GCL A NBL ED18.5 PND3 PND6 B GCL Figure 1: Spatio-temporal expression of cadherin-11 in the murine retina. Cadherin-11 is expressed in the differentiating layer at ED18.5, by migrating cells at PND3 and again highly expressed by cells possibly migrating to their appropriate position in the developing retina at PND6. B. By adult, PND60, cadherin-11 expression is restricted to cell types of the INL with high expression by Müller glia processes throughout the retina. CAN YOU BE MORE EXPLICIT….I DON’T THINK IT SHOWS TOO WELL….WOULD AND INSERT AT HIGHER MAG BE HELPFUL? CAN YOU POINT TO POSITIVE REGIONS? ONLY THE ADULT IS REALLY CLEAR. INL ONL PND60

A B Merge 40x oil GS CDH11 Merge 100x oil Cralbp CDH11 Chx-10 40x oil 100x oil 160 kDa Merge 40x oil CDH11 C Figure 2: Co-expression of cadherin-11 and retinal cell types of the INL. A, C. Cadherin-11 expression co-localizes with Müller glia cell bodies (Cralpb, 100x magnification) and Müller cell processes (GS, 40x magnification) and horizontal cells (160 kDa, 40x magnification), B,D but not with amacrine (HPC-1, 40x magnification) or bipolar (Chx-10, 40x and 100x magnificataion) cell types. IS THE LETTERING REFERRED TO PROPERLY? LOOKS LIKE IT DOES CO-EXPRESS WITH HPC-1, TO ME. AT LEAST SOME CELLS? D Merge 40x oil CDH11 HPC-1

E18.5 P3 P6 P15 P60 A B Figure 3: Retinal histology of developing retina in CDH11+/+, CDH11+/- and CDH11-/- littermates. Hematoxylin and Eosin (H+E) staining of 5 µm sections all cut through the optic nerve and lens. At developmental time points, E18.5, P3, P6, P15 and P60 (adult), no gross retinal phenotype was observed between CDH11+/+ (A), CDH11+/- (B) and CDH11-/- (C) littermates. C

A CDH11+/+ CDH11-/- CDH11+/+ CDH11-/- CDH11+/+ CDH11-/- ED18.5 PND3 Chx- 10 160 kDa HPC-1 Cralbp Figure 4: Immunohistochemical analysis of CDH11+/+ vs. CDH11-/- reitnae. All INL cell types were assayed to detect disruptions in retinal phenotype of CDH11+/+ vs. CDH11-/-. Selections sections were all 5 um think and cut through the optic nerve and lens. A. Retinal cell type markers: Bipolar & progenitor (Chx-10), horizontal (160 kDa), amacrine (HPC-1) and muller glia (Cralbp) showed no gross change at developmental timepoints ED18.5, PND3 and PND6. B, C. As well, expression of S-phase cells via BrdU incorporation and expression of NCDH (cadherin-2) also revealed no change during the 3 developmental time points tested. WHY DO YOU CHANGE THE ORIENTATION OF B AND C FROM THAT SHOWN IN A? CONFUSING AND NOT CLEAR WHY. WHAT ABOUT MULLER CELL MARKER?

B C BrdU CDH11+/+ CDH11-/- NCDH CDH11+/+ CDH11-/- ED18.5 ED18.5 PND3 Muller Marker (Cralbp) E18.5: 542, 546 P3:413, 414 P6:240, 242

Image J quantification Figure 5: Image J analysis

Genotypes TAg+/- ;CDH11+/+ TAg+/- ;CDH11-/- Cell Type Marker Hes-5 Caspase-3 Average of 5 mice: 45 51 56

Total # of T-Antigen positive cells in retina/eye TAg+/- ;CDH11+/+ TAg+/- ;CDH11+/- TAg+/-;CDH11-/- A K-W Test: p=0.0118 B C 10 9 TAg+/- ; CDH11+/+ TAg+/- ; CDH11+/- TAg+/-; CDH11-/- K-W Test: p=0.0078 8 Total # of T-Antigen positive cells in retina/eye 7 Figure 6 (PND8): T-antigen positive cells (cells of origin of RB) are significantly lower in Tag+/-;CDH11-/- vs. Tag+/-;CDH11+/+. Representative sections of the three genotypes - TAg and H+E stained. T-antigen stains single cells in the INL of the retina and these cells are fewer in number with allele dosage of CDH11. H+E staining amongst the three genotypes shows no major phenotypical difference. B. Manual counts were made of T-antigen positive cells of the retina through the entire eye (approximately 7-8 sections, 300 µm apart were counted and tabulated with one eye per mouse analyzed). Numbers reflect the cells of origin of retinoblastoma are far less when each allele of CDH11 is lost, suggesting CDH11 affects the development of this susceptible retinal cell. C. To account for the size of the retina of each genotype, a ratio of T-antigen counts to total retina area were made. This illustration reflects the same as (B), indicating a statistical trend (p=0.0078) of decreasing T-antigen positive cells when one allele of CDH11 is lost each time. THE H&E ARE ALL THE SAME IMAGE!!!!!SURELY YOU MEAN THAT THE H&E WOULD MATCH THE TAG STAINED SECTION!!!!! THE ONE PROVIDED MATCHES NONE OF THEM REWORD THE “B” MORE ACCURATELY; NOT SURE YOU NEED THE TABLE, BUT LEAVE IT THERE FOR NOW. REPHRASE “C” MORE ACCURATELY. 6 Mouse 1 81 57 43 Tag+ve cells / retina [cells/pixelsx10^-4] 5 Mouse 2 132 52 19 4 Mouse 3 71 72 29 3 83 2 Mouse 4 69 40 1 Mouse 5 92 34 46 TAg+/-; CDH11+/+ TAg+/-; CDH11+/- TAg+/-; CDH11-/- Avg: 89 66 33 Genotype

