Sonic Hedgehog promotes proliferation of Notch-dependent monociliated choroid plexus tumour cells

Transcription

1 Soni Hedgehog promotes prolifertion of Noth-dependent monoilited horoid plexus tumour ells Li Li, Ktie B. Grusm,2, Jun Wng 3, Melody P. Lun 4,5, Jsmin Ohli 6, Hrt G. W. Lidov 4, Moni L. Clihio 4, Erling Zeng 7,8, Jeffrey L. Slisury 9,, Roert J. Wehsler-Rey 3, Mri K. Lehtinen 4, Ulrih Shüller 6 nd Hotin Zho,2,,2,3,4 Aerrnt Noth signlling hs een linked to mny ners inluding horoid plexus (CP) tumours, group of rre nd predominntly peditri rin neoplsms. We developed niml models of CP tumours, y induing sustined expression of Noth, tht repitulte properties of humn CP tumours with errnt NOTCH signlling. Whole-trnsriptome nd funtionl nlyses showed tht tumour ell prolifertion is ssoited with Soni Hedgehog (Shh) in the tumour miroenvironment. Unlike CP epithelil ells, whih hve multiple primry ili, tumour ells possess solitry primry ilium s result of Noth-medited suppression of multiilite differentition. A Shh-driven signlling sde in the primry ilium ours in tumour ells ut not in epithelil ells. Linege studies show tht CP tumours rise from monoilited progenitors in the roof plte hrterized y elevted Noth signlling. Anorml SHH signlling nd distint iliogenesis re deteted in humn CP tumours, suggesting the SHH pthwy nd ili differentition s potentil therpeuti venues. Choroid plexus (CP) neoplsms represent rre primry rin tumours found predominntly in hildren. CP ppilloms (CPPs) re more ommon nd enign, wheres CP rinoms (CPCs) re reltively rre nd mlignnt,2. These tumours re elieved to originte from CP epithelium, whih differentites from the roof plte to form the CP, speilized tissue tht produes ererospinl fluid (CSF) in eh ventrile of the rin 3. Surgil resetion remins the primry tretment for CPPs nd is ssoited with exellent prognosis. However, linil outomes for ptients with inompletely reseted tumours, reurrent tumours, metstti spred, or CPCs n e devstting 4,5. NOTCH signlling, tumour protein p53 (TP53) muttions, nd geneti nd epigeneti hnges hve een desried 6 5. Soni hedgehog (Shh) signlling, ruil pthwy in development nd ners, is medited y Pthed (Pth) nd Smoothened (Smo) reeptors in the primry ilium where they orhestrte signlling sde tht tivtes the expression of downstrem trgets, inluding Gli, Myn nd ylin D (Cnd; refs 6,7). By induing sustined Noth expression, we developed mouse models of CP tumours tht losely resemle humn CP tumours with norml NOTCH signlling. We show tht the prolifertion of Noth-indued CP tumours relies on Shh from the tumour miroenvironment through their primry ilium. Aerrnt SHH signlling nd unique ili ptterns found in humn CP tumours my serve s potentil therpeuti trgets. RESULTS Noth pthwy tivtion leds to CP tumours A moleulrly defined oundry exists etween the rhomi lip onsisting of neurl progenitors expressing the trnsription ftor Children s Helth Reserh Center, Snford Reserh, 23 E 6th Street North, Sioux Flls, South Dkot 574, USA. 2 Division of Bsi Biomedil Sienes, Snford Shool of Mediine of the University of South Dkot, 44 E. Clrk Street, Vermillion, South Dkot 5769, USA. 3 Tumor Initition nd Mintenne Progrmme, Snford Burnhm Preys Medil Disovery Institute, 9 North Torrey Pines Rod, L Joll, Cliforni 9237, USA. 4 Deprtment of Pthology, Boston Children s Hospitl, Boston, Msshusetts 25, USA. 5 Deprtment of Pthology nd Lortory Mediine, Boston University Shool of Mediine, Boston, Msshusetts 28, USA. 6 Center for Neuropthology nd Prion Reserh, Ludwig-Mximilns-University, 8377 Munih, Germny. 7 Deprtment of Biology, University of South Dkot, 44 E. Clrk Street, Vermillion, South Dkot 5769, USA. 8 Deprtment of Computer Siene, University of South Dkot, 44 E. Clrk Street, Vermillion, South Dkot 5769, USA. 9 Deprtment of Biohemistry nd Moleulr Biology, Myo Clini (Guggenheim-4), 2 First Street SW., Rohester, Minnesot 5595, USA. Mirosopy nd Cell Anlysis Core, Myo Clini (Guggenheim-4), 2 First Street SW., Rohester, Minnesot 5595, USA. Cner Biology Reserh Center, Snford Reserh, 23 E 6th Street North, Sioux Flls, South Dkot 574, USA. 2 Deprtment of Peditris, Snford Shool of Mediine of the University of South Dkot, 4 W 22nd Street, Sioux Flls, South Dkot 575, USA. 3 Deprtment of Chemistry nd Biohemistry, South Dkot Stte University, Aver Helth Siene Center (SAV) 3, Brookings, South Dkot 577, USA. 4 Correspondene should e ddressed to H.Z. (e-mil: Hotin.Zho@snfordhelth.org) Reeived 9 April 25; epted 9 Ferury 26; pulished online 2 Mrh 26; DOI:.38/n3327 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION

2 Mth enhner X et-gl-min Cre PA R26 STOP NICD IRES/GFP PA R26 Mre;EYFP loxp loxp R26 NICD IRES/GFP PA R26 Mre;NICD; EYFP Lmx Otx2 Aqp Cytokertins e Mre;NICD Mre;NICD GFP d Perentge of EYFP + ells 8 6 NS NS P P7 P4 P2 P9 Mre;NICD Mre;EYFP P4 P f Humn CPP g Reltive gene expression P Hes P7 P4 P2 h Tumour Mre;NICD Lre;NICD Humn Humn CP Reltive gene expression P Hes5 Mre;NICD P7 P4 P2 4th ventrile 4th ventrile Lterl ventrile Figure Constitutive Noth signlling leds to the development of CP tumours. () Shemti illustrtion of the strtegy for Noth signlling tivtion in vivo. Mth-Cre trnsgeni mie hve the Mth enhner frgment together with the β-gloin promoter direting Cre reominse expression in Atoh + progenitors in rhomi lip. NICD expression is loked y the trnsription termintion (STOP) ssette. Cre-medited deletion of the STOP ssette llows trnsription of NICD nd internl riosome entry sequene/green fluoresent protein (IRES/GFP) from the Ros26 (R26) lous. PA, polydenyltion signl. () Brin hemispheres nd CPs from postntl dy 7 (P7) mie re shown. Note tht the enlrged CP from Mth Cre;Ros26 NICD;Ros26 EYFP (Mre;NICD;EYFP) nimls ontins mny EYFP + ells (rrows), wheres EYFP + ells (rrowheds) re sprsely found in the CP from Mth Cre;Ros26 EYFP (Mre;EYFP) nimls. Sle rs, mm. () Anlysis of gene expression in the CP of Mth Cre;Ros26 EYFP mie. EYFP expression (green) lels ells derived from Atoh + progenitors. The expression of Lmx (red), Otx2 (red), ytokertins (red) nd Aqp (red) mrks CP epithelil ells. Sle r, 25 µm. (d) Quntifition of the perentge of EYFP + ells or NICD + /GFP + ells in Otx2 + hindrin CP epithelium of Mre;EYFP mie or Mth Cre;Ros26 NICD (Mre;NICD) mie, respetively, t different postntl dy (P) time points (n =3 tumours from three Mre;NICD nimls per time point; CPs from Mre;EYFP mie: n = 4 (P, P4), n = 6 (P7, P2), n = 5 (P9), dt from single experiment re shown, rw dt re ville in Supplementry Tle 9; men ± s.e.m., two-wy ANOVA, P <.; P <.; NS, not signifint). (e) Hemtoxylin nd eosin (H&E) stining of CPs from Mre;NICD nd wild-type () mie t P nd P4. Note tht CP epithelium exhiits the honil onfigurtion (rrowhed), wheres the enlrged CPs in Mre;NICD mie re flttened on ventriulr surfes (rrow). Vesiulr s with umulted CSF is shown (sterisk). Sle rs, 5 µm (white) nd 25 µm (lk). (f) H&E stining of humn CP ppillom (CPP) nd norml CP. Sle r, 25 µm. (g) qrt PCR nlysis of Hes nd Hes5 expression in CP tumours (lk irles) nd wildtype CPs (white irles) t P, P7, P4 nd P2 (dt from tehnil replites of eh speimen set in single experiment re shown; experiment ws not repeted; rw dt n e found in Supplementry Tle 9). (h) The expression of Ki-67 is shown in CP tumours from Mre;NICD mie, Lmx Cre;Ros26 NICD (Lre;NICD) mie, nd norml CPs from wild-type mie. Dotted lines mrk the oundry of lterl ventriles. The expression of Ki-67 in humn CPP nd norml CP is shown. Sle rs, 5 µm. tonl homologue (Atoh, lso known s Mth) nd the roof plte, hrterized y the expression of Wnt, Gdf7 nd the trnsription ftor Lmx (ref. 8 2). Some Lmx + ells re present in the rhomi lip nd ontriute to the ereellum 2,2. To determine whether rhomi lip progenitors ontriute to the roof plte/cp linege, we used Mth Cre to drive Cre expression in Atoh + progenitors 22 (Fig. ). When rossed with the Ros26 EYFP Cre reporter strin 23, the resulting Mth Cre;Ros26 EYFP mie hve ells expressing enhned yellow fluoresent protein (EYFP) in the CP in ddition to the ereellum (Fig. ). Although these EYFP + ells omprise <.5% of hindrin CP epithelium, they express CP mrkers Lmx, orthodentile homeoox 2 (Otx2), ytokertins nd quporin (Aqp; Fig.,d nd Supplementry Fig. ), inditing tht some Atoh + progenitors ontriute to hindrin roof plte/cp linege. To determine the effets of Noth signlling on CP, Mth Cre;Ros26 EYFP mie were rossed with Ros26 NICD strin tht 2 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION

3 Ki-67 GFP d GFP Lmx Mre;NICD Mre;EYFP P P P4 P7 P Perentge of Ki-67 + tumour ells P P7 P4 P2 Ki-67 p27 Mre;NICD GFP GFP GFP Lmx P7 P7 P9 P7 P7 P2 P7 Otx2 Otx2 Ttr Aqp Aqp Cytokertins Mre:NICD C C C C e Reltive gene expression.5..5 P Ttr P7 P4 P P Aqp Mre;NICD P7 P4 P2 f Aqp β-tin Mre;NICD Otx2 β-tin Mre;NICD Figure 2 Noth-indued CP tumours undergo enhned prolifertion nd exhiit defets in differentition. () The expression of Ki-67 is shown in CP tumours from Mre;NICD mie t P, P7 nd P4; CPs from P wildtype () nd Mre;EYFP mie serve s the ontrol. Ki-67 expression (red) lels proliferting ells, GFP expression (green) mrks NICD + tumour ells, expression of Lmx (green) lels CP epithelium in wild-type mie, nd EYFP expression (green, rrowheds) deteted with GFP ntiody demrtes CP epithelil ells derived from Atoh + progenitors in Mre;EYFP mie. stining (lue) mrks nulei. Sle r, 25 µm. () Quntifition of the perentge of Ki-67 + ells in NICD + /GFP + tumour ells from Mre;NICD mie nd Lmx + wild-type CP epithelil ells t different postntl dy (P) time points (n = 3 CPs from three nimls per genotype per time point, dt from single experiment re shown, rw dt re ville in Supplementry Tle 9; men ± s.e.m., two-wy ANOVA, P <.). () The expression of Ki-67 nd Cdkn (p27 Kip) is shown in tumours from Mre;NICD mie nd wild-type () CPs t P7. Note tht stining for Ki-67 nd p27 is mutully exlusive, nd p27 or Ki-67 expression is sent in wild-type CP epithelium despite positive stining in the ereellum (C). Sle r, 25 µm. (d) The expression of Lmx (red), Otx2 (red), Ttr (red), Aqp (red) nd ytokertins (red) is shown in CP tumours. GFP expression (green) lels NICD + tumour ells. Dotted lines mrk the oundry etween GFP + tumour ells nd epithelium. stining (lue) mrks nulei. Sle r, 25 µm. (e) qrt PCR nlysis of Ttr nd Aqp expression in tumours from Mre;NICD mie (lk irles) nd wild-type () CPs (white irles) t different time points (dt from tehnil replites in single experiment re shown nd ville in Supplementry Tle 9; experiment ws not repeted for Ttr expression; Aqp expression nlysis ws repeted in one independent experiment). (f) Western lot nlysis of Aqp nd Otx2 expression in CP tumours nd wild-type () CPs t P4. β-tin serves s the loding ontrol. Moleulr size mrkers nd representtive unproessed originl sns of lots n e found in Supplementry Fig. 9. onditionlly expresses the intrellulr domin of Noth (NICD) nd green fluoresent protein 24 (GFP, Fig. ). In Mth Cre;Ros26 NICD;Ros26 EYFP nimls, hindrin CP is signifintly enlrged with mny EYFP + ells (Fig. ). Sustined NICD expression in Atoh + linege leds to >5-fold inrese ( 5%) in its ontriution to hindrin CP epithelium t irth tht peks t postntl dy NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 3

