A B GCL NBL ED18.5 PND3 PND6 GCL INL Figure 1: Spatio-temporal expression of cadherin-11 in the murine retina A) Cadherin-11 is expressed in the differentiating layer at (embyronic day) ED18.5, by migrating cells at (post natal day) 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 ONL PND60

A GCL C INL ONL Chx-10 40x oil CDH11 40x oil GS CDH11 Merge 40x oil INL INL Chx-10 100x oil CDH11 Cralbp CDH11 Merge 100x oil D B INL 160 kDa CDH11 Merge 40x oil ONL HPC-1 CDH11 Figure 2: Co-expression of cadherin-11 and retinal cell types of the inner nuclear layer A, B) Cadherin-11 expression co-localizes with Müller glia cell bodies (Cralbp, 100x magnification), Müller glia cell processes (glutamine synthetase, 40x magnification) and horizontal cells (160 kDa, 40x magnification) C, D) but not with bipolar (Chx-10, 40x and 100x magnification) or amacrine (syntaxin, 40x magnification) cell types

E18.5 P3 P6 P15 P60 GCL A INL ONL GCL B INL ONL GCL C Figure 3: Histology of developing retina in Cdh11+/+, Cdh11+/- and Cdh11-/- littermates Hematoxylin and Eosin (H&E) staining of 5 µm sections 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 INL ONL

Cdh11+/+ Cdh11-/- Cdh11+/+ Cdh11-/- Cdh11+/+ Cdh11-/- A B C Cdh11+/+ Cdh11-/- Cdh11+/+ Cdh11-/- Cdh11+/+ Cdh11-/- Chx-10 160kDa HPC-1 Cralbp Brud-U Figure 4: No gross differences are revealed in differentiation of retinal cell types, proliferation or expression of cadherin-2 retinae of Cdh11+/+ vs. Cdh11-/- mice All INL cell types were assayed to detect disruptions in retinal phenotype of Cdh11+/+ vs. Cdh11-/-. Retinal cell type markers: Bipolar & progenitor (Chx-10), horizontal (160 kDa), amacrine (HPC-1) and Müller glia (Cralbp) showed no gross change at developmental time points A) ED18.5, B) PND3 and C) PND6. As well, no changes were seen in expression of S-phase cells by Brd-U incorporation or cadherin-2 Cadherin-2

Manually select retina Program measures retinal area in pixels Cornea Retinal pigment Epithelium (folded over) Lens Figure 5: Using ImageJ software to quantify tumor volume in retinas of mice Retina T-antigen stained tumor Manually select retina Program measures retinal area in pixels Change picture type to 8-bit Threshold tumor region and program measures area of threshold region within retina in pixels Excel : Calculate % tumor/retina

Total # of T-Antigen positive cells in retina/eye TAg+/-;Cdh11+/+ TAg+/-;Cdh11+/- TAg+/-;Cdh11-/- C 10 Ratio of T-Antigen positive cells to retinal area B K-W Test: p=0.0118 9 8 Figure 5 (PND8): Cdh11 allelic loss significantly reduces T-antigen cell number (cells of origin of retinoblastoma) A) Representative sections of TAg+/-;Cdh11+/+, TAg+/-;Cdh11+/-, and TAg+/-;Cdh11-/- genotypes by H&E stain and T-Antigen DAB staining. 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 of T-Antigen positive cells of the retina revealed significantly less (p=0.0118) T-Antigen cells (cells of origin of retinoblastoma) when each allele of Cdh11 is lost. C) To account for retinal size, a ratio of T-Antigen cell numbers 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. K-W Test: p=0.0078 TAg+/-; Cdh11+/+ TAg+/- ; Cdh11+/- TAg+/-; Cdh11-/- 7 6 Total # of T-Antigen positive cells in retina/eye TAg+ve cells / retina [cells/pixelsx10^-4] 5 Mouse 1 81 57 43 4 Mouse 2 132 52 19 3 Mouse 3 71 72 29 2 Mouse 4 69 83 1 40 Mouse 5 92 34 46 TAg+/-;Cdh11+/+ TAg+/-;Cdh11+/- TAg+/-;Cdh11-/- Avg: 89 66 33

Percent tumor in retinae of mice at PND28 TAg+/-;Cdh11+/+ TAg+/-;Cdh11+/- TAg+/-;Cdh11-/- B Percent tumor in retinae of mice at PND28 9 K-W Test: p=0.0167 Figure 6 (PND28): Less multifocal tumors develop when Cdh11 alleles are lost A) A distinct phenotype is observed from representative sections of T-Antigen DAB and H&E stains amongst the three genotypes. T-Antigen DAB staining shows far less multifocal tumors present in mice with mutant Cdh11 alleles; H&E shows first signs of rosette formations in Cdh11+/+ describing more developed tumors in Cdh11+/+ vs. Cdh11-/- mice. B) Mice never regain normal phenotype from PND8 as number of multifocal tumors that emerge are significantly less (p=0.0167) in mice with Cdh11 allele loss. Tumor volume was calculated using image J software measuring tumor area (T-Antigen stained region) as a percentage of retinal area (manually traced) of selected sections (approximately 300 µm apart) through the eye. 8 7 6 5 4 % Tumor/retina [pixles] 3 2 1 TAg+/-;Cdh11+/+ TAg+/-;Cdh11+/- TAg+/-;Cdh11-/-

Percent tumors in retinae of mice at PND84 30 B K-W Test p=0.0935 25 TAg+/-;Cdh11+/+ TAg+/-;Cdh11+/- TAg+/-;Cdh11-/- Percent tumors in retinae of mice at PND84 30 B K-W Test p=0.0935 25 Figure 7 (PND84): Tumor volume in mice with Cdh11 null alleles catches up to mice with normal Cdh11, despite less multifocal tumors to start A) Representative H&E and T-Antigen DAB stained sections show large tumors originating from the INL of the retina. Tumors are composed of disorganized cells, rosette formations and disrupted laminated layers due to tumor growth. No gross phenotypical difference is observed amongst the varying genotypes by H&E. B) Tumor volume in the three genotypes show no statistical difference (p=0.0935) amongst the three genotypes, highly suggestive of faster growing tumors in mice with Cdh11 loss, despite fewer tumors to start. Tumor volume was calculated the same as described in Figure 7. 20 % Tumor per retina 15 10 5 TAg+/-;Cdh11+/+ TAg+/-;Cdh11+/- TAg+/-;Cdh11-/-

A C B CDH11 CDH11 CDH11 CDH11 Figure 8: Loss of cadherin-11 protein expression is seen gradually in TAg-RB tumors A) At 4 weeks of age TAg-RB mice display multifocal tumors (clusters) which stain positive for SV40 T-Antigen (green). Some of these multifocal tumors lose cadherin-11 expression (red) (top panel) while some do not (bottom panel), describing a gradual loss of cadherin-11 expression in tumors at this stage. B) At 5 weeks, entire tumor regions that are positive for SV40 TAg are completely negative for cadherin-11 and regions of no tumor retain cadherin-11 expression (arrow) C) By 5 months of age, entire tumor area shows no cadherin-11 expression.

Add pictures Genotypes TAg+/- ;CDH11+/+ TAg+/- ;CDH11-/- Cell Type Marker Hes-5 Caspase-3 Average of 5 mice: 45 51 56 Hes5 and Caspase-3 Positive cells in retinas of mice 80 70 60 Tag+/-; CDH11+/+ Tag+/-; CDH11-/- 50 Number of Positively Stained Cells 40 30 20 Add pictures 10 Hes 5 Caspase-3

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