nd nlysis of if α f/f (ref. 9), CmKIIlph Cre, nd U Cre ER T (ref. ) hs een desried previously. BAT GAL reporter mie were otined from the Jkson lortory nd genotyped s desried previously 7. To generte glol if α Δ/Δ knokout mie (gif α Δ/Δ ), if α f/f mie were rossed with if α f/f mie expressing U Cre ER T nd offspring were sreened for the presene of lox P using the genotyping primers P: 5ʹ-GCA GTTAAGAGCACTAGTTG-ʹ, P: 5ʹ-GGAGCTATCTCTCTAGACC-ʹ nd P: 5ʹ-TTGGGGATGAAAACATCTGC-ʹ. Cre medited reomintion etween the loxp sites in the loxp llele produes the loxp llele, whih lks exon nd results in mutnt mra trnsript ontining multiple in frme stop odon downstrem of exon sequenes. Tmoxifen free se (MP Biomedils) ws dministered to nursing mothers t onentrtion of mg kg, s desried previously, to indue reomintion. euronl deletion of if α f/f (nif α Δ/Δ ) ws hieved y rossing mie rrying if α f/f lleles with if α f/f mie expressing Cre reominse under the ontrol of the lium/lmodulin-dependent kinse Cm KIIα promoter (CmKII Cre) s desried previously. All mie were mintined in miro-isoltor ges nd treted in ordne with I nd Amerin Assoition of Lortory Animl Cre Stndrds, nd onsistent with the niml re nd use regultions of the University of Pennsylvni, Phildelphi. LZ nd hypoxyproe detetion. Disseted emryos, or setions from dult orgns were wshed in PBS, fixed for min in % prformldehyde, nd inuted in the 5-romo--hloro--indolyl-β-d-gltosidse (X-Gl) stining solution for.5 h. ypoxi regions in developing mouse emryos nd dults were deteted using pimonidzole hydrohloride detetion kit (Chemion), ording to the mnufturer s protool. Briefly, pimonidzole in wter ws dministered intrperitonelly t dosge of mg kg. To minimize hypoxyproe kground, wshout period of h ws llowed. Animls were killed nd rins were removed following rdi perfusion. Antiodies for immunolotting, immunofluoresene nd quntittion. Western lot nlysis ws performed using the following primry ntiodies: Lef, Tf-, IF-α, CREB, GSK β, pgsk β (Cell signling Tehnologies) nd β tenin (BD Trnsdution Lortories) t : dilution, ART (Cell Signlling Tehnologies) t :5 dilution nd tin (Cell Signlling Tehnologies) t :, dilution for h t C. Seondry ntiodies were used t : dilution for h t room temperture. Stining for immunohemistry ws rried out using the following primry ntiody dilutions: β-tuulin III, estin nd Douleortin (Am) t :,, IF-α (Cymn) t :, Lminin (Sigm) t :5, Cre reominse monolonl (Covne), nd Sox (Chemion nd R&D Systems) t :5 for h t C, BrdU (USBiologils) t :, in PBS, nd β-gltosidse polylonl (Fitzgerld) t : for h t room temperture. ypoxyproe (Chemion) ws used t :5 dilution for h t room temperture. Fluoresent onjugted seondry ntiodies were used t : dilution for h t room temperture. To exmine the effets of if α deletion, young dult (7 weeks) neuronl or glol if α Δ/Δ, or ontrol mie lking Cre (if α f/f ), were injeted with BrdU (5-romo-ʹ-deoxyuridine, mg kg, for four onseutive dys) (Rohe) to lel proliferting APs nd their progeny. Mie were perfused fter the finl BrdU injetion. For resue nlysis of neuronl defets, nimls were injeted with either GSK inhiitor SB7 ( mg kg ), or vehile (DMSO) every lternte dy for 7 doses, s desried previously. For tissue stining, orgns were removed from perfused experimentl nimls nd emedded in Tissue Tek optiml utting temperture (OCT) medium (Fisher), then frozen to C for storge. Frozen setions ( μm) were ut using Mirom M 55 ryostt. Setions were fixed with 5% uffered formlin for min, wshed three times in PBS nd inuted in protein loking gent for min t room temperture. Primry ntiody oktils of BrdU nd DCX diluted in % BSA in PBS, or ording to the mnufturer s protool, were then dded to the tissue setions nd inuted t C overnight. Slides were wshed three times in PBS nd then inuted in seondry ntiody oktil in % BSA in PBS for h. Slides were mounted in Vetshield mounting medium (Vetor Lortories) nd visulized on Lei Leitz 5 mirosope. Imges were quired nd proessed using Photoshop CS (Adoe). For quntifition of BrdU +, DCX + nd β-gltosidse + ells, nd for the enumertion of lminin-positive vessels, 5 7 tissue setions offering n nterior to posterior overge of the dentte gyrus from eh niml were stined. Positive ells were ounted per field (t mgnifitions) nd orrelted per mie. The Mmilln Pulishers Limited. All rights reserved. nture ell iology

