Rb suppresses human cone-precursor-derived retinoblastoma tumours

LETTER

doi:10.1038/nature13813

Rb suppresses human cone-precursor-derived retinoblastoma tumours

Xiaoliang L.Xu 1,2,Hardeep P.Singh 3,4,Lu W ang 1,Dong-Lai Qi 3,4,Bradford K.Poulos 5,David H.Abramson 6,Suresh C.Jhanwar 1,7&David Cobrinik 3,4,8,9

Retinoblastoma is a childhood retinal tumour that initiates in response to biallelic RB1inactivation and loss of functional retinoblastoma (Rb)protein.Although Rb has diverse tumour-suppressor functions and is inactivated in many cancers 1–5,germline RB1mutations predis-pose to retinoblastoma far more strongly than to other malignancies 6.This tropism suggests that retinal cell-type-specific circuitry sensitizes to Rb loss,yet the nature of the circuitry and the cell type in which it operates have been unclear 7,8.Here we show that post-mitotic human cone precursors are uniquely sensitive to Rb depletion.Rb knock-down induced cone precursor proliferation in prospectively isolated populations and in intact retina.Proliferation followed the induction of E2F-regulated genes,and depended on factors having strong expres-sion in maturing cone precursors and crucial roles in retinoblastoma cell proliferation,including MYCN and MDM2.Proliferation of Rb-depleted cones and retinoblastoma cells also depended on the Rb-related protein p107,SKP2,and a p27downregulation associated with cone precursor maturation.Moreover,Rb-depleted cone precursors formed tumours in orthotopic xenografts with histological features and protein expression typical of human retinoblastoma.These find-ings provide a compelling molecular rationale for a cone precursor origin of retinoblastoma.More generally,they demonstrate that cell-type-specific circuitry can collaborate with an initiating oncogenic mutation to enable tumorigenesis.

RB1-mutant retinoblastomas can originate from a cellular state found during retinal development in humans but not in other species 9,10.Accord-ingly,to identify the cellular state and corresponding circuitry that sen-sitizes to RB1inactivation,we examined the effects of Rb depletion on human fetal retinal cells.Samples were from post-fertilization weeks 17–19,when all retinal cell types and a range of maturation states are present.Dissociated retinal cells were transduced with RB1-directed or con-trol short hairpin RNAs (shRNAs),followed by co-staining for the proliferation-associated Ki67and cell-type-specific markers.RB1shRNAs abrogated Rb expression in long or medium wavelength (L/M)-opsin 1and thyroid hormone receptor b 21(TR b 21)cone precursors as well as in other cell types (Extended Data Fig.1a).After 2weeks,Ki67was detected in cone-precursor-like cells co-expressing the photoreceptor marker CRX and the cone markers L/M-opsin,cone arrestin and RXR c (Fig.1a and Extended Data Fig.1b–h).Ki671cone-marker 1cells were first detected 9days after transduction,whereas clusters were routinely detected by day 23.Ki67was not detected in cells expressing markers of rods (NRL),bipolar cells (strong CHX10),ganglion cells (BRN-3),or amacrine or horizontal cells (PROX11or PAX61,nestin 2)(Fig.1a and Extended Data Fig.1i,j).Ki67was detected in cells expressing markers ofretinal progenitorcells(RPCs)orMu ¨llerglia(nestinorCRALBP,SOX2),yet in similar proportions after RB1shRNA or control shRNA (Fig.1a and Extended Data Fig.1j).RB1shRNAs also induced incorporation of

5-ethynyl-29-deoxyuridine (EdU),an indicator of S phase entry,increased expression of the mitosis marker phosphohistone H3,suppressed expres-sion of the apoptosis marker cleaved caspase 3(CC3),and induced prolif-eration in cells expressing cone but not other retinal cell markers (Fig.1c,d and Extended Data Fig.1k–n).By contrast,RB1shRNAs induced CC3

1

Department of Pathology,Memorial Sloan-Kettering Cancer Center,1275York Avenue,New York,New York 10021,USA.2Sloan-Kettering Institute for Cancer Research,Memorial Sloan-Kettering Cancer Center,1275York Avenue,New York,New York 10021,USA.3The Vision Center,Division of Ophthalmology,Department of Surgery,Children’s Hospital Los Angeles,4650Sunset Boulevard,Los Angeles,California 90027,USA.4The Saban Research Institute,Children’s Hospital Los Angeles,4650Sunset Boulevard,Los Angeles,California 90027,USA.5Department of Pathology,Albert Einstein College of Medicine,1300Morris Park Avenue,Bronx,New York 10461,USA.6Ophthalmic Oncology Service,Memorial Sloan-Kettering Cancer Center,1275York Avenue,New York,New York 10021,USA.7

Department of Medicine,Memorial Sloan-Kettering Cancer Center,1275York Avenue,New York,New York 10021,USA.8USC Eye Institute,Department of Ophthalmology,Keck School of Medicine of the University of Southern California,1450San Pablo Street,Los Angeles,California 90033,USA.9Norris Comprehensive Cancer Center,Keck School of Medicine of the University of Southern California,1441Eastlake Avenue,Los Angeles,California 90033,USA.

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Figure 1|Proliferation of cone-like cells after Rb depletion in dissociated FW19retina.a ,b ,Responses to RB1-733shRNA (shRB1).D,day;SCR,scrambled control.a ,Percentage Ki671among cells expressing the indicated retinal cell-type-specific markers.b ,Prevalence of DAPI 1(49,6-diamidino-2-phenylindole 1)cells expressing cone (L/M-opsin,CRX),RPC (nestin),or Mu ¨ller glia (CRALBP)markers.c ,d ,Responses to RB1-733and RB1-737shRNAs (shRB1-733and shRB1-737,respectively).c ,Proliferation of cells,of which .90%were cone marker 1at day 60.d ,Percentage CC31among cells expressing the indicated markers at day 14.Values and error bars denote mean and s.d.of triplicate assays;P values are from unpaired Student’s t -test.All results replicated at least twice.

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and decreased the number of cells expressing markers of RPCs and glia (Fig.1b,d and Extended Data Fig.1n).

To assess whether the Rb-deficient proliferating cone-like cells derived from post-mitotic cone precursors,we examined the effects of Rb knock-down in prospectively isolated retinal cell populations.Populations were isolated by sorting for size,for CD133,which is expressed strongly in maturing photoreceptors and weakly in RPCs 11,and for a CD44epi-tope expressed by Mu ¨ller glia and RPCs 12(Fig.2a).Staining for cell-type-specific markers revealed populations enriched for cone precursors,for rod plus cone precursors,for RPCs plus glia,and for a mixture of rod,ganglion,bipolar,amacrine and horizontal cells (Fig.2b and Extended Data Fig.2a–g).In medium and large CD133hi ,CD442populations,96–98%of cells co-stained for CRX and cone arrestin,which is cone-specific at post-fertilization week 19(FW19)(Extended Data Fig.2h).A similar enrichment was observed when cone precursors were identified using CRX and RXR c (Extended Data Fig.2h–k).