4 4 (P4; 8%; Fig. d nd Supplementry Fig. ). Wild-type CP epithelium exhiits n orderly olestone-like pperne with rough, honil onfigurtion on pil surfes, wheres CPs from Mth Cre;Ros26 NICD (Mre;NICD) mie re hrterized y ppillry projetions lined y flttened epithelium with ell rowding, elongtion nd strtifition, reminisent of humn CP ppillom (Fig. e,f nd Supplementry Fig. ). When the Ros26 NICD strin ws rossed with Lmx Cre trnsgeni mie tht express Cre in the roof plte/cp linege 2, norml CP growth with identil hrteristis developed in the lterl ventriles nd hindrin of Lmx Cre;Ros26 NICD (Lre;NICD) nimls (Supplementry Fig. 2). Although these norml CP growths do not invde surrounding regions, CSF umultion nd ventriulr diltion re present in these nimls (Fig. e nd Supplementry Figs nd 2), nd 4 5% of Lre;NICD nimls die from hydroephlus. CP tumours of Mre;NICD mie exhiit inresed expression of Hes nd Hes5, inditing Noth pthwy tivtion (Fig. g nd Supplementry Fig.,d). No Ki-67 expression is deteted in wild-type CP epithelium t P7, wheres undnt Ki-67 + ells re present in CPs from ge-mthed Mre;NICD or Lre;NICD mie, resemling ell prolifertion in humn CP tumours (Fig. h), inditing tht Noth pthwy tivtion uses errnt growth of CP into tumours. Enhned prolifertion in Noth-indued CP tumour Although CP epithelil ells in wild-type nd Mth Cre;Ros26 EYFP mie (EYFP + or EYFP ) remin post-mitoti fter irth, 4% of NICD + /GFP + ells in Mre;NICD mie re Ki This perentge grdully dereses to % fter 3 weeks of ge (Fig. 2, nd Supplementry Fig. 3). EdU (5-ethynyl-2 - deoxyuridine) inorportion ssys lso reveled enhned tumour ell prolifertion in Mre;NICD mie (Supplementry Fig. 4). As tumour ells exit the ell yle, Cnd expression is downregulted, wheres Cdkn (p27 Kip) expression is upregulted (Fig. 2 nd Supplementry Fig. 4,). Cleved spse-3 expression is not deteted (Supplementry Fig. 4d). Together, these results indite tht Nothindued CP tumour undergoes enhned prolifertion trnsiently fter irth. Tumour ells express the CP mrker Lmx, ut not the mesenhyml mrker MfB (ref. 25), nd the expression of Aqp, trnsthyretin (Ttr), ytokertins nd Otx2 is onsistently redued ompred with CP epithelil ells 26 (Fig. 2d f nd Supplementry Figs 3 nd 4e,f), inditing tht sustined Noth signlling interferes with differentition of tumour ells even fter they eome post-mitoti. Anorml Shh signlling in CP tumour ells To identify signls tht drive tumour ell prolifertion, we ompred trnsriptionl profiles of tumours nd wild-type CPs t P (tumour ells re prolifertive) nd P2 (tumour ells re post-mitoti) using RNA-seq. Tumours nd CPs lustered seprtely in prinipl omponent nlysis, inditing distint moleulr profiles (Fig. 3). Tumour ells exhiit gene expression profiles defined y differentil expression of 2,738 (P) nd 4,964 (P2) trnsripts (Supplementry Tle nd Fig. 3). Study of these differentilly expressed trnsripts identified,75 ommon trgets, inluding Hes nd Hes5, Aqp, ytokertins nd Otx2, ll of whih show signifint differentil expression y quntittive PCR with reverse trnsription (qrt PCR) nd immunostining nlyses, vlidting RNA-seq results (Figs g, 2d f nd 3d,e nd Supplementry Figs,d, 3 nd 4e nd Supplementry Tle ). Although tumour ells express higher messenger RNA levels for roof plte mrkers Lmx, Gdf7, Zi3, Zi4 nd Msx2, the expression of mny genes found in CP epithelium is signifintly lower (Fig. 3,e nd Supplementry Tle ). Comprison of tumour expression profiles t P nd P2 unovered 4,9 differentilly expressed trnsripts (Fig. 3 nd Supplementry Tle 2). We resoned tht differentil genes unique to P tumour ells my inlude those involved in prolifertion. To identify these genes, we exluded the,75 ommon differentil trgets etween tumours nd CPs t eh time point to otin,33 differentil trnsripts in tumours t P (Fig. 3). We overlpped these,33 unique trgets with the 4,9 differentil genes etween tumours of P nd P2, further nrrowing it down to 663 genes (Fig. 3 nd Supplementry Tle 3). Interrogting this shortened list of genes using ingenuity pthwy nlysis led us to promising ndidte: Shh signlling, whih is lso identified in nlysis of the lrger dt set (Supplementry Tles nd 3). Tumour ells exhiit inresed expression of Gli, Gli2, Myn nd Cnd ompred with wild-type CP epithelium t P nd P7. After P7, when tumour ells strt to exit the ell yle, the expression of these genes dereses to levels of those in ontrol CPs, suggesting tht deresed prolifertion orreltes with ttenuted Shh signlling (Fig. 3d f,h,i nd Supplementry Fig. 4). In ddition, the expression of p27 nd Cdkn2 is upregulted in nonproliferting tumour ells, wheres Cdkn (p57 Kip2) is expressed t higher levels in mture CP (Fig. 2 nd Supplementry Fig. 4 nd Supplementry Tle ). Among hedgehog lignds, only Shh is undntly expressed in hindrin CP epithelil ells t irth nd delines to undetetle levels fter P4, resulting in lower expression levels in tumours thn in wild-type CPs t P (Fig. 3d,e,g,i). Shh drives CP tumour ell prolifertion To determine the role of Shh signlling in Noth-indued CP tumours, we treted tumour ells with reominnt mino-terminl frgment of Shh (ShhN). Tumour ells formed spheres under serum-free onditions nd ontinued to express Lmx, inditing intt linege hrteristis under these onditions (Fig. 4,). After 96 h, more Ki-67 + ells were deteted in tumour spheres treted with ShhN thn in untreted tumour spheres (Fig. 4). Although epithelil ells formed ggregtes tht remined unresponsive, the size of tumour spheres nd numer of tumour ells were inresed y ShhN, inluding post-mitoti tumours t P2 or lter, wheres the Smo inhiitor ylopmine olished suh effets, inditing tht ShhN stimultes tumour ell prolifertion (Fig. 4,d). To determine whether tumour growth requires Shh, we treted Lre;NICD nd Mre;NICD nimls with the Smo inhiitor vismodegi ( mg kg ) or vehile dily from emryoni dy 7.5 (E7.5) to P7, or from dy E5.5 for 4 dys, respetively 33,34. Vismodegi tretment shrnk hindrin CP tumours, signifintly redued the numer of tumour ells, nd improved the survivl of Lre;NICD mie (Fig. 4e,f). Vismodegi lso deresed tumour ell prolifertion nd suppressed Myn expression in Mre;NICD nimls without ffeting CP development in wildtype littermtes (Fig. 4g i nd Supplementry Fig. 5). Together, these results indite tht Shh drives the growth of Noth-indued CP tumours. 4 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION

5 A 6, 2, 2, 3 2,729 P Mre;NICD P2 Mre;NICD RP mrkers PC3 PC PC2 ( 4 ) ( 4 ),84 C, B CP genes d e f log (P vlue) Medin FPKM vlues (log ) Mre;NICD versus 5 Aqp HesHey Hes5, Shh Myn Cnd Gli Gli log 2 (fold hnge). Lmx Otx2 Reltive gene expression Cnd Hes5 Gli Myn log 2 (fold hnge) P Mre;NICD P P2 Mre;NICD P2 Aqp Gli Gli2 Myn Shh Cnd Hes Hes5 Hey Gli P 6 4 P versus P2 Myn P P7 P4 P2 P P7 P4 P2 Mre;NICD Mre;NICD i Gli2 Shh Shh Myn Gli g h Reltive gene expression β-tin P 3. : Cnd.5..5 P Mre;NICD P P7 P9 Mre;NICD Shh P7 P4 P2 Mre;NICD 3. Mre;NICD Mre;NICD P4 Mre;NICD Figure 3 Noth-indued CP tumours exhiit errnt Shh signlling. () Prinipl omponent (PC) nlysis of CP tumours (lk dots) nd wildtype CPs (, red dots) t P2 (n = 3 speimens per genotype). () Venn digrm of differentil genes (flse disovery rte, FDR <.5) etween tumours nd CPs t P2 (A, n=3 speimens per genotype), t P (B, n=3 speimens per genotype), nd of tumour ells etween P nd P2 (C, n=3 speimens per time point). () Hierrhil lustering of tumours nd CPs (n=3 speimens per genotype per time point) sed on genes expressed in roof plte (RP) nd CP (one-wy ANOVA, FDR <.5, fold hnge is shown). (d) Volno-plot nlysis of gene expression of tumours nd CPs t P (n=3 speimens per genotype), nd of tumours t P nd P2 (n=3 speimens per time point). Differentil trnsripts with sttistil signifine (FDR <.5, log of P vlue, y xis) re shown in red (<two-fold hnge) or green (>2- fold hnge) dots. Non-signifint genes (FDR >.5) re shown s lk or yellow dots (two-fold utoff). Arrows lel selet genes with signifint differentil expression. (e) Medin FKPM (frgments per kilose of exon per million reds mpped) vlues of differentil genes etween tumours nd CPs (n = 3 speimens per genotype per time point, men ± s.e.m., twowy ANOVA, P <.5; P <.; P <.). (f,g) qrt PCR nlysis of the expression of Gli nd Myn (f), nd Shh (g) in tumours (lk irles) nd wild-type () CPs (white irles) t different time points (dt from tehnil replites of eh speimen set in single experiment re shown; experiment ws repeted independently one with similr results; rw dt n e found in Supplementry Tle 9). (h) Western lot nlysis of Cnd expression in tumours nd CPs. β-tin serves s the loding ontrol (n= speimen per genotype per time point, representtive lot imge from one of three independent repeted experiments is shown). Moleulr size mrkers nd representtive unproessed originl sns of lots n e found in Supplementry Fig. 9. (i) In situ hyridiztion nlysis of Shh, Gli nd Myn expression in tumours nd CPs. Shh expression is deteted in epithelium (rrowheds), ut sent in tumour ells (rrows) t P, pttern omplementry to those of Gli nd Myn. Sle r, 25 µm. SHH nd NOTCH signlling in humn CP tumours We exmined NOTCH nd SHH signlling in humn CP tumours. First, we used pulished dt sets nd nlysed tumour trnsriptomes, epigenomes nd genomes. Prinipl omponent nlysis of humn CPPs nd norml CPs reveled distint moleulr profiles for CPPs (ref. 3; Fig. 4j). MetCore enrihment nlysis of differentil NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 5

