A B PND3 ED18.5 PND6 PND60 GCL NBL INL ONL Figure 1: Expression of Cadherin-11 in Developing Murine Retina. A) Cadherin-11 is expressed in the differentiating layer at (embryonic 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 that span the entire retina.

160 kDa Merge 40x oil CDH11 HPC-1 D B GS Merge 100x oil Cralbp A C GCL INL ONL Chx-10 40x oil 100x oil Figure 2: Co-expression of Cadherin-11 and Retinal Cell Types in Adult Retina. 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 (HPC-1, 40x magnification) cell types.

ED18.5 PND3 PND6 PND15 PND60 B C A GCL INL ONL Figure 3: Retinal Histology of 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, ED18.5, PND3, PND6, PND15 and PND60 (adult), no gross retinal phenotype was observed between A) Cdh11+/+, B) Cdh11+/-, and C) Cdh11-/- Littermates.

A B C Cdh11+/-;TAg+/- Cdh11-/-;TAg+/- Cdh11+/+; TAg+/- TAg +ve [cells] Retinal Area [pixels] B 1K-W: p=0.83 2K-W: p=0.01 n=5 20E5 40E5 60E5 80E5 100E5 120E5 140E5 160E5 1000 2000 3000 4000 5000 6000 7000 8000 9000 Cdh11+/+;TAg+/- Cdh11+/-;TAg+/- Cdh11-/-;TAg+/- Average Retinal Area and TAg positive cells per Eye at PND8 1 2 Figure 4: At PND8, Cdh11 supports cells of origin of retinoblastoma (Tag positive cells). A) Representative sections of Cdh11+/+;TAg+/-, Cdh11+/-;TAg+/-, and Cdh11-/-;TAg+/-, genotypes by H&E stain and T-antigen 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 reveals no major phenotypical difference. B) Manual counts of T-antigen positive cells and measured retinal area in pixels of selected sections were tabulated per eye and extrapolated to the entire retina. An average of T-antigen positive cells, of five mice per genotype, revealed that the total numbers of cells of origin of retinoblastoma were 2-fold and 3-fold less (p=0.01) when one allele of Cdh11 is lost each time, when compared to mice with normal Cdh11. On average the retinal size per genotype was found be similar (p=0.83) despite Cdh11 loss. C) To further illustrate the quantity of T-antigen positive cells per retina in each genotype, a percentage of the ratio of T-antigen cell numbers to total retinal area were made (p=0.01). The Kruskal-Wallis Test was used to assess significance and error bars represent standard deviations. % TAg +ve cells / retina (cells/pixels) C Ratio of Total TAg-positive Cells to Total Retinal Area K-W: p=0.01 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.10 Cdh11+/+;TAg+/- Cdh11+/-;TAg+/- Cdh11-/-;TAg+/-

A B Cdh11+/-;TAg+/- Cdh11-/-;TAg+/- Cdh11+/+;TAg+/- Percent Tumor Area per Retinal Area at PND28 % Total Tumor Area per Retina [pixels] K-W: p=0.02 1 2 3 4 5 6 7 8 9 Cdh11+/+;TAg+/- Cdh11+/-;TAg+/- Cdh11-/-;TAg+/- Figure 5: At PND28, Less Multifocal Tumors Develop When Cdh11 Alleles are Lost. A distinct phenotype is observed from representative sections of T-antigen and H&E stains amongst the three genotypes. T-antigen staining shows that far less multifocal tumors present in mice with mutant Cdh11 alleles; H&E staining shows first signs of rosette formations in Cdh11+/+ mice 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=0.02) in mice with Cdh11 allelic loss; notably, due to less tumor initiating cells at PND8. Total 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 and then extrapolated to the entire retina. The Kruskal Wallis Test was used to assess significance and error bars represent standard deviations. Additional Expts Done: P28-PCNA