RB1shRNAs induced similar RB1knockdown in each retinal cell pop-ulation (Extended Data Fig.3a).After 2weeks,Ki67was detected in 80%of cells in the cone-enriched population (Fig.2c),probably reflecting a

high ratio of shRNA-expressing lentivirus to target cells and cone pre-cursor proliferation.After 3weeks,cone precursor numbers increased (Fig.2c).Rb depletion did not induce proliferation in RPCs and glia,but increased the proportion of CC31cells entering apoptosis (Fig.2c).Sorted populations transduced with the scrambled control had higher CC31rates than unsorted cultures,potentially reflecting separation of RPCs and glia from neurons 13,14.Nevertheless,Rb knockdown induced proliferation and apoptosis in cells with the same immunophenotypes as in unsorted cultures.Notably,Rb depletion induced the cell-cycle-related genes CCNE1,SKP2,E2F1,RBL1,CCNB1and CDK1in cone precursors,and induced p53-responsive genes in sorted RPCs and glia (Extended Data Fig.3).Cell-cycle-related genes were induced several days before Ki67,suggesting that further reprogramming was needed for cell cycle entry.

RB1shRNAs also induced cone precursor proliferation in intact retinas.Ki67was detected in L/M-opsin 1cone precursors in the fovea,demar-cated by cones but not rods,15days after transduction (Fig.2d).Ki67was not detected in cells expressing rod,amacrine,horizontal or gan-glion cell markers (Extended Data Fig.4a,d).Ki67was detected in RPCs and glia marked by PAX61,nestin 1or by CHX101,CRX 2,yet in sim-ilar proportions after transduction with RB1-directed and control shRNAs

(Extended Data Fig.4b–d).Moreover,a yellow fluorescent protein (YFP)-expressing RB1shRNA vector selectively induced Ki67in YFP 1cones,

although all cell types were transduced (Extended Data Fig.4e–h).We next determined whether Rb-depleted cone precursors and reti-noblastoma cells depend on similar signalling circuitry.Retinoblastoma cell proliferation requires several proteins that are prominent in cone precursors,including TR b 2,RXR c ,MYCN and MDM2(ref.7).Deple-tion of these factors suppressed Ki67expression and cone precursor proliferation both in dissociated retinal cultures (Extended Data Fig.5a,b)and in isolated populations (Fig.3a).Retinoblastoma cell proliferation also requires SKP2-mediated degradation of Thr 187-phosphorylated p27(ref.15).Concordantly,SKP2depletion suppressed cone precur-sor proliferation and increased CC3(Fig.3a and Extended Data Fig.5a).Notably,maturing cone precursors had exceptionally high Thr 187-phosphorylated p27(Extended DataFig.5c),coincidentwith a maturation-associated decrease in total p27(ref.16),suggesting that SKP2-mediated p27degradation might enable cone precursor proliferation.Consistent with this view,cone precursor proliferation was suppressed by ectopic

p27and enhanced by ectopic SKP2or p27knockdown (Fig.3b and Ex-tended Data Fig.5b),as in retinoblastoma cells 15.Thus,Rb-depleted cone precursors and retinoblastoma cells had similar signalling requirements.We also assessed the roles of the Rb-related p107and p130proteins

(also known as RBL1and RBL2,respectively).In mouse models,retinal

tumorigenesis required loss of Rb combined with loss of p107,p130or p27(refs 10,17).However,in human retinoblastomas,p130(also known

as RBL2)losses are common,whereas p107(also known as RBL1)losses

are rare 18(Extended Data Fig.6a).Moreover,whereas maturing cone precursors had abundant p130and minimal p107,retinoblastomas had barely detectable p130yet prominent p107(Fig.4b and Extended Data Fig.6b),implicating p130but not p107in retinoblastoma suppression.Concordantly,co-knockdown of p130with Rb increased cone precursor proliferation (Fig.3a and Extended Data Fig.5a,d)and p130overex-pression suppressed cone precursor and retinoblastoma cell prolifera-tion (Fig.3b,c,e).Meanwhile,p107knockdown suppressed proliferation both in Rb-depleted cone precursors (Fig.3a and Extended Data Fig.5a,d)and in retinoblastoma cells (Fig.3d,e and Extended Data Fig.5e,g).In Y79cells,p107knockdown decreased expression of MYCN and SKP2,while it increased the SKP2target,p27(Fig.3e).These effects were seen with two shRNAs and were rescued by p107restoration (Fig.3d,e and Extended Data Fig.5d–i).Furthermore,p107overexpression enhanced

proliferation of retinoblastoma cells while suppressing that of neuro-blastoma cells (Extended Data Fig.5h–j).Thus,both in Rb-depleted cone precursors and in retinoblastoma cells,p130suppressed prolif-eration whereas p107had a proliferative role distinct from its function in mouse models.

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FSC Figure 2|Cone precursor response to Rb depletion.a ,Dissociated FW18

retinal cells sorted by size,CD133and CD44,with major populations

designated.FSC,forward scatter;misc,miscellaneous;SSC,side scatter.

b ,Percentage of cone arrestin 1(cArr 1),CRX 1cones,NRL 1rods,nestin

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horizontal,amacrine or ganglion cells in each population.Lg,large;med,medium;sm,small.c ,Responses to RB1-733shRNA .Percentage of Ki671or CC31cells at 14days (top),and cell numbers at days 14and 23(bottom).*P ,0.05,**P ,0.01(compared to scrambled control).Results in a –c are representative of three independent experiments.

d ,Ki671,L/M-opsin 1con

e precursors (white arrows)in FW19fovea

15days after transduction with RB1-733and RB1-737shRNAs;and Ki671,

L/M-opsin 2cells probably representing RPCs or glia (yellow arrows)after

transduction with RB1shRNA or scrambled control.Scale bar,20m m.Values and error bars denote mean and s.d.of triplicate assays;P values are from

unpaired Student’s t -test.RESEARCH LETTER

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After several months,some cone precursor cultures depleted in Rb or in both Rb and p130(Rb/p130-depleted)formed suspension aggre-gates resembling retinoblastoma cells (Extended Data Fig.7a).Rb/p130-depleted cultures proliferated more robustly and longer than those with

Rb depletion alone,consistent with p130losses in many retinoblastoma cell lines (Extended Data Fig.6a).The cultures had properties consistent with Rb/p130-depleted cone precursors (Extended Data Fig.7b–h).When engrafted either 3months or within 1week after knockdown,Rb-or Rb/p130-depleted cone precursors formed retinoblastoma-like tumours in subretinal xenografts (Fig.4a and Extended Data Figs 8and 9).For cells engrafted within 1week,tumours appeared within 6–14months