6 g j n Vehile Vismodegi Reltive gene expression ShhN Control Mre;NICD, GFP Lmx GFP Lmx, H&E ShhN Control 7 k Humn CPC Humn CPP GLI MYCN PTCH HES5 HEY HES ShhN Control ShhN+ylopmine Ki-67 GFP Ki-67 GFP log (P vlue). Development_Hemopoiesis, erythropoietin pthwy 2. Development_Hedgehog signlling 3. Prolifertion_positive regultion ell prolifertion 4. Cytoskeleton_spindle mirotuules 5. Signl trnsdution_notch signlling l A 5, ,58,46,848,87 B d ( 5 ) 2 P Ki-67 GFP C Cell numer P7 h Perentge of Ki-67 + tumour ells P9 4 2 e Control ShhN ShhN+ylopmine P2 NS Perentge of survivl f Cell numer ( 6 ) Vehile Vismodegi i Vismodegi Vehile m (Dys) 8 Vehile Vismodegi log (P vlue) Vismodegi (n = 22) Vehile (n = 9). DNA dmge_hekpoint 2. Cell yle_g2-m 3. Cell yle_mitosis 4. Cell yle_ore 5. Cytoskeleton_regultion of ytoskeleton rerrngement 6. Cytoskeleton_tin filments 7. Cell dhesion_integrinmedited ell-mtrix dhesion 8. Cell yle_g-s 9. Cell yle_s phse. Development_Hedgehog signlling. Cell yle_meiosis 2. Signl trnsdution_ NOTCH signlling Humn Mouse Common Figure 4 Shh drives the prolifertion of Noth-indued CP tumours. (,) Lmx (; red) nd Ki-67 (; red) expression is shown in ultured tumour ells from Mre;NICD mie. GFP (green) mrks tumour ells, wheres stining (lue) lels nulei. Sle rs, µm. () Imges of tumour ells nd wild-type () epithelil ells. Sle r, 5 µm. (d) Quntifition of tumour ells fter 96-h tretments (n=3 speimens per tretment per time point, rw dt re ville in Supplementry Tle 9; men ± s.e.m., two-wy ANOVA, P <.; NS, not signifint). (e) Kpln Meier urve depiting the survivl of Lre;NICD mie treted with vismodegi (n=22 nimls) or vehile (n=9 nimls). (f) Hindrin CPs from dy P7 Lre;NICD mie treted with vismodegi (lower) or vehile (upper). Sle r, mm. Quntifition of tumour ells in treted nimls is shown (n=7 nimls per tretment, rw dt re ville in Supplementry Tle 9; men ± s.e.m., two-tiled unpired t-test, P <.). (g) H&E stining of tumours from treted Mre;NICD mie. Ki-67 expression (red) lels proliferting ells nd GFP (green) mrks tumour ells. stining (lue) lels nulei. Sle r, 25 µm. (h) Anlysis of tumour ell prolifertion in nimls shown in g (n = nimls per tretment; men ± s.e.m., two-tiled unpired t-test, P <.). (i) In situ hyridiztion nlysis of Myn expression in tumour (rrows) or epithelil ells (rrowheds) in nimls shown in g. Sle r, 25 µm. (j) Prinipl omponent nlysis of humn CPPs (red dots, n = 7 tumours from seven individuls) nd norml CPs (lue dots, n = 8 CPs from eight individuls). PC (horizontl): 37%, PC2 (vertil): 9.84%, PC3 (third dimension): 9.27%. (k) MetCore nlysis of differentil genes in humn CPPs. Signifintly enrihed signlling networks inluding the Hedgehog nd Noth signlling pthwys (red) re shown. (l) Venn digrm shows the overlp of differentil genes (FDR <.5) etween humn CPPs nd norml CPs (A), with those etween murine tumours nd wild-type CPs t P2 (B, n = 3 speimens per genotype) nd P (C, n = 3 speimens per genotype). (m) MetCore nlysis of overlpping genes etween mouse nd humn CPPs shown in l. (n) qrt PCR nlysis of gene expression in humn CP tumours (CPP: n=7 tumours from seven individuls; CPC: n = 2 tumours from two individuls). Vlues represent fold hnges reltive to humn CP epithelium. Dt shown re derived from single experiment, ville in Supplementry Tle 9 nd not repeted. 6 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION

7 Arl3 GFP Aqp Mre;NICD Arl3 γ-tuulin At-α-tuulin γ-tuulin GFP Aqp f Hes Hes5 d GFP Aqp Mre;NICD 3. : g Mre;NICD CP CP Tumour Upper RP CP CP Lower RP CP e Reltive gene expression..5 Foxj 2 3. Mids Mre;NICD P P7 P4 P2 P P7 P4 P2 Arl3 γ-tuulin h ARL3B Humn CPP Humn CPC Humn CP i Tissue type Norml horoid plexus (CP) Choroid plexus ppillom (CPP) Choroid plexus rinom (CPC) Cells with single primry ilium (no.) Inidene Cells with multiple primry ili (no.) Inidene Cells with either single or multiple primry ili (no.) Inidene (/6) % (6/6) % (/6) % (/7) 58.82% (3/7) 7.65% (4/7) 23.53% (3/3) % (/3) % (/3) % Figure 5 Noth-indued CP tumours possess solitry primry ilium. () Trnsmission eletron mirogrphs of primry ili in tumour ells (rrow) from Mre;NICD nimls nd wild-type () CP epithelium (rrowheds). Sle r,.5 µm. () The expression of ili mrkers Arl3 (red) nd γ-tuulin (red) in tumour ells is shown. Dotted lines mrk the oundry etween GFP + (green) tumour ells nd Aqp + (yellow) epithelium. stining (lue) lels nulei. Primry ili in epithelil (rrowheds, upper) or tumour ells (rrows, lower) re mgnified in inset pitures. Sle r, µm. () The expression of Arl3 (red), γ-tuulin (red), nd etylted α-tuulin (t-α-tuulin, red) is shown in ultured CP ells. GFP (green) lels tumour ells nd Aqp (yellow) mrks epithelil ells. stining (lue) lels nulei. Sle r, 5 µm. (d) Hierrhil lustering of tumours nd norml CPs t P sed on 47 genes involved in ili differentition (n = 3 speimens per genotype; one-wy ANOVA, FDR <.5, fold hnge is shown). (e) qrt PCR nlysis of the expression of Mids nd Foxj in tumours (lk irles) nd CPs (white irles) t different time points (dt from tehnil replites of eh speimen in single experiment re shown nd ville in Supplementry Tle 9; experiment ws not repeted for Mids expression; Foxj expression nlysis ws repeted in one independent experiment). (f) In situ hyridiztion nlysis of the expression of Hes nd Hes5 is shown in hindrin roof plte (red dotted lines) nd tumour ells (rrows) in Mre;NICD or wild-type nimls t emryoni dy (E) 4.5. Sle r, µm. (g) Primry ili re shown for ells in hindrin roof plte (RP), CP epithelium, nd tumours shown in f. Arl3 (red) nd γ-tuulin (red) expression mrks primry ili nd the sl ody, respetively. stining (lue) lels nulei. Sle r, µm. (h) Representtive imges show ARL3B expression (red) in humn CP tumour ells (rrows) or norml CP epithelium (rrowhed). Primry ili re mgnified in inset pitures. stining (lue) lels nulei. Sle r, µm. (i) Summry of ili pttern in humn CPPs (n=7 tumours from 6 individuls), CPCs (n=3 tumours from 3 individuls), nd norml CPs (n=6 CPs from 6 disese-free individuls). genes in humn CPPs pled NOTCH nd SHH signlling mong signifintly enrihed pthwys (Fig. 4k nd Supplementry Tle 4). Seond, we repeted gene expression profiling of CP tumours in Mre:NICD nimls nd wild-type CPs t P nd P2 using similr mirorry pproh. The differentilly expressed genes in murine CPPs overlpped with those identified in humn CPPs (Fig. 4l). MetCore nlysis of these ommon differentil trnsripts etween murine nd humn CPPs reveled signifint enrihment for genes in oth pthwys, inditing tht murine CPPs exhiit striking resemlne to humn CPPs (Fig. 4m nd Supplementry Tle 5). Third, we exmined dt sets from reently pulished studies of humn CP tumours nd found higher expression levels of roof plte mrkers NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 7

8 in CPPs ompred with CPCs (refs 4,5; Supplementry Fig. 6). MetCore gene expression nlysis showed signifint enrihment for SHH nd NOTCH pthwys in humn CPCs, n oservtion lso supported y nlysis of methyltion nd opy numer vrition dt (Supplementry Fig. 6 d nd Supplementry Tle 6). qrt PCR nlysis of humn CP tumours reveled inresed expression for genes of oth pthwys in CP tumours ompred with norml humn CP epithelil ells (Fig. 4n). In situ hyridiztion demonstrted SHH mrna in oth CPPs nd CPCs (perentge of SHH-positive ells in CPP: ± 3.32, n = speimens exmined from individuls; CPC: ± 5.4, n = ; disese-free ontrol: %; n = 5; of note, signl strength vried mong smples, most likely owing to rin regions smpled, speimen ge nd storge; Supplementry Fig. 6e). Together, these results indite tht humn CP tumours exhiit errnt NOTCH nd SHH signlling, suggesting tht oth pthwys my ply similrly importnt role in humn CP tumorigenesis. Noth-indued CP tumour ells re monoilited In vertertes, Shh-driven signlling ours through the primry ilium 6,7. To understnd the mehnisms y whih tumour ells with sustined Noth signlling respond to Shh, we exmined primry ili in CP tumour nd epithelil ells. Trnsmission eletron mirosopy nd stining for the ili mrkers ADP-riosyltion ftor-like 3 (Arl3; ref. 35), γ-tuulin nd etylted α-tuulin reveled multiple short primry ili in wild-type nd EYFP + epithelil ells in Mth Cre;Ros26 EYFP mie, wheres single, longer primry ilium is present in tumour ells (epithelil ells:.7 ±. µm, n=; tumour ells: 3.78 ±.3 µm, n = 2; two-tiled unpired t-test, P <.; dt from single experiment re shown, rw dt re ville in Supplementry Tle 9; Fig. 5 nd Supplementry Fig. 7,). Gene expression profiling showed tht mny genes involved in iliogenesis re downregulted in tumour ells (Fig. 5d). The expression of forkhed ox J (Foxj) nd multiilite differentition nd DNA synthesis ssoited ell yle protein (Mids), two ruil regultors of the differentition of ells with numerous motile ili 39 4, is onsistently redued in tumour ells (Fig. 5e nd Supplementry Fig. 7), inditing tht Noth signlling suppresses Mids nd Foxj expression, nd loks multiilite differentition of tumour ells. Similr to tumour ells, progenitors in hindrin roof plte exhiit inresed expression of Hes nd Hes5 t dy E4.5, nd possess single primry ilium (Fig. 5f,g nd Supplementry Fig. 7d,e), suggesting tht tive Noth signlling preserves the single primry ilium of the progenitors during development. We hrterized ili pttern in humn CP tumours (7 CPPs nd 3 CPCs). Compred with norml humn CP epithelil ells with multiple ili, most CPPs omprise either monoilited tumour ells lone or mixed popultions of monoilited nd multiilited ells. In ll CPCs exmined, ili oserved in tumour ells were solitry primry ili (Fig. 5h,i nd Supplementry Fig. 7f). Monoilited CP tumour ells re uniquely ple of trnsduing Shh signls To determine whether the distint ili pttern of tumour ells ffets Shh signlling, we hrterized Shh-driven Smo iliry trnslotion. Both CP epithelil nd tumour ells express Lmx when grown with serum; however, only tumour ells n proliferte (Fig. 6, nd Supplementry Fig. 7g). After serum removl, ells were treted with ShhN or SAG, Shh pthwy gonist 42 (Fig. 6). Although suh tretment filed to promote iliry umultion of Smo in epithelil ells, it led to trnslotion of Smo into the solitry primry ilium of tumour ells (ShhN: n=3, 7.78 ± 6.93%, P <.; SAG: n=3, ± 3.3%, P <.; two-wy ANOVA; Fig. 6,d), even though oth ell types express similr levels of Pth nd Smo (Fig. 6e), inditing tht Shh signlling in the primry ilium is preserved in tumour ells, ut lost in epithelil ells despite their multiple primry ili. Indeed, ShhN or SAG restored the perentge of Ki-67 + tumour ells fter serum removl, n effet tht n e reversed y ylopmine, inditing tht tumour ells re uniquely ple of responding to Shh through prolifertion (Fig. 6f h). Noth-indued CP tumour rises from roof plte progenitors The similrity in gene expression etween tumour nd roof plte ells, together with elevted Noth signlling nd solitry primry ilium in the ltter, suggests tht Noth-indued CP tumour is relted to the roof plte. To delinete the developmentl origin of CP tumour, we first nlysed the distriution of Atoh + progenitors in hindrin roof plte using Mth MGFP mie with enhned green fluoresent protein (EGFP) fused to the roxy terminus of Atoh (ref. 43). In ddition to the rhomi lip, Atoh:EGFP + ells re present in the Lmx + /Otx2 + upper roof plte ut lrgely sent from the lower roof plte (Fig. 7 nd Supplementry Fig. 8). Seond, we nlysed tumour formtion in Mre;NICD nimls during development. At dy E2.5, lthough mny NICD + /GFP + ells re loted in the rhomi lip ordering the Lmx + roof plte, some NICD + /GFP + ells re present within CP forming into ppillry strutures (Fig. 7). These prospetive Lmx + tumour ells undergo prolifertion (Ki ), nd remin undifferentited (Aqp ; Fig. 7 nd Supplementry Fig. 8). At dy E4.5, most NICD + /GFP + tumour ells re found in Lmx + upper roof plte nd the rostrl hlf of the CP (Fig. 7). Third, prolifertive progenitors (Lmx + /Ki-67 + /Aqp ) in hindrin roof plte differentite into post-mitoti epithelil ells tht form the CP epithelium (Lmx + /Ki-67 /Aqp + ) during emryogenesis 44,45. The shpe nd size of the roof plte re similr etween wild-type nd Mre;NICD mie; however, tumour ells remin prolifertive nd undifferentited (Ki-67 + /Aqp ) even fter their inorportion into CP epithelium (Fig. 8 nd Supplementry Fig. 8). Together, these results indite tht CP tumours in these nimls rise from roof plte progenitors nd migrte into the CP where they ontinue to undergo Shh-driven prolifertion throughout development (Fig. 8,). DISCUSSION In this study, we exmined Noth signlling in CP tumours, group of rre rin neoplsms most ommonly found in hildren. Consistent with role for NOTCH pthwy in these tumours 8, nlysis of humn CP tumour dt sets reveled signifint enrihment for NOTCH signlling. Using niml models, we showed tht sustined Noth signlling leds to CP tumours tht, similr to humn CP tumours, express CP mrkers nd undergo inresed prolifertion. Cross-speies moleulr nlysis demonstrtes tht these CP tumours losely resemle their humn ounterprt, vlidting our niml models s urte representtions of humn disese. CP tumours re thought to originte from CP epithelium 4,5. Anlysis of Mth Cre;Ros26 EYFP mie indites tht EYFP + 8 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION

