1. 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.

2. 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.

3. 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.

4. 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+/-

5. 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

6. 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+/-

7. 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

8. 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.

9. 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.

10. Hes5 and Caspase-3 Positive cells in retinas of mice10 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

11. ? 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# , Lot# 06247
1:2000
BrdU (purified anti-bromodeoxyuridine)
Cat# , 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.

12. 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

13. 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]

14. 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

15. 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+/-

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

17. 200
400
600
800
1000
1200