(Extended Data Fig.8b),similar to the time needed to form tumours in children.Cone-precursor-derived tumours had differentiated histology,little Rb or p130,many Ki671cells,and prominent p107and SKP2,consis-tent with robust proliferation (Fig.4a,b and Extended Data Fig.9).They expressed the photoreceptor-related CRX,CD133and IRBP and the cone-specific cone arrestin,L/M-opsin and RXR c ;all at levels similar to reti-noblastomas and developing retinas (Fig.4b and Extended Data Fig.9).This is consistent with the many cone-specific proteins in retinoblas-toma tumours 7(Supplementary Table 1).As in human retinoblastomas 7,

cone-precursor-derived tumour cells lacked numerous markers of other retinal cell types and had rare S-opsin and rhodopsin expression (Ex-tended Data Fig.10).Rb-depleted and Rb/p130-depleted cone precursor tumours also had structures resembling Flexner–Wintersteiner rosettes and fleurettes (Fig.4a),which are retinoblastoma hallmarks 19.Trans-mission electron microscopy confirmed the rosettes,with mitochon-dria positioned between the nuclei and rosette lumens (Fig.4c).Dense core vesicles were not seen in two Rb-depleted cone precursor tumours nor in two retinoblastomas,consistent with the reported rarity of such structures 20,21.Finally,single nucleotide polymorphism (SNP)-array ana-lyses of two tumours revealed no megabase-size gains or losses,whereas quantitative PCR (qPCR)analyses revealed a partial RB1loss but no other frequently reported changes (Extended Data Fig.8c–e),consistent

withthe lackof DNAcopynumberalterationsin someretinoblastomas 22–24.Thus,cone precursor tumours resembled human retinoblastomas at the histological,ultrastructural,retinal marker and molecular cytoge-netic levels.

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or Figure 4|Rb-depleted or Rb/p130-depleted cone precursor tumours.a ,Haematoxylin-and-eosin-stained Rb-depleted and Rb/p130-depleted cone xenograft tumours and human retinoblastoma (n 54).KD,knockdown.Dashed lines denote Flexner–Wintersteiner rosettes;solid lines denote

fleurettes;asterisks mark rosette cavities.b ,Cone-and cell-cycle-related protein expression in human retinoblastoma and cone xenografts (n 56).Scale bars,40m m (a ,b ).c ,Transmission electron microscopy of Flexner–Wintersteiner

rosettes in a human retinoblastoma and a cone-derived tumour,with

mitochondria (arrows)between nuclei and rosette cavity (n 52).The 325,000images are from the boxed area (top)or from a rosette not shown (bottom).Results are representative of at least two experiments.d ,Model of cone-precursor retinoblastoma origin highlighting proteins that suppressed (blue)or promoted (red)the proliferative response.

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Figure 3|Effects of cone precursor circuitry on response to Rb depletion.

a ,Prevalence of Ki671or CC31cells (top)and cell numbers (bottom)after

shRNA transduction of isolated cone precursors.b ,Isolated cone precursor

response to co-transduction with RB1shRNA and BE-Neo (BN)vector,

BN-p130or BN-SKP2.c –e ,Effect of p130overexpression or p107knockdown on Y79cell proliferation (c ,d )and protein expression (e ).*P ,0.05,**P ,0.01(compared to scrambled and vector controls);{P ,0.05,{{P ,0.01(compared to RB1shRNA plus SCR (a ),or to RB1shRNA plus BN

vector (b )).Results represent at least two independent experiments.Values and

error bars denote mean and s.d.of triplicate assays;P values are from

unpaired Student’s t -test.

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This study examined collaboration between Rb loss and retinal cell-type-specific circuitries.We found that the circuitry of maturing L/M-cone precursors was uniquely conducive to proliferation and development of retinoblastoma-like tumours.Although we cannot exclude the pos-sibility that Rb loss could induce a cone program and proliferation in other cell types,the robust responses of the most highly enriched cone precursor populations and of cells in an intact fovea suggest that cone precursors are the primary if not the sole responding cell type.Cone precursor features that collaborated with Rb loss included cone lineage factors(TR b2and RXR c),highly expressed oncoproteins(MYCN and MDM2),and p27downregulation probably mediated by SKP2.Some of these features may be interdependent,as RXR c promoted MDM2 expression7,yet the larger program encompassing these features and its developmental purpose are unknown.Importantly,Rb-depleted cone precursor tumours had differentiated histology and lacked gross DNA aberrations,similar to putative early retinoblastoma elements25.These findings support a model in which Rb-deficient cone precursors form differentiated retinoblastomas,then dedifferentiate(Fig.4d)and pos-sibly acquire non-cone features8,22.Much of the circuitry implicated in cone precursor tumour initiation was also needed for retinoblastoma cell proliferation7,15,suggesting that tumour cells can be addicted to the cancer-predisposing circuitry of their originating cell types.

Online Content Methods,along with any additional Extended Data display items and Source Data,are available in the online version of the paper;references unique to these sections appear only in the online paper.

Received31October2013;accepted1September2014.

Published online24September2014.

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Supplementary Information is available in the online version of the paper. Acknowledgements We thank P.MacLeish,D.Forrest,C.Craft,G.Chader,

C.Gregory-Evans,R.Molday,P.Hargrave,Y.Imanishi,K.Palczewsk,E.Weiss,

A.Swaroop,T.Li,R.Lee and J.Saari for antibodies.We thank T.Baumgartner and P.Byrne for FACS assistance,https://www.sodocs.net/doc/1b13788925.html,mpen for electron microscopy assistance,N.Zhou, T.Patel and J.Wang for technical assistance,S.Puranik and Z.Li for DNA constructs, and J.Aparicio for critical reading of the manuscript.Funding was received from The Gerber Foundation(X.L.X.),The Fund for Ophthalmic Knowledge(D.H.A.),the Research and Development Funds of the MSKCC Department of Pathology(S.C.J.),The Larry& Celia Moh Foundation(D.C.),and National Institutes of Health grant1R01CA137124 (D.C.).

Author Contributions X.L.X.,S.C.J.and D.C.designed the study.X.L.X.conducted most of the experiments in S.C.J.’s laboratory,supported in part by D.C.H.P.S.and D.-L.Q. quantified Rb knockdown and confirmed effects at different time points.H.P.S. transduced retina with YFP-labelled constructs,and analysed them with X.L.X.L.W. analysed SNP arrays.D.H.A.provided retinoblastoma samples.B.K.P.provided fetal retina.D.C.wrote the manuscript with assistance from X.L.X.and review by S.C.J. Author Information SNP array data have been deposited with NCBI GEO under accession number GSE60720.Reprints and permissions information is available at https://www.sodocs.net/doc/1b13788925.html,/reprints.The authors declare no competing financial interests. Readers are welcome to comment on the online version of the paper.Correspondence and requests for materials should be addressed to S.C.J.(jhanwars@https://www.sodocs.net/doc/1b13788925.html,)or D.C.(dcobrinik@https://www.sodocs.net/doc/1b13788925.html,).