9 DOI:./n METODS results were pooled to generte men vlues. For neurite quntifition, DCX + ells with proesses longer thn μm extensions were ounted s positive. eurl stem ell isoltion, ulture, differentition nd trnsfetion. Mouse ventrl mesenephlon E neurospheres were purhsed from STEMCELL Tehnologies. Isolted neurl stem ells nd emryoni neurospheres (STEMCELL Tehnologies) were ultured using the STEMCELL Tehnologies kit (5). Briefly, 5 hippompi disseted from week young dult mie were olleted in dish ontining neuroult tissue olletion solution. The tissue ws hopped into smll piees using rzor, sujeted to rief enzymti dissoition in euroult dissoition solution t 7 C, mehnilly dissoited y gentle pipetting, nd resuspended in euroult resuspension solution. Primry ells were seeded t density of 5 ells in T-5m flsks, nd growth of neursopheres monitored under the mirosope. For trnsfetion, ultures of dy-old neurospheres generted from seeding density of ells ml were diluted :, plted into or -well dish (.5 ml) nd trnsfeted with.5 μg plsmid using Lipofetmine (Invitrogen). Smples were nlysed for mra or reporter tivity h fter trnsfetion. For differentition ssy, neurospheres were seeded on poly-ornithine-oted glss overslips nd ultured in euroult differentition medi (STEMCELL Tehnologies). For immunostining of neurospheres, the floting spheres were first tthed to glss overslips using Cell-Tk ell tissue dhesive (BD) nd then proessed similrly to dherent ells. Genertion of -lef ells. Lef DA (OpenBiosystems: MMM-97) ws suloned into plko. neo (Addgene: Plsmid 5, provided y S. Stewrt, Wshington University, USA) with Age nd EoR. LEF--overexpressing lentivirus ws prepred in EK9T ells using the pmdlg/prre, prsv-rev nd envelope system. ells were trnsdued with virus superntnt nd seleted with. mg ml genetiin. ells trnsdued with empty plko. neo virus (VC) served s ontrol. Genertion of SA-β-tenin lentivirus nd stereotti injetion. The ontrol vetor ws piv-ir-zsgreen (Addgene: Plsmid 9, provided y Z. Wer nd B. Welm, UCSF, USA). ΔGSK β-tenin insert hs een desried efore. The insert ws suloned into piv-ir-zsgreen. igh-titre lentivirus (~ 9 trnsduing units ml, μl) ws injeted ilterlly into the dentte gyrus of week-old mie (AP -, ML ±.5, DV. from Bregm, n = 5 per group) s desried previously. Virl spred ws ssessed y visulizing the presene of Zsgreen-positive ells long the nterior posterior xis of the dentte gyrus (out.. mm). Following weeks of reovery, mie were injeted intrperitonelly with BrdU ( mg kg ) dily for dys. The mie were then perfused with % prformldehyde nd hippompl setions proessed for BrdU nd DCX stining. Sttistil nlyses. Sttistil signifine ws omputed using Student s t-test or one-wy AOVA, nd signifine ws set t P <.5.. Mltepe, E., Shmidt, J. V., Bunoh, D., Brdfield, C. A. & Simon, M. C. Anorml ngiogenesis nd responses to gluose nd oxygen deprivtion in mie lking the protein ART. ture, 7 (997).. Mltepe, E., Keith, B., Arshm, A. M., Brorson, J. R. & Simon, M. C. The role of ART in tumor ngiogenesis nd the neurl response to hypoxi. Biohem. Biophys. Res. Commun. 7, ().. Ruznkin, Y. et l. Deletion of the developmentlly essentil gene ATR in dult mie leds to ge-relted phenotypes nd stem ell loss. Cell Stem Cell, (7).. Gruer, M. et l. Aute postntl ltion of if-α results in nemi. Pro. tl Ad. Si. USA, (7).. Mo, Y. et l. Disrupted in shizophreni regultes neuronl progenitor prolifertion vi modultion of GSKβ/β-tenin signling. Cell, 7 (9). nture ell iology Mmilln Pulishers Limited. All rights reserved.