TAg+/- ;CDH11+/+ TAg+/- ;CDH11+/- TAg+/-;CDH11-/- B Percent tumors in retinas of mice at PND28 K-W Test: p=0.0167 9 Figure 7(PND28): Less multifocal tumors develop when CDH11 is lost. A. A distinct phenotype is observed from representative sections of T-antigen and H+E stained sections amongst the three genotypes. T-antigen staining shows far less multifocal tumors present in CDH11-/- vs. CDH11+/+ mice, as well, H+E staining shows first signs of potential rosette formations in CDH11+/+ describing perhaps more developed tumors in CDH11+/+ vs. CDH11-/- genotype. B. Mice never regain normal phenotype from PND8 as multifocal tumors that emerge are significantly less (p=0.0167) in CDH11-/- vs. CDH11+/+. Tumor volume is calculated using image J software by measuring tumor area (T-antigen stained region) per retinal area (manually traced) of the entire eye. 8 7 6 % Tumor/retina [pixles] 5 4 3 2 1 TAg+/- ;CDH11+/+ TAg+/- ;CDH11+/- TAg+/-;CDH11-/- Genotype

TAg+/- ;CDH11+/+ TAg+/- ;CDH11+/- TAg+/-;CDH11-/- B Percent tumors in retinas of mice at PND84 K-W Test p=0.0602 30 Figure 8 (PND84): Tumor volume of CDH11-/- catches up to CDH11+/+ despite less multifocal tumors to start. A. Representative H+E and T-antigen stained sections show large tumors originating from the INL of the retina. Tumors show disorganized cells, rosette formations and disrupted laminated layers due to tumor growth. No gross phenotypical difference is observed amongst the varying genotypes. B. Tumor volume of CDH11+/+ genotype is 13% vs. 13.5% and 10% in CDH11+/- and CDH11-/- respectively, describing no statistical difference (p=0.0602) amongst the genotypes. This is highly suggestive of faster growing, perhaps more aggressive tumors in CDH11-/- and CDH11+/- genotypes. Tumor volume is calculated using image J software by measuring tumor area (T-antigen stained region) per retinal area (manually traced) of the entire eye. 25 20 % Tumor per retina 15 10 5 TAg+/- ;CDH11+/+ TAg+/- ;CDH11+/- TAg+/-;CDH11-/- Genotype

Antibody Name Company Dilution Used SV40 Tag (Pab 101) mouse monoclonal Santa Cruz Biotechnology Cat# SC-147, Lot# A2506, 1:200 CDH11 - clone CDH113H From St. John at ICOS Corp. 1:2,500 CDH2 (N-cadherin) BD Biosciences Pharmigen Cat# 610920, Lot# 06247 1:2000 Brd-U (purified anti-bromodeoxyuridine) Cat# 555627, Lot#52817, Progenitors and Bipolars: Chx-10 sheep polycolonal gift from Rod Bremner, UHN 1:1000 Apoptosis: activated caspase-3: anti-h/m Caspase 3 (active) rabbit polyclonal R&D Systems Cat# AF835, Lot# CFZ326011 1:500 Early Müller: Hes-5 gift from John Saari Müller: Vimentin goat polyclonal Cat# Lot# 1:100 Ganglion - Brn3b Sant Cruz Biotechnology Cat# sc-6026 Amacrine – Syntaxin clone HPC-1 mouse monoclonal Sigma Cat# S0664 Horizontal – 160 kDa 1:40 Table 1: Antibody List

CDH11 is gradually lost in tumors of Tag-RB mice Anti-SV40 lg(Tag) Anti-CDH11 4 wks Tag-RB 129/C57Bl-6 4 wks Tag-RB 129/C57Bl-6 5 wks Tag-RB 129/C57Bl-6 P28: TH21 5 mth Tag-RB C57Bl-6

Staining for Cralbp, Muller cell marker 6 litters analyzed Birth:P3 CDH11+/+ CDH11-/- P6 NBL P6 cralph, CDH11+/+ is C240, -/- is C242