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METHODS

Retinoblastoma and retinal cell culture.Retinoblastoma cell lines Y79and Weri-RB1were obtained from the ATCC.RB177was from an early passage culture and its identity confirmed by RB1mutation sequencing.Retinoblastoma cells were con-firmed free of mycoplasma and cultured in RB culture medium(IMDM,10%FBS, 55m M b-mercaptoethanol,with glutamine,penicillin,streptomycin,fungizone and 10m g ml21plasmocin(Invivogen)7,26).Fetal eyes were obtained with informed con-sent from the Human Fetal Tissue Repository of the Albert Einstein College of Med-icine and from Advanced Bioscience Resources under protocols approved by the Memorial Sloan-Kettering Cancer Center(MSKCC)Institutional Review Board, the Albert Einstein College of Medicine Institutional Review Board,and the Children’s Hospital Los Angeles Committee on Clinical Investigations.After transport in IMDM with10%FBS on ice,eyes were rinsed in70%ethanol for3s and washed in sterile PBS. Eyes were opened using a sterile scalpel and lens removed.Retinas were detached using forceps and incubated in papain solution(Worthington Tissue Dissociation Kit)for 10–30min at37u C and5%CO2,with pipette mixing every5min.After dissociation to,20-cell clusters,cells were diluted with10volumes of PBS and collected by cen-trifugation at2,000r.p.m.(Sorvall,Legend RT),re-washed in PBS(all centrifuga-tions at2,000r.p.m.unless otherwise stated),suspended in RB culture medium as above,incubated at37u C with5%CO2overnight,and frozen in RB culture medium containing10%dimethylsulphoxide(DMSO).Supernatant was transferred into a sterile container after each centrifugation and re-spun to prevent retinal cell losses. For lentivirus infections,cells were recovered from liquid nitrogen,cultured over-night,washed with PBS,suspended in0.05%trypsin/EDTA(Cellgro)for3–10min with gentle pipetting,re-centrifuged,suspended in RB culture medium as above,and immediately infected.Cultures were maintained at high density,typically50,000 cells per well(24-well dish)for unsorted cultures,with media changes every3days. FACS.Approximately10million dissociated retinal cells(,5million per retina)were cultured for18h after thawing in RB culture medium,collected by centrifugation, washed with PBS,digested with5ml warm0.05%trypsin/EDTA for5–15min while triturating in a24-well culture plate20–30times per minute using a1,000-m l tip and checking every,3min,to produce90–95%single cells,centrifuged as above (retaining the supernatant to prevent cell loss),suspended in400m l5%FBS in PBS, and incubated at room temperature for10min.Cells(100m l)were combined with 100m l4m g ml21of mouse IgG(Sigma,I-8765),300m l of cells were combined with 300m l of pre-mixed anti-CD133-phycoerythrin(PE)(Miltenyi Biotec,130-080-801) at1:6and anti-CD44fluorescein isothiocyanate(FITC)(clone IM7,Abcam ab19622 or BD Biosciences,BDB553133)at1:25,to give1:12CD133and1:50CD44final dilutions.After1h at room temperature,cells were diluted with900m l5%FBS in PBS,centrifuged as above,suspended in500m l5%FBS/PBS with300ng ml21DAPI and held on ice until sorting.Cells were sorted using a Becton-Dickenson FACSAria SORP with100mW488-nm laser,the triple bandpass filter removed in the FITC channel,FACSDiva v8.0software,and selecting live single cells based on FSC width, SSC width,and DAPI exclusion.On FSC/SSC plots,cells were divided into small, medium,and large size groups and evaluated for CD133-PE and CD44-FITC.Eight populations collected into500m l complete medium as above were small,medium and large CD133hi,CD442;small,medium,and large CD133lo,CD441;small CD1332, CD442;and ungated live single cells.Each population was cultured in50%Y79-conditioned medium with fungizone(50%fresh RB culture medium combined with50%filtered Y79-conditioned RB culture medium),and half of the volume changed with fresh50%Y79-conditioned medium every3days.Sorted popula-tions were characterized by adhering cells to poly-L-lysine-coated coverslips(1,000–2,000cells each)for3h,fixing in4%paraformaldehyde(PFA)for5min,washing in PBS four times,and storing at220u C until immunostaining.Lentivirus infec-tion was performed within24h after sorting.

Lentiviral shRNA and cDNA expression constructs.pLKO lentiviral shRNA vectors from the TRC library(Open Biosystems/Thermo Scientific or MSKCC SKI High-Throughput Drug Screening and RNAi Core Facility)27were designated by ‘sh’followed by the name of the target gene and last3–4digits of the TRC or SKI identification numbers(Supplementary Table2).shRNAs directed specifically against THRB variant2(also known as TR b2)were designed using Invitrogen BLOCK-iT RNAi Designer(https://www.sodocs.net/doc/1b13788925.html,/rnaiexpress/)and siDirect(http:// sidirect2.rnai.jp/doc/)andcloned usingtheTRCcloning strategy(http://www.addgene. org/pgvec1?f5v&cmd5showfile&file5protocols)with deoxyoligonucleotides for DNA-directed RNAi(Integrated DNA Technologies).They are designated accord-ing to the position of the first shRNA target nucleotide after the translation ini-tiation site(Supplementary Table2).The pLKO scrambled control was Addgene plasmid1864(ref.28).pLKO-YFP-shRB1-733,pLKO-YFP-shRB1-737and pLKO-YFP-SCR control virus were produced by replacement of the puromycin resistance gene with YFP complementary DNA using In-Fusion cloning(Clontech),and pro-vided by Z.Li.The lentiviral cDNA expression BN vector was created by replacing the EGFP gene of the BE-GFP lentiviral vector29with the neomycin resistance gene between the EcoRI and BamHI sites(with assistance of S.Puranik).BN-p130was produced by inserting human p130cDNA between the BE-Neo PshAI and XbaI site.BN-SKP2and BN-p107were produced by inserting human SKP2and p107 cDNA,respectively,between the BsiWI and PspXIsites of BE-Neo.Because shRBL1-2621(shp107-1)targets the39untranslated region,only the RBL1open reading frame was cloned into BE-Neo to produce shRBL1-2621-resistant BN-p107.To produce shRBL1-2623(shp107-2)-resistant BN-p107-2r,the shRBL1target sequence GC AGTGAATAAGGAGTATGAA was mutated to GCAGTAAACAAAGAATAT GAA without amino acid sequence changes using

BE-p27was as described15.