A B C Cdh11+/-;TAg+/- Cdh11-/-;TAg+/- Cdh11+/+;TAg+/- Average Retinal and Tumor Area per Eye at PND84 B 140E5 1K-W p=0.07 2K-W p=0.42 120E5 100E5 Retinal and Tumor Area [pixels] 80E5 Figure 6: At PND84, total tumor volume is similar in all three genotypes. Representative H&E and T-antigen 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) Measured retinal area and tumor area of selected sections were tabulated and extrapolated to the entire retina. On average, total tumor volume in mice per genotype was not statistically different, p=0.42, however, retinal areas appear to be larger when Cdh11 is lost (p=0.07). C) To accommodate for varying retinal size per genotype, total tumor volume was represented as a percentage total retinal area in all mice. Again, we observe no statistical difference (p=0.93) in tumor volume amongst the three genotypes, suggestive of faster growing tumors in mice with Cdh11 loss, despite fewer tumors to start (PND28). Tumor volume was calculated as described in Figure 5. The Kruskal Wallis Test was used to assess significance and error bars represent standard deviations. Additional Expts Done: P84-PCNA P84-Caspase-3 60E5 1 40E5 20E5 2 Cdh11+/+;TAg+/- Cdh11+/-;TAg+/- Cdh11-/-;TAg+/- Percent Tumor Area per Retinal Area at PND84 C 3 2 5 K-W: p=0.93 2 % Total Tumor Area per Retina [pixels] 1 5 1 5 Cdh11+/+;TAg+/- Cdh11+/-;TAg+/- Cdh11-/-;TAg+/-

Boxplot of Tumor Volume From a Single Cell at PND8 to PND84 1200 K-W: p=0.008 1000 800 Tumor Volume per cell 600 400 200 Cdh11+/+;TAg+/- Cdh11+/-;TAg+/- Cdh11-/-;TAg+/- Figure 7: Tumors grow faster in mice with Cdh11 allelic loss. Tumor volume from a single tumor-initiating cell at PND8 to PND84 in all mice of each genotype is illustrated. On average (large grey blocks) tumor volume is approximately 3x more in mice with Cdh11 loss compared to mice with both copies of Cdh11 (p=0.008). B) Boxplot of tumour volume represented by PND84 Tumor Pixels / PND8 Average Tag+ Cells.  The center line within the box represents the median value, while the bottom and top of the box are the 1st and 3rd quartiles (Q1, and Q3) respectively.  The stems extend out to the furthest datapoint within 1.5 Interquartile Range (Q3 - Q1), and outliers are marked by small points. Boxplot of Tumor Volume From a Single Cell at PND8 to PND84 Cdh11+/+;TAg+/- Cdh11+/-;TAg+/- Cdh11-/-;TAg+/- 1000 800 600 400 200 B K-W: p=0.008 Fold increase in tumor growth

Chx-10 160kDa HPC-1 Cdh11+/+ A Cdh11-/- B C Cralbp Brd-U Cadherin-2 Supplementary Figure 1: No gross differences are observed in differentiation of retinal cell types, proliferation or expression of cadherin-2 retinae of Cdh11+/+ Cdh11+/- and Cdh11-/- Littermate Mice. All INL cell types were assayed to detect disruptions in retinal phenotype of Cdh11+/+ vs. Cdh11-/- Littermates. 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. Additionally, no changes were observed in expression of S-phase cells by BrdU incorporation or cadherin-2 expression.

cadherin-11 TAg A B C Supplementary Figure 2: Gradual Loss of Cadherin-11 Expression in TAg-RB Tumors. A) At 4 weeks of age TAg-RB mice display multifocal tumors (clusters) which stain positive for T-antigen (green). Some of these multifocal tumors lose cadherin-11 expression (red) while some retain expression (magnified picture), describing a gradual loss of cadherin-11 expression in tumors at this stage. B) At 5 weeks, entire tumor regions that are positive for T-antigen 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.