9 Dissoited CP tumour ShhN /SAG /PBS.5 dys Signlling nlysis % FBS 3 dys FBS (-) 2 dys FBS (-) 3 dys Prolifertion nlysis Lmx Lmx e Reltive gene expression.5..5 Pth Mre;NICD Smo f Ki-67 GFP Aqp Arl3 Smo GFP Ki-67 Merge Aqp Ki-67 g Ki-67 GFP d Ctrl ShhN SAG SAG ShhN Ctrl Arl3 Smo Merge SAG+ ShhN+ ylopmine SAG ylopmine ShhN Ctrl h Perentge of Ki-67 + tumour ells ShhN ShhN+ylopmine SAG SAG+ylopmine % FBS % FBS Figure 6 Monoilited CP tumour ells re uniquely ple of trnsduing Shh signls. () Dissoited CP tumour ells from Mre;NICD nimls were initilly ultured in the presene of % fetl ovine serum (FBS). Cultured CP tumour ells were then swithed to serum-free onditions for 2 dys followed y tretment with ShhN or SAG for.5 or 3 dys for signlling nd prolifertion nlysis, respetively. () The expression of Lmx (red) nd Ki-67 (red) is shown in CP tumour ells ultured with FBS. GFP (green) lels NICD + CP tumour ells, wheres Aqp mrks CP epithelil ells. stining (lue) lels nulei. Sle r, 25 µm. (,d) After tretment with ShhN, SAG or ontrol vehile (Ctrl), Smo (red) loliztion is shown in CP tumour () or epithelil ells (d). Arl3 expression (green) mrks primry ili, wheres stining (lue) lels nulei. Sle r, µm. (e) qrt PCR nlysis of Pth nd Smo expression in CP tumours from Mre;NICD nimls (lk irles) nd wild-type CPs (, white irles) t P7 (dt from tehnil replites of eh set of speimen in single experiment re shown; experiment ws not repeted; rw dt n e found in Supplementry Tle 9). (f) The expression of Ki-67 (green) in CP tumour ells from Mre;NICD nimls treted with ShhN for 48 h is shown. Arl3 expression (red) lels primry ili; stining (lue) mrks nulei. Note tht Ki-67 expression is present in monoilited CP tumour ell, wheres multiilited CP epithelil ell lks Ki-67 expression. Sle r, µm. (g) The expression of Ki-67 (red) in ultured CP tumour ells from Mre;NICD nimls whih were treted with ShhN, SAG or ontrol vehile (Ctrl). GFP (green) lels NICD + tumour ells, wheres stining (lue) mrks nulei. Sle r, 25 µm. (h) Quntifition of the perentge of Ki-67 + ells in NICD + /GFP + tumour ells fter 72-h tretment s indited (n = 4 speimens per tretment, dt from single experiment re shown, rw dt re ville in Supplementry Tle 9; men ± s.e.m., two-wy ANOVA, P <.; P <.). NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 9

10 Atoh:GFP Upper roof plte Lower roof plte Lmx GFP CP RP E2.5 H&E GFP Lmx GFP Ki-67 GFP Aqp Lmx GFP Upper roof plte Lower roof plte Tumour Figure 7 Noth-indued CP tumours rise from progenitors in the roof plte. () Atoh:EGFP expression in Mth MGFP mie t dy E4.5 is deteted with GFP ntiody nd shown s green signls. Lmx expression (red) in the roof plte (mrked y dotted lines) is shown, stining (lue) lels nulei. Note tht Atoh:EGFP + progenitors (rrows) re present only in the Lmx + upper roof plte. Sle r, 5 µm. () Shemti illustrtion of hindrin roof plte (RP) nd CP t dy E2.5. The solid line mrks the trnsverse plne ross the roof plte shown in the H&E stining imge nd digrm. The red outlined region in the digrm representing the interfe of the rhomi lip nd roof plte is shown in Mre:NICD mie t dy E2.5. GFP (green) mrks Atoh + rhomi lip progenitors nd prospetive tumour ells (rrows) in the roof plte nd CP. The expression of Lmx (red) mrks the roof plte/cp linege. Ki-67 expression (red) lels proliferting ells, wheres Aqp expression (red) mrks differentited epithelil ells. stining (lue) lels nulei. Sle rs, mm (lk) nd 3 µm (white). () Gene expression in the roof plte nd CP in Mre:NICD mie t dy E4.5. Outlined regions of progenitors within the upper (, 2) nd lower (, 2) roof plte s well s tumour ells () in CP re shown t higher mgnifition. GFP expression (green) mrks NICD + tumour ells in the roof plte nd CP. Lmx expression (red) lels the roof plte nd CP linege, wheres stining (lue) mrks nulei. Sle r, 3 µm. epithelil ells derived from Atoh + progenitors in the roof plte exhiit properties similr to the rest of the CP epithelium. Indeed, the identil CP tumours in Lre;NICD nd Mre:NICD mie indite tht oth Atoh + nd Atoh lineges in CP re sensitive to Noth signlling tivtion. However, despite the expression of CP mrkers, expression of mny genes found in mture epithelium fils to e upregulted in tumour ells, suggesting relted ut distint developmentl origin, or lok in differentition, or oth. The presene of Atoh + progenitors nd nsent tumour ells from Mre:NICD mie in the roof plte suggests tht CP tumours rise from progenitors within this region 44,45. In greement, tumour ells nd these progenitors exhiit similr hrteristis: inresed expression of roof plte mrkers, elevted Noth signlling, nd single primry ilium. The development of CP tumours indued y sustined Noth signlling my reflet its inherent role in roof plte/cp morphogenesis : the Noth pthwy suppresses multiilite nd epithelil differentition of progenitors, therey preserving the primry ilium-sed Shh signlling (Fig. 8). Compred with NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION

11 Ki-67 Lmx Aqp Lmx i ii iii iv Mre;NICD i ii iii iv v vi v vi Tumour RP Shh CP RP Shh CP RL Renewl Wild type RL Renewl Mutnt Figure 8 Noth-indued CP tumour ells retin properties of roof plte progenitors. () Anlysis of gene expression nd ell prolifertion in tumour ells nd progenitors within hindrin roof plte in Mre;NICD nd wild-type () nimls, respetively, t dy E2.5. The expression of Lmx (red) mrks the roof plte nd CP linege. Ki-67 expression (green) lels proliferting ells, nd Aqp expression (green) mrks differentited epithelil ells. White dotted lines demrte the domin of progenitors in hindrin roof plte. Outlined regions of Lmx + /Ki /Aqp progenitors (i, iii, v, vi), tumour ells (ii, iv) (rrows), nd Lmx + /Ki-67 /Aqp + differentited epithelil ells (ii, iv) (red dshed lines) re shown t higher mgnifition. Sle rs, µm. () Shemti digrm of intertion etween Noth nd Shh pthwys during the roof plte/cp morphogenesis nd tumour formtion. Progenitors in the roof plte (RP, ornge) next to the rhomi lip (RL) exhiit tive Noth signlling, possess solitry primry ilium, nd proliferte in response to Shh (green dots) sereted from multiilited epithelil ells (grey). The self-renewl will ese s Noth pthwy tivity in progenitor ells is ttenuted to llow for multiilite differentition, therey olishing the response of differentiting progenitors to Shh tht drives their expnsion during development. () Roof plte progenitors with onstitutive Noth pthwy tivity (red) remin monoilited nd undergo errnt prolifertion to eome tumour ells tht retin the ility to respond to Shh signls in the lol environment nd undergo Shh-driven prolifertion. CPCs, humn CPPs express higher levels of roof plte mrkers (Supplementry Fig. 5), onsistent with their developmentl origin. We provide evidene tht CP epithelil ells serete Shh tht drives the prolifertion of distint popultion of Shh-responsive progenitors in the upper roof plte. Consistent with previous reports, we showed Shh expression in hindrin CP epithelium t irth tht grdully disppers within two weeks 5,5. Interestingly, low-level ell prolifertion nd Myn expression is deteted in tumour ells from Mre;NICD nimls t P4, presumly driven y residul or trnsvesiulr Shh from other rin regions, s shown for CP-derived signls regulting distnt ell popultions in the rin Tissuespeifi deletion my delinete the role of Shh from these soures in tumour growth 25,5. Anlysis of pulished dt sets demonstrted enrihment for SHH signlling, suggesting role for deregulted SHH expression in humn CP tumours, inluding CPCs (refs 3 5). Consistently, tumours from Mre;NICD or Lre;NICD mie exhiit tive prolifertion similr to tht oserved in CPCs t P when Shh is roustly expressed. Our results suggest tht Shh pthwy inhiition my represent vile strtegy for trgeted therpy for CP tumours. SHH pthwy inhiitors, linilly pproved for treting other more ommon ner types, my e repurposed for treting ptients with CP tumours exhiiting norml SHH signlling. Indeed, vismodegi tretment interferes with tumour progression in our models, lthough further vlidtion with xenogrft models would e neessry. The tumour eomes quiesent s Shh expression dereses, suggesting tht dditionl hnges re present to support ggressive growth of CP tumours s seen in humn CPCs with errnt NOTCH signlling. Tumour ells proliferte in the presene of serum (Fig. 6 nd Supplementry Fig. 7g), inditing tht serum my ontin ftors ple of driving tumour growth. Amplifitions of TAF2, NFYC nd RAD54L ply n importnt role in CPCs (ref. 5). Their expression is not signifintly hnged in NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION

12 our CP tumours; however, deregultion of these genes my lter the ehviour of Noth-indued CP tumours. TP53 ltertions ply ruil role in humn CPCs (ref. 6,4). CP tumours in our models retin the wild-type Tp53 nd exhiit signs of Tp53 signlling nd DNA dmge response (Supplementry Tles 3 nd 5 nd Fig. 4m), suggesting tht Tp53 loss my filitte mlignnt trnsformtion of Noth-indued CP tumours. Aerrnt NOTCH signlling hs een linked to different ners in humns nd therpeutilly trgeted The single primry ilium on tumour ells with onstitutive Noth signlling proves to e essentil for Shh signlling, wheres multiple primry ili my interfere with ilium-dependent signlling tivities A Noth-regulted signlling sde involving Mids, My nd Foxj plys n importnt role in the differentition of ells with multiple motile ili 39 4, Our results revel tht the intertion etween Noth signlling nd primry ili is ruil for CP tumorigenesis (Fig. 8): sustined Noth signlling preserves the single primry ilium y suppressing Mids nd Foxj expression to lok multiilite differentition, trnsforming progenitors into tumour ells tht undergo Shh-driven prolifertion. Trgeting Nothmedited multiilite differentition my represent rtionl strtegy for CP tumour tretment. Humn CP epithelil ells exhiit honil ontour on the pil side, wheres the surfe of CP tumour ells is more flttened 9,65. Our results suggest tht the ili pttern of tumour ells my medite this distint morphology. Indeed, most humn CP tumours (inluding ll CPCs) exhiit solitry primry ilium. Understnding the intertion etween Noth signlling, iliogenesis nd epithelil differentition in CP tumours is essentil to vlidte the therpeuti potentil of Noth inhiitors. METHODS Methods nd ny ssoited referenes re ville in the online version of the pper. Note: Supplementry Informtion is ville in the online version of the pper ACKNOWLEDGEMENTS We thnk ll memers of the lortory for helpful disussions. We re grteful to K. Millen (Settle Children s Hospitl Reserh Institute, USA), J. Kim (University of Texs Southwestern Medil Center, USA) nd R. Kgeym (Institute for Virus Reserh Kyoto University, Jpn) for providing the Lmx Cre trnsgeni mouse strin, Smo ntiody, nd Hes ntiodies, respetively, nd C. Eerhrt (Johns Hopkins University Shool of Mediine, USA), M. Tylor (The Hospitl for Sik Children, Cnd) nd S. Sntgt (Boston Children s Hospitl, USA) for providing humn CP tumour smples. We wish to knowledge the Ltt Brin Tumour Reserh Centre Tumour nd Tissue Repository, whih is supported y.r..i.n hild nd Megn s Wlk. We re indeted to C. Evns, A. Kelsh nd E. Grndprey for exellent tehnil ssistne. We thnk W.K. Miskimins nd K. Surendrn for helpful suggestions, nd J. To nd D. Mher for ritil reding of the mnusript nd helpful disussions. This projet is supported y: Boston Children s Hospitl IDDRC P3 HD8655, Snford Reserh, nd Institutionl Development Awrds (IDeA) from the Ntionl Institute of Generl Medil Sienes of the Ntionl Institutes of Helth (NIH) under grnt numer 5P2GM3548 (Cner), whih lso supports Cores t Snford Reserh together with NIH grnt P2GM362- A (Peditris). The RNA In Situ Hyridiztion Core fility t Bylor College of Mediine is supported y Shred Instrumenttion grnt from the NIH (SOD667). Additionl support ws provided y the Ntionl Brin Tumor Soiety (R.J.W.-R.). AUTHOR CONTRIBUTIONS L.L. nd H.Z. oneived nd plnned the projet, nd wrote the mnusript. H.G.W.L. nd M.L.C. reviewed dignoses of humn tissue smples. J.W., J.O., R.J.W.-R. nd U.S. nlysed morphologil hrters nd gene expression ptterns of humn tumour smples. M.P.L. nd M.K.L. performed ili nd gene expression nlyses in humn tissue smples. K.B.G. provided ssistne with gene expression nlysis. E.Z. ondued RNA-seq dt proessing nd nlysis. J.L.S. provided tehnil dvie, support nd dt nlysis for eletron mirosopy studies. COMPETING FINANCIAL INTERESTS The uthors delre no ompeting finnil interests. Pulished online t Reprints nd permissions informtion is ville online t Gopl, P., Prker, J. R., Deski, R. & Prker, J. C. Jr Choroid plexus rinom. Arh. Pthol. L. Med. 32, (28). 2. Ogiwr, H., Diptri, A. J. Jr, Alden, T. D., Bowmn, R. M. & Tomit, T. Choroid plexus tumors in peditri ptients. Br. J. Neurosurg. 26, (22). 3. Lun, M. P., Monuki, E. S. & Lehtinen, M. K. Development nd funtions of the horoid plexus-ererospinl fluid system. Nt. Rev. Neurosi. 6, (25). 4. Sun, M. Z. et l. Current mngement of horoid plexus rinoms. Neurosurg. Rev. 37, (24). 5. Sfee, M. et l. Surgil outomes in horoid plexus ppilloms: n institutionl experiene. J. Neuroonol. 3, 7 25 (23). 6. Tori, U. et l. TP53 ltertions determine linil sugroups nd survivl of ptients with horoid plexus tumors. J. Clin. Onol. 28, (2). 7. Nupponen, N. N. et l. Pltelet-derived growth ftor reeptor expression nd mplifition in horoid plexus rinoms. Mod. Pthol. 2, (28). 8. Beshorner, R., Widelih, J., Trutmnn, K., Psrs, T. & Shittenhelm, J. Noth reeptors in humn horoid plexus tumors. Histol. Histopthol. 28, (23). 9. Dng, L. et l. Noth3 signling initites horoid plexus tumor formtion. Onogene 25, (26).. Fouldi, M. et l. Phse I tril of MK-752 in hildren with refrtory CNS mlignnies: peditri rin tumor onsortium study. J. Clin. Onol. 29, (2).. Sfee, M. et l. Choroid plexus ppilloms: dvnes in moleulr iology nd understnding of tumorigenesis. Neuro-onol. 5, (23). 2. Rulnd, V. et l. Choroid plexus rinoms re hrterized y omplex hromosoml ltertions relted to ptient ge nd prognosis. Genes Chromosomes Cner 53, (24). 3. Hsselltt, M. et l. TWIST- is overexpressed in neoplsti horoid plexus epithelil ells nd promotes prolifertion nd invsion. Cner Res. 69, (29). 4. Merino, D. M. et l. Moleulr hrteriztion of horoid plexus tumors revels novel linilly relevnt sugroups. Clin. Cner Res. 2, (25). 5. Tong, Y. et l. Cross-speies genomis identifies TAF2, NFYC, nd RAD54L s horoid plexus rinom onogenes. Cner Cell 27, (25). 6. Brkt, M. T., Humke, E. W. & Sott, M. P. Lerning from Jekyll to ontrol Hyde: Hedgehog signling in development nd ner. Trends Mol. Med. 6, (2). 7. Jing, J. & Hui, C. C. Hedgehog signling in development nd ner. Dev. Cell 5, 8 82 (28). 8. Mhold, R. & Fishell, G. Mth is expressed in temporlly disrete pools of ereellr rhomi-lip neurl progenitors. Neuron 48, 7 24 (25). 9. Wng, V. Y., Rose, M. F. & Zoghi, H. Y. Mth expression redefines the rhomi lip derivtives nd revels novel lineges within the rinstem nd ereellum. Neuron 48, 3 43 (25). 2. Chizhikov, V. V. et l. The roof plte regultes ereellr ell-type speifition nd prolifertion. Development 33, (26). 2. Chizhikov, V. V. et l. Lmx regultes ftes nd lotion of ells originting from the ereellr rhomi lip nd telenephli ortil hem. Pro. Ntl Ad. Si. USA 7, (2). 22. Mtei, V. et l. Smller inner er sensory epitheli in Neurog null mie re relted to erlier hir ell yle exit. Dev. Dynm. 234, (25). 23. Srinivs, S. et l. Cre reporter strins produed y trgeted insertion of EYFP nd ECFP into the ROSA26 lous. BMC Dev. Biol., 4 (2). 24. Murtugh, L. C., Stnger, B. Z., Kwn, K. M. & Melton, D. A. Noth signling ontrols multiple steps of pnreti differentition. Pro. Ntl Ad. Si. USA, (23). 25. Nielsen, C. M. & Dymeki, S. M. Soni hedgehog is required for vsulr outgrowth in the hindrin horoid plexus. Dev. Biol. 34, (2). 26. Johnsson, P. A. et l. The trnsription ftor Otx2 regultes horoid plexus development nd funtion. Development 4, (23). 27. Liu, Y., Helms, A. W. & Johnson, J. E. Distint tivities of Msx nd Msx3 in dorsl neurl tue development. Development 3, 7 28 (24). 28. Elsen, G. E., Choi, L. Y., Millen, K. J., Grinlt, Y. & Prine, V. E. Zi nd Zi4 regulte zerfish roof plte speifition nd hindrin ventrile morphogenesis. Dev. Biol. 34, (28). 29. MMhon, A. R. & Merzdorf, C. S. Expression of the zi, zi2, zi3, nd zi4 genes in erly hik emryos. BMC Res. Notes 3, 67 (2). 3. Mrques, F. et l. Trnsriptome signture of the dult mouse horoid plexus. Fluids Brriers CNS 8, (2). 2 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION

13 3. Bowyer, J. F. et l. Comprison of the glol gene expression of horoid plexus nd meninges nd ssoited vsulture under ontrol onditions nd fter pronouned hyperthermi or mphetmine toxiity. BMC Genomis 4, 47 (23). 32. Jnssen, S. F., Gorgels, T. G., Ten Brink, J. B., Jnsonius, N. M. & Bergen, A. A. Gene expression-sed omprison of the humn seretory neuroepitheli of the rin horoid plexus nd the oulr iliry ody: potentil implitions for gluom. Fluids Brriers CNS, 2 (24). 33. Xie, J., Brtels, C. M., Brton, S. W. & Gu, D. Trgeting hedgehog signling in ner: reserh nd linil developments. OnoTrgets Ther. 6, (23). 34. De Smele, E., Ferretti, E. & Gulino, A. Vismodegi, smll-moleule inhiitor of the hedgehog pthwy for the tretment of dvned ners. Curr. Opin. Investig. Drugs, (2). 35. Cspry, T., Lrkins, C. E. & Anderson, K. V. The grded response to Soni Hedgehog depends on ili rhiteture. Dev. Cell 2, (27). 36. Choksi, S. P., Bu, D., Lu, D., Yu, X. & Roy, S. Systemti disovery of novel iliry genes through funtionl genomis in the zerfish. Development 4, (24). 37. Thoms, J. et l. Trnsriptionl ontrol of genes involved in iliogenesis: first step in mking ili. Biol. Cell. 2, (2). 38. Hoh, R. A., Stowe, T. R., Turk, E. & Sterns, T. Trnsriptionl progrm of ilited epithelil ells revels new ilium nd entrosome omponents nd links to humn disese. PLoS ONE 7, e5266 (22). 39. Stus, J. L., Vldr, E. K., Axelrod, J. D. & Kintner, C. Multiilin promotes entriole ssemly nd iliogenesis during multiilite ell differentition. Nt. Cell Biol. 4, 4 47 (22). 4. Yu, X., Ng, C. P., Hher, H. & Roy, S. Foxj trnsription ftors re mster regultors of the motile iliogeni progrm. Nt. Genet. 4, (28). 4. Gomperts, B. N., Gong-Cooper, X. & Hkett, B. P. Foxj regultes sl ody nhoring to the ytoskeleton of ilited pulmonry epithelil ells. J. Cell Si. 7, (24). 42. Chen, J. K., Tiple, J., Young, K. E., Miti, T. & Behy, P. A. Smll moleule modultion of smoothened tivity. Pro. Ntl Ad. Si. USA 99, (22). 43. Rose, M. F. et l. Mth is essentil for the development of hindrin neurons ritil for perintl rething. Neuron 64, (29). 44. Hunter, N. L. & Dymeki, S. M. Moleulrly nd temporlly seprle lineges form the hindrin roof plte nd ontriute differentilly to the horoid plexus. Development 34, (27). 45. Awtrmni, R., Sorino, P., Rodriguez, C., Mi, J. J. & Dymeki, S. M. Crypti oundries in roof plte nd horoid plexus identified y intersetionl gene tivtion. Nt. Genet. 35, 7 75 (23). 46. Broom, E. R., Gilthorpe, J. D., Butts, T., Cmpo-Pys, F. & Wingte, R. J. The roof plte oundry is i-diretionl orgniser of dorsl neurl tue nd horoid plexus development. Development 39, (22). 47. Imyoshi, I., Shimogori, T., Ohtsuk, T. & Kgeym, R. Hes genes nd neurogenin regulte non-neurl versus neurl fte speifition in the dorsl telenephli midline. Development 35, (28). 48. Bill, B. R. et l. Development nd Noth signling requirements of the zerfish horoid plexus. PLoS ONE 3, e34 (28). 49. Gri-Lee, M., Kondryhyn, I., Fong, S. H., Ye, Z. R. & Korzh, V. In vivo nlysis of horoid plexus morphogenesis in zerfish. PLoS ONE 3, e39 (28). 5. Hung, X. et l. Soni hedgehog signling regultes novel epithelil progenitor domin of the hindrin horoid plexus. Development 36, (29). 5. Lun, M. P. et l. Sptilly heterogeneous horoid plexus trnsriptomes enode positionl identity nd ontriute to regionl CSF prodution. J. Neurosi. 35, (25). 52. Hung, X. et l. Trnsventriulr delivery of Soni hedgehog is essentil to ereellr ventriulr zone development. Pro. Ntl Ad. Si. USA 7, (2). 53. Lehtinen, M. K. et l. The horoid plexus nd ererospinl fluid: emerging roles in development, disese, nd therpy. J. Neurosi. 33, (23). 54. Sptzz, J. et l. Choroid-plexus-derived Otx2 homeoprotein onstrins dult ortil plstiity. Cell Rep. 3, (23). 55. South, A. P., Cho, R. J. & Aster, J. C. The doule-edged sword of Noth signling in ner. Semin. Cell Dev. Biol. 23, (22). 56. Koh, U. & Rdtke, F. Noth signling in solid tumors. Curr. Top. Dev. Biol. 92, (2). 57. Groth, C. & Fortini, M. E. Therpeuti pprohes to modulting Noth signling: urrent hllenges nd future prospets. Semin. Cell Dev. Biol. 23, (22). 58. Mhjou, M. R. & Sterns, T. Supernumerry entrosomes nulete extr ili nd ompromise primry ilium signling. Curr. Biol. 22, (22). 59. Mhjou, M. R. The importne of single primry ilium. Orgnogenesis 9, 6 69 (23). 6. Stsiulewiz, M. et l. A onserved role for Noth signling in priming the ellulr response to Shh through iliry lolistion of the key Shh trnsduer Smo. Development 42, (25). 6. Kong, J. H. et l. Noth tivity modultes the responsiveness of neurl progenitors to soni hedgehog signling. Dev. Cell 33, (25). 62. Mret, B. et l. Control of verterte multiiliogenesis y mir-449 through diret repression of the Delt/Noth pthwy. Nt. Cell Biol. 3, (2). 63. Morimoto, M. et l. Cnonil Noth signling in the developing lung is required for determintion of rteril smooth musle ells nd seletion of Clr versus ilited ell fte. J. Cell Si. 23, (2). 64. Tn, F. E. et l. My promotes entriole mplifition nd lter steps of the multiiliogenesis progrm. Development 4, (23). 65. Imi, M., Toming, J. & Mtsume, M. Choroid plexus ppillom originting from the ererum prenhym. Surg. Neurol. Int. 2, 5 (2). NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 3