Lentivirus production and infections.Lentiviruses were produced by reverse transfection of suspended23107293T cells using20m g lentiviral vector,10m g pVSV-G,20m g pCMV-dR8.91(ref.30)and100m l Polyjet(SignaGen)or Lipofec-tamine2000(Life Technologies)in15cm3dishes.The3-ml plasmids–Polyjet com-plex and1.5ml293T cell suspension were mixed in50ml centrifuge tubes and shaken for30min before being transferred to dishes.Virus collected48and72h after transfection was combined,concentrated50–100-fold by centrifugation at 25,000r.p.m.for90min,and suspended in RB culture medium.Concentrated virus (500–2,000m l)was used to infect53105Y79,Weri-1or RB177retinoblastoma cells, or to infect53105total retinal cells or13105of each sorted retinal cell population in500m l of filtered conditioned RB culture medium in the presence of4m g ml21 polybrene(Sigma-Aldrich)followed by gentle pipetting25times and shaking for 10min in the hood.After18h,cells were diluted in an equal volume of conditioned RB culture medium and maintained at37u C with5%CO2.For co-infections,100m l of each concentrated virus was used to infect either13104total retinal cells or13103 sorted retinal cells suspended in100m l of conditioned RB culture medium with 4m g ml21polybrene in a total volume of300m l,and medium was replaced with150m l 50%Y79and other RB cell conditioned medium24h after infection.Infected cells were selected starting48h after infection with1.4–3m g ml21puromycin for48–72h or with50–100m g ml21G418for4–7days,and fed every2–3days by replacing two-thirds of the media with50%Y79and other RB cell-conditioned medium. Intact FW19retinas were either infected within the globe,by cutting a cross-section through the cornea,removing the lens and most of the vitreous,and pipetting500m l of concentrated pLKO versions of shRB1-733and shRB1-737or scrambled control lentivirus into the sub-retinal space and vitreous(causing retinal detachment)in a 24-well plate with the globe submerged in RB culture medium with1ml lentivirus suspension,followed after2days by addition of2ml of freshly prepared concen-trated lentivirus;or infected after removal of the intact retina and residual vitreous in a12-well plate,by addition of1ml of803concentrated pLKO-YFP-shRB1-733 or scrambled control lentivirus,reinfection with the same viruses1and3days later, and changing50%of mediumwith a1:1mixture of fresh and ocular globe-conditioned medium daily thereafter.Displaced retinal tissue was fixed with2%PFA in PBS for 2h at4u C,and eyes with remaining tissue were fixed in2%PFA in PBS overnight at4u C.Tissue samples were washed with PBS,transferred to30%sucrose in PBS, and embedded in30%sucrose/PBS:OCT at a2:1ratio,and cryosectioned at8–10m m. Real-time quantitative PCR.Total RNA was isolated using StrataPrep total RNA microprep kit(Stratagene)for,1,000cells(in FACS isolated populations)or GenElute Mammalian Total RNA Miniprep Kit(Sigma)for all other analyses.cDNA was synthesized using ImProm-II Reverse Transcription System(Promega).Primers were designed by Beacon Designer software(Premier Biosoft International)or Primer3 (https://www.sodocs.net/doc/1b13788925.html,/primer3/)(Supplementary Table3).Relative messenger RNA levels were determined by qPCR using QuantiTect SYBR Green PCR Kit (Qiagen)or Maxima SYBR Green qPCR Master Mix(Fermentas)on an Applied Biosystems ABI7900HT Sequence Detection System or ViiA7Real-Time PCR System using95u C10min followed by40cycles of95u C20s,54u C30s,72u C30s. Each sample was evaluated in triplicate and normalized to ACTB and GAPDH. Values represent the averages of both normalized results and error bars the stan-dard deviation.

Immunostaining.Antibodies are described in Supplementary Table4.Eyes were prepared and cryosectioned as described7,16.Cultured retinal cells were dissociated by gentle triturating,spread on poly-L-lysine-coated slides,incubated in a humidi-fied incubator at5%CO2and37u C for3h,fixed in4%PFA and PBS for5min, gently rinsed with PBS four times,vacuum-dried for5min,and stored at220u C. The following co-staining combinations and orders were used to assess Ki67expres-sion in different retinal cell types.For cones:1a.Mouse anti-cone arrestin31,anti-mouse-biotin,streptavidin-FITC,rabbit anti-Ki67,anti-rabbit-Cy3,rabbit anti-CRX and anti-rabbit-Cy5.1b.Mouse anti-Ki67,anti-mouse-Cy3,rabbit anti-CRX,anti-rabbit-FITC,rabbit anti-human conearrestin32and anti-rabbit-Cy5.2.Mouse anti-Ki67, anti-mouse-biotin,streptavidin-FITC,rabbit anti-CRX,anti-rabbit Cy3,rabbit anti-L/M-opsin and anti-rabbit-Cy5.3.Mouse anti-RXR c,anti-mouse-biotin,streptavidin-FITC,rabbit anti-Ki67,anti-rabbit-Cy3,rabbit anti-CRX and anti-rabbit-Cy5.For progenitors,Mu¨ller,and horizontal amacrine cells:mouse anti-Pax6,anti-mouse-biotin,streptavidin-FITC,rabbit anti-Ki67,anti-rabbit-Cy3,rabbit anti-nestin and anti-rabbit-Cy5.Forotherretinalcelltypes:mouseanti-human Ki67,anti-mouse-biotin,

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streptavidin-FITC,and rabbit antibodies for retinal specific markers,anti-rabbit Cy3.For BrdU labelling,10m M BrdU was added to medium for2h on day23after Rb knockdown and cells were stained with rat anti-BrdU,anti-rat-FITC,rabbit anti-CRX,anti-rabbit-Cy3,rabbit anti-L/M-opsin and anti-rabbit-Cy5.

For co-staining with mouse antibodies,sections or cells were treated with1mM EDTA/PBS for5min at room temperature and washed with PBS.Sections were treated with ABC kit reagent A(Vector Laboratories)in PBS for15min,washed in PBS,treated with ABC kit reagent B(Vector Laboratories)in PBS for15min,washed in PBS,blocked and permeabilized for20min in super block(2.5%horse serum, 2.5%donkey serum,2.5%human serum,1%BSA,0.1%Triton-X-100and0.05% Tween-20in PBS;filtered with0.22m m filter),incubated in mouse primary anti-body in super block overnight at4u C,washed in PBS,incubated in biotinylated horse anti-mouse antibody in super block for30min,washed in PBS,incubated with FITC-conjugated streptavidin in PBS,and washed with PBS.

For co-staining with antibodies of other species,after completing the first stain-ing reaction as above,sections were incubated in super block for20min,incubated overnight with primary antibody in super block,washed in PBS,incubated with Cy3-or Cy5-conjugated secondary antibody in super block for30min,and washed in PBS.Sections were then stained with1m g ml21DAPI in PBS,dried,mounted in Vectashield(Vector Labs)and imaged using an Axioplan2(Carl Zeiss MicroImaging, LLC)or confocal DMIRE2(Leica)microscope.Antibody specificity was confirmed by staining in parallel with control IgG or no primary antibody.