10 supplementry informtion DOI:./n TOP-Flsh reporter tivity (fold) P9, % O,.5% O FOP-Flsh reporter tivity (fold) ,.5% O P9 pgsk-β GSK-β tin Figure S ypoxi tivtes Wnt/-tenin signlling in emryoni ells., nd P9 EC ells trnsiently trnsfeted with TOP-Flsh reporter plsmid were ultured under different low O levels (.5% nd %) for h (n=)., o signifint enhnement of FOP-Flsh reporter tivity ws oserved in hypoxi ells (.5% O or other low O levels [dt not shown]) over ells ultured under normoxi (n=). Luiferse tivity from prl-sv reporter o-trnsfeted with TOP-Flsh or FOP- Flsh reporter plsmids ws used for normliztion., Western lot nlysis of whole ell extrts of ells ultured under normoxi or hypoxi for phosphorylted GSK-, nd totl GSK-. Atin served s the loding ontrol. = P <.5, = P <.5., Student s t-test. Error rs represent S.D. Mmilln Pulishers Limited. All rights reserved.

11 supplementry informtion Wnt- CM P LiCl P9 d G/S rtio.5.5 h h % TUEL + ell/field (Fold)..... h h Figure S ypoxi inreses Wnt/-tenin tivity in stimulted ells.,, TOP-Flsh reporter tivity in nd P9 EC ells treted with either Wnt- CM or LiCl nd ultured either under % or.5% O for h (n=9). Wheres Wnt- CM nd LiCl stimultes TOP-Flsh tivity, exposure to.5% O further inreses TOP-Flsh tivity in stimulted ells., Cellyle nlyses for ells fter exposure to normoxi or hypoxi (.5% O ) for h or h. Cells were lelled with BrdU, stined with propidium iodide (PI) nd nlysed y flow ytometry (n= independent experiments). Inresed G/S rtio of hypoxi ells indites inresed umultion in G stge, nd delyed S-phse entry. ote G/S phse rtio dereses in ells exposed to hypoxi for h, s ompred to ells ultured under hypoxi for h. d, Deresed ell deth in ells ultured under hypoxi s ompred to normoxi ontrol ells (n= pltes, 9 rndom fields in eh plte) ssessed y TdT medited dutp nik end lelling (TUEL) ssy. Inresed ell survivl likely ounts for ell expnsion under prolonged hypoxi (Fig. g). = P <.5., Student s t-test. Error rs represent S.D. Mmilln Pulishers Limited. All rights reserved.

12 supplementry informtion Fold hnge in mra (normlized to s), Arnt +/+, Arnt +/+, Arnt -/-, Arnt -/-, Arnt Res, Arnt Res Lef- Tf- Dkk- Pgk o. of ells x if-α +/+ 5 5 Dys in ulture o. of ells x if-α -/- Dys in ulture o. of ells x Arnt +/+ 5 5 Dys in ulture o. of ells x Arnt -/- Dys in ulture o. of ells x Arnt Res 5 5 Dys in ulture Figure S IF- nd ART re required for hypoxi tivtion of Wnt/tenin signlling in emryoni ells., qrt-pcr nlysis of Wnt- trget genes in Arnt +/+, Arnt -/- nd Arnt Res ells show diret dependene of Wnt indution on hypoxi ART tivity (n=).,, Cells were plted t density of ells per mm nd ultured under normoxi or.5% O for dys. umers were ssessed y ell ounts in hemoytometer t indited time points. if- +/+ (), Arnt +/+ nd Arnt Res () ells displyed ell numer expnsion under hypoxi. To the ontrry, hypoxi if- -/- () nd Arnt -/- () ells grew t rtes omprle to normoxi ontrol ells. = P <.5, = P <.5., Student s t-test. Error rs represent S.D. Mmilln Pulishers Limited. All rights reserved.

13 supplementry informtion DCX DAPI Merge - B7 + 5µm IP: WB: β-tenin IF-α CREB % Input IF-α o. of ells x VC Lef- VC+Dkk Lef-+Dkk Dys in ulture Figure S ell differentition, IF-/-tenin intertion nd LEF- modultion of ell growth., ells treted with (+) or without (-) B7 neuronl growth nd differentition supplements differentite into neurons s indited y the expression of the neuronl mrker douleortin (DCX) in green. The nulei re stined with DAPI (lue)., Immunopreipittion with IF- ntiody ws performed on nuler extrts of normoxi or hypoxi (.5% for h) ells. CREB served s the loding ontrol., -Lef- ells were plted t density of ells per mm, ultured under normoxi nd ell numers ssessed over dys. Empty virus trnsdued (VC) ells served s ontrol. Both ell lines were lso treted with DKK- ( ng ml - ). = P <.5., Student s t-test. Error rs represent S.D. Mmilln Pulishers Limited. All rights reserved.