14 M E T H O D S DOI:.38/n3327 METHODS Mie. Gt(ROSA)26Sor tm(noth)dm /J (Ros26 NICD) mie, B6.29X Gt(ROSA)26Sor tm(eyfp)cos /J(Ros26 EYFP) mie, B6.29S Atoh tm4.hzo /J (Mth MGFP ) mie, B6.Cg Tg(Atoh-re)Bfri/J (Mth Cre) trnsgeni mie, nd C57BL/6 mie (ll from Jkson Lortory), nd Tg(Lmx re)kjmi (Lmx Cre) trnsgeni mie were mintined y reeding with C57BL/6 mie. Experimentl proedures on nimls housed t Snford Reserh were pproved y Snford Reserh Institutionl Animl Cre nd Use Committee nd performed in ompline with ntionl regultory stndrds. No sttistil method ws used to predetermine smple size in niml experiments. The niml experiments were not rndomized. The investigtors were not linded to group llotion during experiments nd outome ssessment. Experimentl nimls were dministered mg kg vismodegi (LC lortories, V-45) or vehile following two regimens: dily tretment from dy E5.5 to E8.5 (Mre;NICD mie: nimls for eh tretment); or from dy E7.5 to dy P7 (Lre;NICD mie: 29 nimls for vismodegi, 26 nimls for vehile), y gstri gvge of pregnnt or nursing femles. Humn smples. Humn CP speimens were proured with informed onsent from humn sujets following the requirements y institutionl review ords t Snford Reserh, Snford Burnhm Preys Medil Disovery Institute, nd Ludwig-Mximilns-University. All CP speimens from Boston Children s Hospitl were otined under n pproved institutionl review ord protool (Supplementry Tle 7). Norml humn CP epithelil ells (SienCell) were used s the ontrol in gene expression nlysis. All tissues were hndled in ordne with guidelines nd regultions for the reserh use of humn rin tissue set forth y the NIH ( Dignoses of humn CP speimens from Boston Children s Hospitl were reviewed y two neuropthologists (H.G.W.L., S. Sntgt) using stndrd WHO riteri 66. Isoltion nd ulture of primry CP ells. Multiple sets of CP speimens from Mre;NICD nd/or wild-type mie were olleted. To otin suffiient numers of ells, eh set of speimens inluded tissues pooled from multiple nimls of the sme genotype. Gender informtion is not ville for nimls t P nd P7. Both mle nd femle nimls were used t other time points (see Supplementry Tle 9 for informtion on nimls used for eh experiment). Disseted CP speimens were dissoited with foreps under stereosope followed y enzymti digestion t 37 C for 2 min in.7 mg ml of hyluroni id (H356, Sigm- Aldrih),.2 mg ml of kynureni id (Sigm-Aldrih K3375), nd mg ml of trypsin in Hnk s lned slt solution (HBSS, 47-2; Life Tehnologies) supplemented with 2 mm gluose. The trypsin inhiitor ovomuoid (LS385, Worthington Biohemil Corportion) ws dded to stop enzymti digestion nd dissoited CP ells were entrifuged t 2g for 5 min t 4 C. Cell pellets were resuspended in Duleo s modified Egle s medium/nutrient Mixture F-2 Hm s- Liquid Medi (DMEM/F2, SH327; Thermo Fisher Sientifi), nd ultured in DMEM/F2 supplemented with 3 ng ml of EGF (Sigm-Aldrih E427), 3 ng ml of FGF2 (Sigm-Aldrih F29), B27 supplement, 2 mm glutmine, nd U ml peniillin/streptomyin (ll from Life Tehnologies). After tretment under serum-free onditions for 96 h, ells were dissoited mehnilly y pipetting nd quntified. For ulture with serum, medium ws supplemented with % fetl ovine serum. Cytosine β-d-rinofurnoside (Ar-C, 2 µm; Sigm-Aldrih C768) ws dded the following dy to eliminte ontminting firolsts. Cultured ells were treted with ShhN (ref. 67; 2 ng ml ), or SAG (2 nm; 94, Cymn Chemil Compny), with or without ylopmine ( µm; LC lortories C-87). Primry CP tumour or epithelil ells were not listed in the dtse of ommonly misidentified ell lines mintined y ICLAC nd NCBI Biosmple. Anlyses of gene expression, prolifertion nd signl trnsdution were performed in ultured primry CP ells (Figs 4 d nd 6 d,f h nd Supplementry Fig. 7g). Results from these studies onfirmed their identity. Given the short time (<8 dys) during whih CP ells were mintined s primry ultures supplemented with ntiiotis, we did not test for myoplsm ontmintion. Histology, immunohistohemistry, immunofluoresene nd immunoytohemistry. Whole rins were fixed overnight in 4% prformldehyde (PFA) nd proessed for prffin emedding nd setioning. Tissue setions were deprffinized with CitriSolv (Deon Ls), nd then rehydrted with grded ethnol series. For frozen tissues, smples were further equilirted in 2% surose t 4 C for h, emedded in Tissue-Tek Optiml Cutting Temperture (O.C.T.) ompound (Skur Finetek), nd setioned (5 2 µm thikness) on ryostt. Cultured ells were fixed in 4% PFA t room temperture for min nd stored in phosphte-uffered sline (PBS). For primry ili stining, ells were fixed with old methnol for min nd stored in PBS t 4 C. No rndomiztion ws used to determine how smples were lloted to experimentl groups nd proessed. Immunostining ws rried out s desried previously 67. Het-indued epitope retrievl ws performed for prffin-emedded tissue setions in Rodent Deloker (Biore Medil). For immunohistohemistry, endogenous peroxidse tivities were intivted in 3% H 2 O 2 for min t room temperture. Tissue setions were loked with % norml ser in PBS-.% Triton X- for h t room temperture nd inuted with primry ntiodies for h. After wshes with PBS, iotinylted seondry ntiodies were pplied for h, followed y tretment with vidin/iotinylted enzyme omplex nd sustrte/hromogen inution (Vetor lortories). Slides were ounterstined with hemtoxylin. For immunofluoresene, tissue setions or ells were sequentilly proed with primry ntiodies nd fluoresently lelled seondry ntiodies (Jkson Immunoreserh). Primry ntiodies used nd dilution rtios re: mouse monolonl nti-etylted α-tuulin (:5, 246, lone 6-B-, m), mouse monolonl nti-etylted α-tuulin (:5, Sigm- Aldrih T745, lone 6-B-), mouse monolonl nti-arl3 (:5, lone N295B/66, NeuroM), rit nti-arl3 (:5, 77--AP, Proteinteh), mouse monolonl nti-quporin (:,, lone /22, m 9566), rit ntiquporin (:,, AB229, EMD Millipore), rit monolonl nti-leved spse-3 (Asp75; lone 5AE; :8, 9664, Cell Signling Tehnology), mouse monolonl nti-cnd (:, s-45, lone 72-3G, Snt Cruz Biotehnology), mouse monolonl nti-cdkn (p27 Kip; :, 6242, lone 57/Kip/p27, BD Biosienes), rit nti-ytokertins (:, Z622, Dko, Crpinteri), mouse monolonl nti-foxj (:5, , Clone 2A5, ebiosiene), hiken nti-gfp (:,, GFP-, Aves L), mouse monolonl nti-γ-tuulin (Sigm-Aldrih T6557, lone GTU-88, :,), guine pig nti-hes (:5, from R. Kgeym), rit monolonl nti-ki-67 (m 6667, lone SP6, :), got nti- Lmx (Snt Cruz s-54273, :), rit nti-mfb (:2, IHC-35, Bethyl Lortories), rit nti-otx2 (EMD Millipore AB9566, :5), rit nti-smo (ref. 68; :), sheep nti-trnsthyretin (m 95, :5). The investigtor ws linded to group llotion in the following two experiments. First, EYFP + or GFP + ells in 3 Otx2 + ells were ssessed y nlysing three distint tissue regions of eh smple. The perentge of EYFP + or GFP + ells ws otined y verging the numers of EYFP + or GFP + ells per Otx2 + ells of ll smples for eh genotype t eh time point. Seond, in nlysis of ell prolifertion, the numers of Ki-67 + ells in 3 Lmx + or GFP + ells were ssessed from three distint tissue regions for eh niml. The perentge of Ki ells in the totl Lmx + or GFP + ell popultion ws lulted y verging the numers of Ki-67 + ells per Lmx + or GFP + ells of ll smples for eh genotype t eh time point or eh tretment. For prolifertion nlysis of ultured ells, Ki-67 expression ws ssessed y nlysing three distint fields: the perentge of Ki-67 + ells ws lulted y verging the numers of Ki-67 + ells per ells of ll smples for eh tretment. For primry ili stining of CP speimens from mie, the ili pttern ws ssessed y nlysing three distint tissue regions of eh niml. To mesure the length of primry ili, 2 primry ili from tumour ells nd primry ili from epithelil ells were exmined. Smo iliry trnslotion ws ssessed y nlysing three distint fields: the perentge of ells with iliry umultion of Smo ws lulted y verging the numer of ells with Smo trnslotion per ells of ll smples for eh tretment. Informtion on nimls used for eh experiment n e found in Supplementry Tle 9. For stining of primry ili in humn CP tumours nd norml humn CPs, the investigtor ws linded to group llotion. Tissues used inlude: norml CP: 6 disese-free individuls; CPP: 7 tumour speimens from 6 individuls; CPC: 3 tumours from individuls (Supplementry Tle 9). Cili stining ws ssessed y nlysing 5 distint tissue regions. EdU (5-ethynyl-2 -deoxyuridine) inorportion ssy. One hour fter intrperitonel injetion of mg kg EdU (78, Setreh Bioteh), whole rins were olleted from nimls. Tissue setions were rinsed with Tris-uffered sline (TBS) nd then inuted for 2 min with solution onsisting of mm ph 8.5 Tris, mm CuSO 4, mm Cy5 zide (B33, Lumiproe) nd mm sori id. Informtion on nimls used is provided in Supplementry Tle 9. Imge quisition. Whole-mount imges were otined using Nikon SMZ stereomirosope. Light nd fluoresent mirosopi imges were otined y Nikon Elipse 9i mirosope system or Nikon onfol mirosope system A +. Trnsmission eletron mirosopy. CP speimens were olleted t different time points. To ensure olletion of suffiient mount of norml CP speimens, eh norml CP speimen inludes tissues pooled from two wild-type nimls for P7, P4 nd P22. Informtion on nimls used is ville in Supplementry Tle 9. The investigtor ws linded to group llotion. Tissues were fixed in 4% PFA, % glutrldehyde in. M odylte uffer (ph 7.4) t 4 C overnight. After wshing NATURE CELL BIOLOGY