Antibody-dependent immunofluorescence signals were distinguished from auto-fluorescence by virtue of signal detected in only one colour channel.Cells with auto-fluorescence in multiple channels or with DNA condensation,fragmentation,or degradation were excluded.Cytoplasmic autofluorescence common in astrocytes, Mu¨ller glia,and ganglion cells was distinguished from authentic antigens by its detection at multiple wavelengths in cells with characteristic glial cytoplasmic and nuclear morphology.Nonspecific cytoplasmic staining by concentrated nestin anti-body was distinguished from authentic nestin staining by its homogeneous rather than fibre-like structure.Nonspecific nuclear staining of L/M-opsin was avoided by using reduced antibody concentration.

EdU labelling and detection.Click-iT EdU Alexa Fluor488Imaging Kit was used for EdU labelling to detect proliferation.Dissociated or sorted retinal cells were infected with shRB1or control lentivirus.After14days,20m g ml21of EdU was added into medium and incubated for1h;the cells were attached on poly-L-lysine-coated coverslips for2h and fixed for5min.

The cells were blocked and permeabilized for20min in super block as above and EdU was detected by addition of Click-iT reaction cocktails containing2m M Alexa Fluor488azide for1h.Co-staining was performed after EdU labelling with different combination of antibodies for retinal cell markers and secondary antibodies conjugated with Cy3or Cy5,described as above.For cones,cone arrestin-Cy3plus CRX-Cy5,RXR c-Cy3plus CRX-Cy5,and L/M-opsin-Cy3plus CRX-Cy5were used for co-staining with EdU.

Immunoblotting.Cells were washed in PBS,lysed in ELB1(150mM NaCl,50mM HEPES,pH7.4,0.1%NP40,5mM EDTA,2mM dithiothreitol(DTT),1mM phe-nylmethylsulfonyl fluoride,10mM NaF,1mM sodium orthovanadate,Thermo Scientific Halt phosphatase inhibitor cocktail and protease inhibitor cocktails),sep-arated on4–20%Ready Gel polyacrylamide gels(Jule Biotechnologies INC)or8% polyacrylamide(for Rb western),and transferred to Hybond-ECL nitrocellulose membrane(Amersham).Membranes were probed with antibodies(Supplementary Table4)and developed using horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies and the ECL Advance Western Blotting Detection Kit(Amer-sham Biosciences)or Thermo Scientific SuperSignal-West Femto Chemilumines-cent Substrate,and HyBlot CL X-Ray film(Denville Scientific).Xenografts.All animal experiments complied with ethical regulations and were approved by the MSKCC Institutional Animal Care and Use Committee.Xenografts were performed on6-week-old male athymic(Foxn12/2)mice(Taconic)or6-week-old male non-obese diabetic–severe combined immunodeficient(NOD–SCID) Il2rg2/2mice(Jackson Laboratories).Cultured cells were dissociated by pipetting, suspended in RB growth medium at53104cells per microlitre(day90)or2.53103 cells per microlitre(days3or7),held on ice,and2m l injected into the subretinal space as described7.Irradiated5053rodent diet with amoxicillin was provided from2days before to2weeks after injection to prevent infection.Some tumour-bearing eyes were fixed and embedded as described7,16.

DNA copy number analyses.Genomic DNA from retinoblastomas,cone-derived retinoblastoma-like cells,and cone-derived xenograft tumours were isolated with QiaAMP DNA Mini kit(Qiagen).Genomic DNA of cone-derived cells was digested with XhoI to separate pLKO DNA hairpin structures.Relative DNA levels were determined in triplicate by qPCR using QuantiTect SYBR Green PCR Kit(Qiagen) on an Applied Biosystems ABI7900HT Sequence Detection System or ViiA7Real-Time PCR System,using primers listed in Supplementary Table5and normalizing to the average of the HNF4A and BRCA1genes.Integrated pLKO-shRB1-733,shRB-737and shRBL2-923copy numbers were analysed using primers corresponding to the pLKO.1U6promoter and RB1-or RBL2-specific shRNA sequences.High resolution SNP-array DNA copy number analyses were performed using CytoScan HD(Affymetrix,901835).Data were analysed using Chromosome Analysis Suite 2.0(Affymetrix).

Statistical analyses.Measurements were performed in triplicate and differences between means assessed for significance using unpaired Student’s t-tests.Sample sizes were chosen based on the maximum cell numbers that could be used for indi-vidual experiments given sample availability.

Transmission electron microscopy.Human retinoblastomas and cone-derived xenograft tumours were fixed with4%PFA in PBS,rinsed in0.1M sodium caco-dylate buffer,post-fixed in2%osmium tetroxide for1h,rinsed in distilled water, dehydrated in a graded series of50%,75%,95%and100%ethanol,followed by two 10-min incubations in propylene oxide and overnight incubation in1:1propylene oxide/Poly Bed812.The samples were embedded in Poly Bed812and cured at 60u C.Ultra-thin sections were obtained with a Reichert Ultracut S microtome. Sections were stained with Uranyl Acetate and Lead Citrate and photographed using

a Jeol1200EX transmission electron microscope.

26.DiCiommo,D.P.,Duckett,A.,Burcescu,I.,Bremner,R.&Gallie,B.L.

Retinoblastoma protein purification and transduction of retina and

retinoblastoma cells using improved alphavirus vectors.Invest.Ophthalmol.Vis.

Sci.45,3320–3329(2004).

27.Moffat,J.et al.A lentiviral RNAi library for human and mouse genes applied to an

arrayed viral high-content screen.Cell124,1283–1298(2006).

28.Sarbassov,D.D.,Guertin,D.A.,Ali,S.M.&Sabatini,D.M.Phosphorylation and

regulation of Akt/PKB by the rictor-mTOR complex.Science307,1098–1101 (2005).

29.Cobrinik,D.,Francis,R.O.,Abramson,D.H.&Lee,T.C.Rb induces a proliferative

arrest and curtails Brn-2expression in retinoblastoma cells.Mol.Cancer5,72 (2006).

30.Zufferey,R.,Nagy,D.,Mandel,R.J.,Naldini,L.&Trono,D.Multiply attenuated

lentiviral vector achieves efficient gene delivery in vivo.Nature Biotechnol.15,

871–875(1997).

31.Wikler,K.C.,Rakic,P.,Bhattacharyya,N.&Macleish,P.R.Early emergence of

photoreceptor mosaicism in the primate retina revealed by a novel cone-specific monoclonal https://www.sodocs.net/doc/1b13788925.html,p.Neurol.377,500–508(1997).