Hes5 and Caspase-3 Positive cells in retinas of mice 10 20 30 40 50 60 70 80 Hes-5 Caspase-3 Number of Positively Stained Cells CDH11+/+;TAg+/- CDH11-/-;TAg+/- p = 0.3 p = 0.35 Supplementary Figure 3: Quantitation of Hes-5 and Activated Caspase-3 Positive Cells in Retinae of Cdh11+/+;TAg+/- and Cdh11-/-;TAg+/- mice at PND8. Cells positive for early Müller differentiating marker, Hes-5 and activated caspase-3 were counted in 3 random sections taken from 5 mice per genotype. These counts revealed no significant difference amongst genotypes with Student’s t-test p-values: p=0.3 for Hes-5 counts and p=0.35 for Caspase-3. Error bars reveal standard deviations. Additional Expts Done: P8: Hes 5, Caspase 3, PCNA, Cralbp PCNA Clone PC10; Dako, Denmark

? Antibody Name Company Dilution Used SV40 Tag (Pab 101) mouse monoclonal Santa Cruz Biotechnology Cat# SC-147, Lot# A2506, 1:200 CDH11 - clone CDH113H Gift from Dr. St. John at ICOS Corp. 1:2500 CDH2 (N-cadherin) BD Biosciences Pharmigen Cat# 610920, Lot# 06247 1:2000 BrdU (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 Glia: Hes-5 ABCAM Cat# ab25374 1:50 Müller Glia: CRALBP Rabbit polyclonal Vimentin goat polyclonal Gift from John Saari Cat# Lot# 1:6000 1:100 Ganglion - Brn3b Cat# sc-6026 Amacrine – Syntaxin clone HPC-1 mouse monoclonal Sigma Cat# S0664 Horizontal – 160 kDa ? 1:40 Table 1: Antibody List: This table is informative of all antibodies, corresponding company names and dilutions used in this study.

C Tumor growth from PND28 - PND84 Cdh11+/-;TAg+/- Cdh11-/-;TAg+/- Fold increase tumor growth Tumor growth from PND28 - PND84 C Cdh11+/-;TAg+/- Cdh11-/-;TAg+/- Cdh11+/+;TAg+/- 2 4 6 8 10 12 Two-way ANOVA p=0.0972

C C Ratio of Total TAg-positive cells to total retinal area K-W Test: TAg +ve cells / retina (cells/pixels) C Ratio of Total TAg-positive cells to total retinal area K-W Test: p=0.01* Cdh11+/-; TAg+/- Cdh11-/-; Cdh11+/+; 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.10 C Percent Total Tumor Volume in Retinas of Mice at PND84 K-W Test P=0.42 Cdh11+/-; TAg+/- Cdh11-/-; Cdh11+/+; 5 1 2 3 % Total Tumor Volume per Retina [pixels]

C Tumor growth from PND28 - PND84 Cdh11+/-;TAg+/- Cdh11-/-;TAg+/- Fold increase tumor growth Tumor growth from PND28 - PND84 C Cdh11+/-;TAg+/- Cdh11-/-;TAg+/- Cdh11+/+;TAg+/- 2 4 6 8 10 12 Two-way ANOVA p=0.0972 Tumor Growth from a single cell at PND8 to PND84 450 400 350 300 Fold increase tumor growth 250 200 150 100 50 Cdh11+/+;TAg+/- Cdh11+/-;TAg+/- Cdh11-/-;TAg+/- 200 400 600 800 1000 1200 Fold increase tumor growth Cdh11+/+;TAg+/- Cdh11+/-;TAg+/- Cdh11-/-;TAg+/- Tumor Growth from a single cell at PND8 to PND84

Avg Retinal Area [pixels] Avg Tag +ve [cells] 20E5 40E5 60E5 80E5 100E5 120E5 140E5 160E5 1000 2000 3000 4000 5000 6000 7000 8000 9000 Avg Retinal Area [pixels] Cdh11+/+;TAg+/- Cdh11+/-;TAg+/- Cdh11-/-;TAg+/-

B K-W Test p=0.42*, K-W Test p=0.07**

200 400 600 800 1000 1200