14 supplementry informtion e f g β-gl BAT-GAL β-gl egtive ontrol β-gl, Sox DG 5µm 5µm 5µm 5µm d 5µm l Pimonidzole-FITC m egtive ontrol-pbs n Pimonidzole-DAB.5µm.5µm 5µm 5µm 5µm h Indi Ink i DAPI, CD o CAIX p VEGF q IF-α Oriens lyer j Indi Ink 5µm k DAPI, CD 5µm 5µm 5µm r s t Cre, DAPI egtive ontrol Cre, Sox 5µm Dentte gyrus 5µm 5µm 5µm 5µm 5µm Figure S5 ypoxi regions in emryoni nd dult rin.,, Wnt /-tenin tivity mrked y -gltosidse enzyme stining (lue) in E.5 BAT- GAL reporter mie () is losely ssoited with hypoxi regions mrked y pimonidzole stining (rown) in n djent -gltosidse enzyme stined (lue) emryoni setion (). (, d) Mgnifitions of the oxed region in () nd (). Blk line in (d) demrtes highly hypoxi region from the djent light rown re. e-g, -gltosidse (-gl) immunostining in BAT-GAL reporter line (e) nd wildtype ontrol (f) identifies the GCL s tive for Wnt/tenin signlling. g, Co-expression of -gltosidse nd Sox in neurl stem ells in the SGZ. h-k, Indi lk ink nd CD mrk fewer lood vessels in the GCL of the DG (j, k) s ompred to the Oriens lyer of the hippompus (h, i). l-n, Pimonidzole immunofluoresene stining (l) nd enzymti immunodetetion (n) indites the presene of hypoxi pokets in the GCL. The hippompus of PBS injeted nimls served s negtive ontrol (m). o-q, Expression of CAIX (o), VEGF (p) nd stiliztion of nuler IF- (q) within the GCL of the DG. r-t, uler (rrows) nd ytoplsmi distriution of Cre in the GCL of CmKII-Cre R line (r). Cre negtive mie served s negtive ontrol (s). Cololiztion with Sox + ells (rrows) indites the expression of Cre in neurl stem nd progenitor ells in the SGZ (t). In e-t, rrowheds point towrds the SGZ, nd rrows indite the relevnt ells. 5 Mmilln Pulishers Limited. All rights reserved.

15 supplementry informtion Fold hnge in mra (normlized to s). gif-α f/f gif-α /.... if-α gif-α f/f gif-α / Cre if-α loxp if-α loxp X-gl stining d e DCX, BrdU gif-α f/f gif-α / 7 µm % of DG Xgl + ells/field gif-α f/f gif-α / gif-α f/f gif-α / µm f DCX, DAPI nif-α f/f nif-α / µm g % of DCX + ells with neurite/field nif-α f/f nif-α / h nif-α f/f Dentte gyrus IV- GSK β-tenin-ir-zsgreen PBS injeted µm i o. of BrDU + ells/ field nif-α f/f + CV nif-α / + CV nif-α / + GSK--β-tenin # o. of DCX + ells/ field 5 # Figure S In vivo deletion of if- impirs dult hippompl neurogenesis nd Wnt/-tenin signlling., qrt-pcr onfirmtion of if- deletion in the dult hippompl extrts of gif- D/D mie (n=- in eh group)., PCR genotyping inditing Cre medited deletion of if- mrked y the sene of loxp nd nd the presene of Cre nd loxp nds (gif- D/D mie). The reomintion effiieny of Cre is vrile. Wek loxp nd ws deteted in some if- D/D mie (dt not shown)., -gltosidse enzyme (X-gl) stining of the dentte gyrus of if- f/f, BAT-GAL Tg ( upper pnel) nd gif- D/D, BAT-GAL Tg ( lower pnel). d, fold redution in Wnt tivity s ssessed y X-gl stining in the dentte gyrus of dult gif- D/D ompred to gif- f/f ontrol nimls (n=, 5 setions per niml). e, DCX (green) nd BrdU (red) doule positive ells (white rrows) revel post-mitoti neurons in the SGZ nd GCL of gif- f/f (upper) nd gif- D/D mie (lower). f, g, In vivo deletion of neuronl if- impirs dult hippompl neuronl morphology (n=). = P <.5, = P <.5., Student s t-test. Error rs represent S.E.M.. h, Stereotti injetion of D-GSK--tenin lentivirus (IV- DGSK--tenin-IR-Zsgreen) into the dult DG (- weeks) is deteted y green fluoresene, whih is sent in PBS injeted dult DG. i, Quntifition of BrdU + (i left pnel) nd DCX + (i right pnel) ells in nif- D/D DG trnsdued with high titer D-GSK-tenin lentivirus. Following weeks of reovery, mie were injeted with BrdU ( mg Kg - ) i.p dily for dys. Control virus treted nif- D/D nd nif- f/f mie served s ontrols (n=-5 per group). Sttistil signifine (i) ws omputed using one-wy AOVA. nif- D/D nimls trnsdued with high titer D-GSK--tenin lentivirus displyed remrkle inrese in BrdU + nd DCX + ell ounts s ompred to ontrol virus treted nif- D/D nimls, nd is pprohing signifine (# indites P=.). Error rs represent S.E.M. Mmilln Pulishers Limited. All rights reserved.