32.Li,A.,Zhu,X.&Craft,C.M.Retinoic acid upregulates cone arrestin expression in

retinoblastoma cells through a Cis element in the distal promoter region.Invest.

Ophthalmol.Vis.Sci.43,1375–1383(2002).

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Extended Data Figure1|Proliferation of cone-like cells after Rb depletion in dissociated FW19retina.a,Decreased Rb protein in L/M-opsin1or TR b21 cells(arrows)on days5or23,and decreased RB1RNA or Rb protein on day4after shRB1-733transduction.b,Cone arrestin1,CRX1cells(arrows) with or without Ki67co-expression.c,Ki671and cone arrestin1cells first detected9or14days after transduction in two experiments.d–f,Co-staining of Ki67with RXR c and CRX at14days(d),with cone arrestin and CRX at 14days(e),or with L/M-opsin and CRX at23days(f)after transduction with shRB1-733or a scrambled control.g,Percentage of cells co-expressing Ki67 with L/M-opsin and CRX,RXR c and CRX,or cone arrestin and CRX,23days after transduction.h,Prevalence of cells co-staining for L/M-opsin and CRX, RXR c and CRX,or cone arrestin and CRX,23days after transduction.

i,Ki67not detected in cells expressing markers of rods(NRL),ganglion cells (BRN-3),bipolar cells(strong CHX10),or horizontal cells(PROX1)14days after transduction.j,Co-expression of Ki67with markers of RPCs(nestin, white arrows)or Mu¨ller glia(CRALBP or SOX2),but not in PAX61, nestin2ganglion,amacrine or horizontal cells(yellow arrows)14days after transduction.k,l,EdU incorporation in cells expressing markers of cones(cone arrestin and CRX or RXR c and CRX,yellow arrows in l)but not in cells expressing markers of rods(CNGA1,CNGB1),bipolar cells(CHX10,CRX),or ganglion,horizontal or amacrine cells(syntaxin)(white arrows in l)14days after transduction.Black lines above labels demarcate distinct fields.

m,Co-staining of phosphohistone H3(PH3)with cone arrestin and CRX

23days after transduction.n,Apoptosis marker CC3in cells expressing RPC and glial marker nestin14days after transduction with RB1-directed shRNAs (yellow arrow)but not with scrambled control(white arrow).Values and error bars are mean and s.d.of triplicate assays.Scale bars,20m m.Data are representative of at least two independent experiments.

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Extended Data Figure2|FACS isolation of retinal cell populations.Retinal cells were isolated according to size,CD133and CD44staining.In study1,cell type compositions in each fraction(a)were determined by immunostaining with cone arrestin and CRX(b,e),NRL(c),and nestin and PAX6(d,f).In study2,cell type compositions(i)were determined by immunostaining with RXR c and CRX,nestin and CHX10,nestin and PAX6,and CRALBP(j,k).The percentages of the predominant cell types in each population(a,i)and marker specificities(g)are indicated.h,Cone-specific co-staining of cone arrestin and GNAT2(top)and cone-specific co-staining of RXR c and CRX(bottom)in FW19retina.GCL,ganglion cell layer;INL,inner nuclear layer;ONL,outer nuclear layer.l,Co-staining of cells for EdU with cone arrestin and CRX or with RXR c and CRX14days after transduction of the cone-enriched medium plus large CD133hi CD442population isolated as in i–k with two RB1shRNAs (yellow arrows)but not with the scrambled control(white arrows).In both studies,CD133hi CD442medium and large size populations mainly consisted of cells expressing cone markers(CRX and cone arrestin,or CRX and RXR c). The CD133hi CD442small population mainly consisted of cells expressing a rod marker(NRL)with a variable proportion expressing cone markers.All CD133lo CD441populations mainly consisted of cells co-expressing RPC and glial markers(nestin and PAX6,nestin and CHX10,or CRALBP).The CD133lo CD442small size population consisted of cells with diverse immunophenotypes.Values and error bars are mean and s.d.of triplicate assays.Scale bars,30m m.

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Extended Data Figure3|Cone precursor gene expression response to Rb depletion.a–c,Fold change in RNA level relative to day0uninfected cells for RB1(a),or the indicated E2F-responsive genes(b),or the indicated

p53-regulated genes(c),3and6days after transduction of each population with a mixture of shRB1-733and shRB1-737,or with scrambled control.*P,0.05, **P,0.01(comparing shRB1and scrambled control).Data are representative of two sets of qPCR analyses.Values and error bars are mean and s.d.of triplicate assays.

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Extended Data Figure4|Proliferation status of retinal cells other

than cones15days after shRB1transduction of intact FW19retinas.

a–d,Combined transduction with pLKO-shRB1-733and-737.a,Ki67not detected in NRL1or rhodopsin1rod photoreceptors or in calbindin1 horizontal cells.b,Ki67detected in PAX6lo,nestin1RPCs(white arrows) but not in PAX6hi,nestin2horizontal,amacrine or ganglion cells(yellow arrows).c,Ki67detected in CHX101,CRX2RPCs(white arrows)but not in CHX101,CRX1bipolar cells(yellow arrows).d,Percentage of cells

co-expressing Ki67and retinal cell markers.e–h,Transduction with

YFP-marked pLKO-YFP-shRB1-733.e,Ki67detected in YFP1,L/M-opsin1or YFP1,cone arrestin1cone precursors(white arrows)and in an undefined YFP2cell(yellow arrow).f,Ki67not detected in YFP1,calbindin1horizontal cells,YFP1,syntaxin1or YFP1,PAX61amacrine cells,or in YFP1,NRL1 rod precursors.g,Ki67detected(white arrows)or not detected(yellow arrows) in YFP1,nestin1RPCs or glia,or in YFP1,CHX101RPCs or bipolar cells. h,Proportion of Ki671cells co-expressing YFP and retinal markers after transduction with pLKO-YFP-shRB1-733or scrambled control.Values and error bars are mean and s.d.of triplicate assays.Scale bars,20m m.Analyses in a–d and in e–h represent two independent experiments.All immunostaining was performed at least twice.

Extended Data Figure5|Effect of cone-and Rb-related circuitry on cone precursor response to Rb depletion.A,Percentage of Ki671cells among

L/M-opsin1,CRX1cells(a),among RXR c1,CRX1cells(b),or among cone arrestin1,CRX1cells(c);and percentage of L/M-opsin1,CRX1cells among all cells with DAPI1nuclei(d)after transduction of dissociated FW18retina with shRB1-733and shRNAs against p130,p107,TR b2,SKP2,MDM2and MYCN.B,Percentage of Ki671cells among L/M-opsin1,CRX1cone-like cells (top)and proliferative response(bottom)after transduction of dissociated FW18retina with shRB1-733and with shRNAs against RXR c and p27 (shRNAs8561930),or with overexpression of p27and p27-T187A.