15 DOI:.38/n3327 M E T H O D S with odylte uffer supplemented with % surose, tissues were post-fixed with % osmium tetroxide (OsO 4 ), followed y inution with % urnyl ette in 3% ethnol. Tissue smples were dehydrted, then trnsferred to propylene oxide, nd emedded in Eponte-2 epoxy resin (Ted Pell). Tissue smples were setioned (85 nm thikness) with Lei UC-6 ultrmirotome. Setions were ounterstined with urnyl ette nd led itrte, nd oserved under the JEOL 4 trnsmission eletron mirosope (JEOL). Imges were tken with Gtn UltrSn CCD (hrge-oupled devie) digitl mer (Gtn). Immunolotting. Immunolotting ws rried out s desried previously 67. Multiple sets of CP speimens were olleted t eh time point. To otin suffiient quntity of protein, eh set of speimens my ontin tissues pooled from severl nimls of the sme genotype. Gender informtion is not ville for nimls t P nd P7 (see Supplementry Tle 9 for informtion on nimls used). Tissues were homogenized in protein lysis uffer (Tris/HCl ph 7.4, mm EDTA, 5 mm NCl, % NP4,.25% sodium deoxyholte, protese inhiitor oktils (Reserh Produt Interntionl)). Protein onentrtions were mesured y stndrd BCA ssy (Life Tehnologies). Equl mounts of protein were resuspended in redued smple uffer, seprted y SDS PAGE, nd then trnsferred to polyvinylidene difluoride memrne. After inution with 5% non-ft milk in TBST (TBS,.5% Tween 2) for h, the memrne ws inuted with primry ntiodies for h. The memrne ws wshed nd inuted with horse rdish peroxidse (HRP)-onjugted seondry ntiodies (GE Helthre) for h. After wshes, hemiluminesene detetion ws performed using Western Lightning Plus-ECL (PerkinElmer). β-tin ws used s protein loding ontrol. Primry ntiodies used inlude: mouse monolonl nti-β-tin (Sigm-Aldrih A544, lone AC- 5, :2,), rit nti-quporin (EMD Millipore AB229, :,), mouse monolonl nti-cnd (Snt Cruz s-45, lone 72-3G, :2), rit nti-hes (R. Kgeym, :,), rit nti-otx2 (EMD Millipore AB9566, :5). RNA preprtion, quntittive RT PCR, in situ hyridiztion, mirorry, nd RNA-seq. For murine tissues, three independent sets of tumour speimens from Mre;NICD mie nd three sets of CP speimens from wild-type mie were olleted t eh time point. To ensure tht suffiient mount of RNA n e extrted, eh set of speimens inluded tissues pooled from severl nimls of the sme genotype. Gender informtion ws not ville for nimls t P nd P7 (see Supplementry Tle 9 for informtion on nimls used for eh set of speimens). For humn tissues, seven CPPs from seven individuls, two CPCs from two individuls, nd norml humn CP epithelil ells (SienCell) were used (Supplementry Tle 9). Totl RNA ws extrted using Trizol nd PureLink RNA Mini Kit (Life Tehnologies) ording to the mnufturer s instrutions. For quntittive PCR with reverse trnsription (qrt PCR), totl RNA smples were onverted to DNA using the GoSript Reverse Trnsription System (Promeg). All retions were set up in triplite with ABsolute Blue QPCR Mix (Thermo Fisher Sientifi) nd run on n ABI 7 Sequene Detetion System (Life Tehnologies). Gene-speifi primers nd proes for mouse Aqp, Ttr, Gli, Myn, Shh, Pth, Smo, Hes, Hes5, Foxj, Mids nd ontrol Atin were used (Supplementry Tle 8). The following Tqmn ssys for humn genes were used (Life Tehnologies): GLI (Hs766_m), PTCH (Hs87_m), MYCN (Hs23274_m), HES5 (Hs387463_g), HES (Hs72878_m), HEY (Hs43_m), ACTB (Hs _m). Dt were nlysed using ABI Fst System SDS softwre. Trnsript levels were determined s the numer of trnsripts for eh gene reltive to those of At (mouse) or ACTB (humn). The results for eh set of speimens were otined y verging trnsript levels of tehnil triplites nd used for susequent nlyses. Dt from qrt PCR experiments n e found in Supplementry Tle 9. Exlusion ws pplied only on rre osions when one of the three wells of the triplite ws signifint outlier; however, for eh smple in question, qrt PCRs were repeted in independent experiments, whih vlidted the exlusion. In situ hyridiztion ws performed y RNA In Situ Hyridiztion Core fility t Bylor College of Mediine s desried previously 69. For nlysis of Shh, Myn nd Gli expression, CP speimens from multiple nimls were olleted t P nd P4. For nlysis of Myn expression in Mre;NICD nimls treted with vismodegi or vehile, three tumours from three vismodegi-treted mie, nd five tumours from five vehile-treted mie were exmined t P. For Hes nd Hes5 expression nlysis, two Mre;NICD mie nd one wild-type mouse were olleted t dy E4.5. Gender informtion ws not ville for emryos, P nimls, nd the P8 niml for sense proe ontrols (see Supplementry Tle 9 for informtion on nimls used for eh experiment). For humn tissue smples, SHH (proe no. 695), PPIB (positive ontrol, proe no. 339), dpb (negtive ontrol, proe no. 343) RNA were visulized using RNAsope 2. HD detetion kit-brown (Advned Cell Dignostis) ording to the mnufturer s protool. Humn tissues used inlude: norml CP: 5 disese-free individuls; CPP: tumour speimens from individuls; CPC: tumours from individuls (Supplementry Tles 7 nd 9). SHH expression ws ssessed in five distint tissue regions: perentge of SHH-expressing ells ws lulted y verging the numers of SHH + ells per ells in five distint tissue regions of eh speimen. Mirorry nd RNA-seq studies were performed t the DNA Anlysis Core t Snford Burnhm Preys Medil Disovery Institute. For mirorry experiments, lelled RNA ws prepred from 5 ng RNA using the Illumin RNA Amplifition Kit from Amion (Life Tehnologies). The lelled RNA (,5 ng) ws hyridized overnight t 58 C to the Sentrix MouseWG-6 Expression BedChip (>46, gene trnsripts; Illumin) ording to the mnufturer s instrutions. BedChips were susequently wshed nd developed with fluorolink streptvidin Cy3 (GE Helthre). BedChips were then snned with n Illumin BedArry Reder. tumourpthwy nlysis using the GeneGo MetCore Anlytil Suite (http: //genego.om; GeneGo) ws used to sore nd rnk pthwys enrihed in dt sets y the proportion of pthwy-ssoited genes with signifint expression vlues. For RNA-seq experiments, totl RNA smples were rio-depleted using the Riominus Eukryote System (Life Tehnologies), nd used to generte sequening lirries of roded frgment using the Ion Totl RNA-Seq Kit V2 (Life Tehnologies). Lirries were sequened on the Ion Proton sequener, three lirries per Ion Proton PI Chip, using 2 p sequening regents. Reds were ligned to the mouse genome (mm) using omintion of Topht2, nd Bowtie2 (http: //topht..umd.edu). Differentilly expressed trnsripts were deteted using the Cufflinks Cuffdiff pkge ( nd trnsripts with q vlue <.5 (Cuffdiff) were nlysed y ingenuity pthwy nlysis (Ingenuity Systems). Prinipl omponent nlysis ws performed using R pkge rgl ( rn.r-projet.org/we/pkges/rgl/index.html), three-dimensionl visuliztion system sed on OpenGL. Hierrhil lustering ws performed using Genesis ( Sttistil nlysis nd reproduiility. Sttistil nlyses were performed with GrphPdPrism 6. (GrphPd Softwre). All pooled dt were expressed s the men ± s.e.m. Differenes etween two groups were ompred using n unpired two-tiled t-test. Differenes etween multiple groups were nlysed with one-wy or two-wy ANOVA followed y Tukey s multiple omprisons test. Results were onsidered signifint t P <.5( );P <.( );P <.( );P <.( ). For western lotting, nlyses of the expression levels of Aqp nd Otx2 t P4, nd Hes expression t P nd P7, were repeted in one independent experiment. Anlyses of the expression levels of Hes t P9, nd Cnd expression were repeted in two independent experiments with similr results. RNA-seq nd mirorry studies were performed in single experiments. For in situ hyridiztion, nlyses of Myn expression in vismodegi-treted nimls, nd Myn nd Gli expression t P nd P4, were repeted in one independent experiment with similr results. Anlysis of Shh, Hes nd Hes5 expression ws performed in single experiments (Supplementry Tle 9). SHH expression nlysis in humn tissues ws onduted in one experiment with smples divided into two groups. For qrt PCR nlysis, the expression of Hes, Hes5, Ttr, Pth, Smo nd Mids ws exmined in single experiments, wheres Aqp, Gli, Myn, Shh nd Foxj expression nlysis ws repeted in one independent experiment with similr results. qrt PCR nlysis of humn smples ws performed in single experiment. Anlyses of ell prolifertion (Ki-67 or EdU stining), perentge of EYFP + or GFP + ells in CP epithelium, tumour ell numers, nd Smo trnslotion were performed in single experiments. The mesurement of ili length ws repeted in one independent experiment with similr results. Representtive imges were seleted from two independent experiments. Aession numers. Pulished humn CP tumour dt sets (GSE498, GSE6886) were downloded from GEO dtse for nlysis. Dt sets from our studies were deposited t NCBI. Sequening: BioProjet ID, PRJNA282889; Mirorry: GSE Louis DN, O. H., Wiestler, O. D. & Cvenee, W. K. (eds) WHO Clssifition of Tumours of the Centrl Nervous System (IARC Press, 27). 67. Zho, H., Ayrult, O., Zindy, F., Kim, J. H. & Roussel, M. F. Post-trnsriptionl down-regultion of Atoh/Mth y one morphogeni proteins suppresses medullolstom development. Genes Dev. 22, (28). 68. Kim, J., Lee, J. J., Kim, J., Grdner, D. & Behy, P. A. Arseni ntgonizes the Hedgehog pthwy y preventing iliry umultion nd reduing stility of the Gli2 trnsriptionl effetor. Pro. Ntl Ad. Si. USA 7, (2). 69. Yyloglu, M. B. et l. Comprehensive expression tls of firolst growth ftors nd their reeptors generted y novel rooti in situ hyridiztion pltform. Dev. Dyn. 234, (25). NATURE CELL BIOLOGY

16 SUPPLEMENTARY INFORMATION DOI:.38/n3327 P P7 P4 P2 P9 Mre;NICD Mre;EYFP GFP Otx2 Mre;NICD Mre;NICD P P7 P4 P2 GFP d Hes Mre;NICD Mre;NICD Mre;NICD β-tin P P7 P9 Hes Hes GFP Supplementry Figure Morphologil nd gene expression nlysis of CPs in Mre;NICD mie. () EYFP expression (green, rrowheds) deteted with GFP ntiody demrtes CP epithelil ells derived from Atoh + progenitors in Mre;EYFP mie, while GFP expression (green) lels NICD + tumour ells in Mre;NICD mie t P, P7, P4, P2, nd P9. The expression of Otx2 (red) mrks CP epithelil ells. stining (lue) lels nulei. Sle r: 25 µm. () H&E stining of CPs from Mre;NICD mie nd wild type () littermtes t P, P7, P4, nd P2. Boxed regions of the norml growth of CP in the enlrged 4 th ventrile of Mre;NICD nimls re shown in higher mgnifition. Notie tht wild type CP epithelium exhiits the honil onfigurtion (rrowheds), while norml CP growth in Mre;NICD mie displys flttened pperne on ventriulr surfes (rrow). Sle rs: white, 25 µm; lk: 4 µm. () The expression of Hes (red) in CPs from Mre;NICD mie. GFP expression (green) lels NICD + CP ells. Dotted lines mrk the oundry etween CP epithelil ells nd NICD + ells. stining (lue) lels nulei. Sle r: 25 µm. (d) Western lot nlysis of Hes expression in CP tumours from Mre;NICD mie nd wild type () CPs t P, P7 nd P9. β-tin serves s loding ontrol. Moleulr size mrker nd representtive imge of unproessed lots n e found in Supplementry Figure 9.

17 SUPPLEMENTARY INFORMATION 4th ventrile lterl ventrile Lre;NICD * * * P4 P7 4th ventrile lterl ventrile Lre;NICD Lre;NICD P3 * * * Supplementry Figure 2 Morphologil nlysis of CPs in Lre;NICD mie. () Bright field imges of rin hemispheres in Lre;NICD mie nd wild type () littermtes t P4. Notie tht Lre;NICD mie develop norml growth of CP (dotted red lines) nd disply enlrged lterl ventrile (rrows). Vesiulr ss with umulted ererospinl fluid re shown (sterisks). Sle r: mm. () H&E stining of CPs in hindrin nd lterl ventriles of Lre;NICD nimls nd wild type littermtes t P7, P4, nd P3. Notie tht ompred to CPs in wild type () littermtes (rrowheds), Lre;NICD mie develop norml CP growth (rrows) nd exhiit enlrged 4 th nd lterl ventriles. Vesiulr ss with umulted ererospinl fluid re shown (sterisks). Sle rs: white, µm; lk: 3 µm. 2

18 SUPPLEMENTARY INFORMATION 4th ventrile lterl ventrile P P7 P P7 P4 P9 P4 P9 4th ventrile lterl ventrile Lre;NICD Lre;NICD Otx2 Aqp Cytokertins Lmx i j k l Supplementry Figure 3 Anlysis of prolifertion nd gene expression in CP tumours from Lre;NICD mie. () The expression of Ki-67 in tumour ells in the hindrin nd lterl ventriles of Lre;NICD nimls t P, P7, P4, nd P9, respetively. Dotted lines mrk the oundry of tumours in lterl ventriles. Notie tht the expression of Ki-67 in tumour ells is grdully redued over time. Sle r: 25 µm. () The expression of Lmx, Otx2, Aqp, nd ytokertins in CP tumours in the hindrin nd lterl ventriles of Lre;NICD mie t P7. CPs t these sites from P7 wild type () littermtes serve s ontrol. Dotted lines mrk the oundry of the 4 th nd lterl ventriles. Notie tht tumour ells disply redued expression of Aqp nd ytokertins. Sle r: 25 µm. 3

19 SUPPLEMENTARY INFORMATION EdU GFP Merge d Cleved Cspse-3 P Mre;NICD P7 P4 e Merge Aqp Cnd Merge Lmx P Otx2 P4 Aqp EdU p27 Merge f Mre;NICD GFP C C C C MfB Merge Supplementry Figure 4 Anlysis of CP tumours from Mre;NICD mie nd CPs of wild type () mie. () Results of 5-ethynyl-2'-deoxyuridine (EdU) inorportion ssys re shown for tumours t P, P7, nd P4, respetively. EdU stining (mgent) lels ells in S-phse of the ell yle. GFP expression (green) lels NICD + tumour ells. Dotted lines mrk the oundry etween GFP + tumour ells nd CP epithelium. stining (lue) lels nulei. Sle r: 25 µm. () The expression of ylin D (Cnd) in tumours from Mre;NICD mie t P nd P4, respetively. Aqp expression (green) lels CP epithelium, while Cnd expression (red) lels proliferting ells. Dotted lines mrk the oundry etween prolifertive tumour ells nd Aqp + epithelil ells. stining (lue) lels nulei. Sle r: 25 µm. () The presene of EdU-inorporting ells (mgent) nd Cdkn + (p27 Kip, green) ells in tumours nd t P7. Though tumours ontin undnt ells positive for EdU or p27 expression, stining for EdU or p27 is mutully exlusive. Notie tht p27 + or EdU-inorporting ells re sent in wild type CPs despite positive stining in neighoring ereellum (C). Sle r: 25 µm. (d) The expression of Cleved Cspse-3 in CP tumours nd CPs t P7. Cleved Cspse-3 expression (red) is undetetle in tumour nd CPs. stining (lue) mrks nulei. Sle r: 25 µm. (e) Gene expression in CP epithelium t P7. The expression of Lmx (red), Otx2 (red), nd Aqp (red) lels CP epithelil ells. stining (lue) mrks nulei. Sle r: 25 µm. (f) The expression of mesenhyme mrker MfB in CPs from Mre;NICD nimls t P7. GFP (green) lels NICD + tumour ells. Dotted lines mrk the oundry etween GFP + tumour ells nd CP epithelium. MfB expression (red) is deteted in norml CP tissue, ut sent from GFP + tumour ells. stining (lue) mrks nulei. Sle r: 25 µm. 4

20 SUPPLEMENTARY INFORMATION Vismodegi Vehile Lmx Ki-67 H&E C C C C C C C C merge C C Supplementry Figure 5 Morphologil nd gene expression nlysis of CPs from treted wild type nimls. H&E stining of CPs from wild type mie treted with either vismodegi or vehile from dy E5.5 for 4 dys. The expression of Ki-67 (red) lels proliferting ells. Lmx expression (green) mrks CP epithelil ells. stining (lue) lels nulei. Notie tht Ki-67 + prolifertive ells re present in neighoring ereellum (C); however, there is no Ki-67 + ells in CP epithelium. Sle r: 25 µm. 5

21 SUPPLEMENTARY INFORMATION CPC CPP reltive -2 2 WNT28 ZIC ZIC4 FZD3 WNT9A ZIC3 ZIC2 LMXA LMXB GDF Networks -log (p vlue). Cytoskeleton_Regultion of ytoskeleton rerrngement 2. Development_Blood vessel morphogenesis 3. Development_Neurogenesis _Axonl guidne 4. Cell dhesion_attrtive nd repulsive reeptors 5. Development_Neurogenesis_ Synptogenesis 6. Cell dhesion_cdherins 7. Signl trnsdution_esr- nuler pthwy 8. Cytoskeleton_Atin filments 9. Signl trnsdution_wnt signling. Development_Hedgehog signling d log (p vlue). Reprodution_Feeding nd Neurohormone signling 2. Signl trnsdution_neuropeptide signling pthwys 3. Cytoskeleton_Regultion of ytoskeleton rerrngement 4. Reprodution_Progesterone signling 5. Cytoskeleton_Atin filments 6. Cell dhesion_pltelet ggregtion 7. Reprodution_FSH-et signling pthwy 8. Development_Neurogenesis_ Axonl guidne 9. Development_Ossifition nd one remodeling e norml CP Networks. Development_Hedgehog signling CPP CPC Supplementry Figure 6 Trnsriptome, epigenome, nd genome nlysis of humn CP tumours. () Hierrhil lustering of humn CPPs nd CPCs sed on the expression of roof plte genes (CPP: n=24 tumours from 24 individuls; CPC: n=5 tumours from 5 individuls; one-wy ANOVA, FDR <.5, fold hnge is shown). () MetCore gene enrihment nlysis of differentilly methylted genes etween humn CPPs nd CPCs (CPP: n=5 tumours from 5 individuls; CPC: n=5 tumours from 5 individuls). () Shemti illustrtion of results from MetCore network nlysis of differentilly methylted genes etween humn CPPs nd CPCs shown in (). The red olored nodes indite reltively higher expression nd green nodes indite reltively lower expression in CPPs ompred to CPCs. (d) MetCore gene enrihment nlysis of opy numer vritions in humn CP tumours (CPP: n=37 tumours from 37 individuls; CPC: n=38 tumours from 38 individuls). (e) Representtive imges show in-situ hyridiztion nlysis of SHH expression in norml humn CP (n=5 CPs from 5 disesefree individuls) nd humn CP tumours (CPP: n= tumour speimens from individuls; CPC: n= tumours from individuls). Notie tht SHH expression is present in norml hindrin CP epithelium (rrowheds) nd tumour tissue (rrows). Signl intensity vried mong smples, likely s result of rin regions exmined, onditions under whih speimens were olleted nd stored, nd the length of time of speimens in storge. Representtive imges re shown. Sle r: 5 µm. 6

22 SUPPLEMENTARY INFORMATION Mre;NICD Arl3 γ-tuulin GFP Aqp Foxj Aqp d e upper roof plte lower roof plte CP E4.5 H&E Arl3 CB CP LRL LRP URP URL γ-tuulin f Humn CP Humn CPP Humn CPC t-α-tuulin Aqp g Lmx Lmx GFP Aqp GFP Aqp GFP Ki67 GFP Ki67 Aqp Ki67 Aqp Ki67 Supplementry Figure 7 Anlysis of primry ili nd gene expression. () Trnsmission eletron mirogrph of primry ili on CP tumour ells (rrow) or CP epithelium (rrowheds). Sle r:.5 µm. () The expression of Arl3 (red) nd γ-tuulin (red) in EYFP + CP epithelil ells of Mre;EYFP mie. EYFP (green) deteted with GFP ntiody demrtes epithelil ells derived from Atoh + progenitors in Mre;EYFP mie, while Aqp (yellow) mrks differentited epithelil ells. stining (lue) lels nulei. Inset pitures show high mgnifition imges of EYFP + (rrows, upper) or EYFP - epithelil ells (rrowheds, lower). Sle r: µm. () The expression of Foxj (red) in CP tumour ells from Mre;NICD mie t P4. Dotted lines mrk oundry etween tumour ells nd Aqp + (green) CP epithelium. stining (lue) lels nulei. Notie tht Foxj expression is redued in tumour ells ompred to epithelil ells. Sle r: 25 µm. (d) Shemti illustrtion of hindrin roof plte nd CP in mouse emryo t dy E4.5. Solid line mrks the trnsverse plne ross the ereellum, roof plte, nd CP. H&E stining nd the digrm show strutures in this plne. LRL: lower rhomi lip; URL: upper rhomi lip; LRP: lower roof plte; URP: upper roof plte; CB: ereellum. Sle r: mm. (e) Cili pttern in ells within the roof plte nd CP epithelium of wild type nimls is shown t dy E4.5. Arl3 (red) nd γ-tuulin expression (red) mrks primry ili nd sl odies, respetively. stining (lue) lels nulei. Sle r: µm. (f) Representtive imges show the expression of etylted α-tuulin (t-α-tuulin, red) nd AQP (red) in norml humn CP nd humn CP tumours. solitry ilium (rrows) or multiple ili (rrowheds) re mgnified in inset pitures. Hoehst stining (lue) lels nulei. Sle r: µm. (g) The expression Lmx (red) nd Ki-67 (red) in CP ells from Mre;NICD mie ultured in the presene of serum. GFP (green) lels NICD + tumour ells, while Aqp mrks epithelil ells. stining (lue) lels nulei. Sle r: 25 µm. 7

23 SUPPLEMENTARY INFORMATION lower roof plte upper roof plte lower roof plte Atoh:GFP upper roof plte Otx2 GFP GFP Ki-67 GFP Aqp Ki-67 Lmx Aqp Lmx Mre;NICD Mre;NICD Supplementry Figure 8 Noth-indued CP tumours rise from progenitors in the roof plte. () Atoh:EGFP expression in Mth MGFP mie t dy E4.5 is deteted with GFP ntiody nd shown in green signls. Otx2 expression (red) lels the roof plte (mrked y dotted lines), while stining (lue) mrks nulei. Notie tht Atoh:EGFP + progenitors (rrows) re present in Otx2 + upper roof plte, ut sent in lower roof plte. Sle r: 5 µm. () Gene expression in CPs from Mre:NICD mie t dy E4.5. GFP (green) mrks tumour ells. Ki-67 expression (red) lels proliferting ells, nd Aqp expression (red) mrks differentited epithelil ells. stining (lue) lels nulei. Sle r: 3 µm. () Anlysis of gene expression nd prolifertion of tumour ells nd progenitors within hindrin roof plte in Mre;NICD nd wild type () mie, respetively, t dy E4.5. The expression of Lmx (red) mrks the roof plte nd CP linege. While Ki-67 expression (green) lels proliferting ells, Aqp expression (green) mrks differentited epithelil ells. White dotted lines mrk domin of progenitors (Lmx + /Ki-67 + / Aqp - ) in hindrin roof plte. Boxed regions in upper roof plte re shown in higher mgnifition. Red rket lines mrk differentited epithelil ells (Lmx + /Ki-67 - /Aqp + ). Strs demrte tumour ells (Lmx + /Ki /Aqp - ). Sle r: 25 µm. 8

24 Mre;NICD Mre;NICD Mre;NICD Mre;NICD Mre;NICD Mre;NICD Mre;NICD Mre;NICD N/A N/A N/A Mre;NICD Mre;NICD Mre;NICD Mre;NICD Mre;NICD Mre;NICD Mre;NICD Mre;NICD N/A N/A N/A SUPPLEMENTARY INFORMATION Hes Aqp Otx2 Cnd 75 kd 75 kd 75 kd 75 kd 8 kd 58 kd 46 kd 3 kd 23 kd 8 kd 58 kd 46 kd 3 kd 23 kd 8 kd 58 kd 46 kd 3 kd 23 kd 8 kd 58 kd 46 kd 3 kd 23 kd 7 kd 7 kd 7 kd 7 kd P P7 P9 β-tin β-tin β-tin P P7 P9 β-tin 75 kd 75 kd 75 kd 75 kd 8 kd 58 kd 46 kd 3 kd 23 kd 7 kd P P7 P9 8 kd 58 kd 46 kd 3 kd 23 kd 7 kd 8 kd 58 kd 46 kd 3 kd 23 kd 7 kd 8 kd 58 kd 46 kd 3 kd 23 kd 7 kd P P7 P9 Supplementry Figure 9 Representtive unproessed snned imges of lots. Western lot nlysis of Hes (), Aqp nd Otx2 (), nd Cnd () expression in CP tumours from Mre;NICD mie nd CPs from wild type () mie t different time points. β-tin serves s loding ontrol. N/A: protein smples for unrelted study run on the sme gel (). 9