C,High-level Thr187phosphorylated p27(p-p27(T187),top)coinciding with downregulation of total p27(bottom)and prominent Rb during cone precursor maturation.C,a,Perifoveal region of FW18retina.C,b,Enlarged view of boxed regions in C,a.Arrows,cone precursors identified by large, strongly Rb1nuclei and lack of p27signal in characteristic outer nuclear layer position7,16.D,Effect of two RBL1-p107or two RBL2-p130shRNAs on proliferation of Rb-depleted isolated cone precursors.E,Knockdown efficacy of two RBL1-p107or two RBL2-p130shRNAs in Y79and RB177retinoblastoma cells.F,Impaired proliferation of Weri-RB1retinoblastoma cells after transduction with BN-p130compared to vector control.G,Impaired proliferation of RB177retinoblastoma cells following transduction with two p107shRNAs.H,I,Impaired proliferation and MYCN expression in Y79cells after p107knockdown with two p107-directed shRNAs,and rescue by shRNA-resistant BN-p107constructs.J,p27accumulation and growth suppression following p107knockdown with shp107-2rescued by BN-p107-2r in RB1wild type SKN-BE(2)neuroblastoma cells.p107overexpression impaired SKN-BE(2)growth,contrary to its effects in Y79.*P,0.05,**P,0.01(compared to SCR or vector control);{P,0.05,{{P,0.01(compared to RB1-KD plus SCR or RB1-KD plus BN vector);{P,0.05,{{P,0.01(compared to

shp107-2plus BN vector)(H–J).Data are representative of more than two independent experiments except for SKN-BE(2)analyses.Values and error bars are mean and s.d.of triplicate assays.

Extended Data Figure6|p130copy number in retinoblastomas and cone precursor expression.a,DNA copy number of p130,other16q genes implicated in retinoblastoma(CDH11,CDH13),and p107determined by qPCR (n56).The percentage of retinoblastomas with copy number(CN),1.5 was higher for p130than for other16q genes(summarized at right;P values relative to p130using Fisher’s exact test).b,p130in peripheral,lateral and central FW19retina.Boxed region in maturing central retina(top)and enlarged view(bottom)show prominent p130in weakly DAPI-stained cone precursor nuclei(arrows).Scale bars,40m m.Data are representative of at least two independent experiments.Values and error bars are mean and s.d.of triplicate assays.

Extended Data Figure7|Characterization of Rb/p130-depleted retinoblastoma-like cells.a,Similar appearance of Rb/p130-depleted cones and early passage retinoblastoma cells.Scale bar,40m m.b,c,DNA copy number of shRNA vectors(b)or selected genes(c)in cell lines derived from Rb/p130-depleted cone precursors(Cone1,Cone2,Cone5)or from

Rb/p130-depleted unsorted retinal cells(All3,All4),in Rb-depleted unsorted retinal cells4days after transduction(All-RB1-KD-d4),in Y79cells,or in FW21retina(normal)(n56).All cell lines retained RB1and p130shRNA vectors and lacked RB1or p130copy number alterations.The Y79MYCN copy number(,78)is not shown(asterisk).d–g,qPCR gene expression analyses in the indicated cell lines relative to cones transduced with scrambled control or FW21retina(n56).d,All cell lines had diminished RB1and p130 expression.e–g,Altered expression of cell-cycle-related(e),cone-related(f) and apoptosis-related(g)genes.h,SNP-array analysis of two Rb/p130-depleted cone precursor cell lines(1,2),revealing no megabase-size loss of heterozygosity(LOH)or copy number alterations(CNA).Data are representative of at least two analyses(b–g)or analyses of two cell lines(h). Values and error bars are mean and s.d.of triplicate assays.

Extended Data Figure8|Characterization of Rb-and Rb/p130-depleted cone precursor tumours.a,Intraocular tumour4months after Rb-depleted cone precursor xenograft.b,Summary of subretinal xenograft groups1,2and3. Sample size was as needed to assess tumour phenotypes.Mice were randomly assigned to different xenograft regimens and the investigator blinded to the assignment until the tumour analyses.Two mice with early death were excluded from the analyses.c,SNP-array analysis of one Rb/p130-depleted (tumour1)or one Rb-depleted(tumour2)cone-precursor-derived tumours from xenograft group3,revealing no megabase-size loss of heterozygosity or copy number alterations.d,qPCR analysis of pLKO shRNA vector copy number in tumours derived from Rb/p130-depleted cone precursors

(m-Cone1,m-Cone2)or from Rb/p130-depleted unsorted retinal cells(m-All3, m-All4),or in mouse ocular tissue(m-Cone-SCR),Y79cells,or FW19retina (normal).All tumours retained RB1and/or p130shRNA vector sequences, confirming their engineered cone precursor origin.e,qPCR analysis of MDM2, MDM4,RB1and MYCN copy number in three cone-derived tumours and normal retina(n56).DNA copy number data(d,e)are representative of two analyses.Values and error bars are mean and s.d.of triplicate assays.

Extended Data Figure9|Cone and cell-cycle-related proteins in Rb-or Rb/p130-depleted cone precursor tumours engrafted3days after transduction.Most tumour cells expressed human nuclear antigen(HuNU), confirming their xenograft origin.They also expressed cone-related proteins(CRX,cone arrestin,L/M-opsin,RXR c,CD133and IRBP)and proliferation-related proteins(Ki67,SKP2,p107and cytoplasmic p27)but lacked Rb.Tumours had elements resembling Flexner–Wintersteiner rosettes (asterisks)and fleurettes(daggers).Scale bars,40m m.Data are representative of three independent experiments.

Extended Data Figure10|Analysis of non-cone cell markers in cone-precursor-derived tumours and retinoblastomas.a,Proteins detected in normal retina but not in cone-derived tumour or human retinoblastoma cells included markers of rods(rhodopsin and CNGB1),RPCs and Mu¨ller glia (nestin,GFAP and PAX6),bipolar cells(CHX10),ganglion,amacrine and horizontal cells(calbindin and PAX6),and ganglion cells(nuclear BRN-3,thin arrows in mouse retina).PAX61,nestin1cells detected in human retinoblastoma were previously found to be Rb1non-tumour cells from tumour-associated retina7.An uncharacterized cytoplasmic BRN-3signal(bold arrows)was detected in mouse photoreceptor outer segments and in cone-derived tumour and retinoblastoma rosettes.b,L/M-opsin was detected in most cone-derived tumour cells.However,rare cells co-expressed S-opsin and L/M-opsin(arrows),as in immature L/M-cone precursors and human retinoblastomas7.c,One tumour had rare rhodopsin1,Ki672cells but no detected rhodopsin1,Ki671cells,as in a previously characterized retinoma-like regions7.Scale bars,40m m.Data are representative of three independent xenograft experiments.