1. A
GCL
NBL
ED18.5
PND3
PND6 B
GCL
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.
INL
ONL
PND60

2. A
GCL
INL
ONL GS
CDH11
Merge 40x oil
INL
Cralbp
CDH11
Merge 100x oil B
INL
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) (white arrow) cell types.
160 kDa
CDH11
Merge 40x oil
ONL
GCL C
INL
ONL
Chx-10
40x oil
CDH11
40x oil
Chx-10
100x oil
CDH11
INL D
INL
HPC-1
CDH11
ONL

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 phenotypic differences were observed between A) Cdh11+/+, B) Cdh11+/-, and C) Cdh11-/- Littermates.

4. A B C Cdh11+/+; TAg+/- Cdh11+/-;TAg+/- Cdh11-/-;TAg+/-
Average Retinal Area and TAg positive cells per Eye at PND8 B
20E5
40E5
60E5
80E5
100E5
120E5
140E5
160E5
1K-W: p=0.83
2K-W: p=0.01
n=5 1
Retinal Area [pixels]
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 reduced in number with reduced Cdh11 allele dosage . H&E staining reveals no major phenotypical difference between the three genotypes. 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. The total number of T-antigen positive cells cells of origin of retinoblastoma were 2-fold and 3-fold less (p=0.01) when one or two alleles of Cdh11 were lost respectively, compared to mice with normal Cdh11. The retinal size per genotype was similar (p=0.83) between the Cdh11 geneotypes. C) The ratio of numbers of T-antigen postive cells to total retinal area was significantly reduced with reduced Cdh11 gene dose (p=0.0098). The Kruskal-Wallis Test was used to assess significance and error bars represent standard deviations.
9000
8000 2
7000
6000
TAg +ve [cells]
5000
4000
3000
2000
1000
Cdh11+/+;TAg+/-
Cdh11+/-;TAg+/-
Cdh11-/-;TAg+/- C
Ratio of Total TAg-positive Cells to Total Retinal Area
0.10
K-W: p=0.01
0.09
0.08
0.07
% TAg +ve cells / retina
(cells/pixels)
0.06
0.05
0.04
0.03
0.02
0.01
0.00
Cdh11+/+;TAg+/-
Cdh11+/-;TAg+/-
Cdh11-/-;TAg+/-

5. A B Cdh11+/+;TAg+/- Cdh11+/-;TAg+/- Cdh11-/-;TAg+/-
Percent Tumor Area per Retinal Area at PND28
Figure 5: At PND28, fewer multifocal tumors developed when Cdh11 alleles were lost.
A) A distinct Cdh11 loss phenotype is observed from representative sections of T-antigen and H&E stains. Fewer T-antigen positive multifocal tumors were present in mice with mutant Cdh11 alleles; H&E showed more advanced tumors in Cdh11+/+ mice than in Cdh11+/- or Cdh11-/- mice. B) The number of multifocal tumors was significantly less (p=0.0155) in mice with Cdh11 allelic loss, correlating with fewer 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 9 8
K-W: p=0.02 7 6
% Total Tumor Area
per Retina [pixels] 5 4 3 2 1
Cdh11+/+;TAg+/-
Cdh11+/-;TAg+/-
Cdh11-/-;TAg+/-

6. A B C Cdh11+/+;TAg+/- Cdh11+/-;TAg+/- Cdh11-/-;TAg+/-
Average Retinal and Tumor Area per Eye at PND84
Retinal and Tumor
Area [pixels]
1K-W p=0.07
2K-W p=0.42
20E5
40E5
60E5
80E5
100E5
120E5
140E5
Cdh11+/+;TAg+/-
Cdh11+/-;TAg+/-
Cdh11-/-;TAg+/- 1 2 B
Figure 6: At PND84, total tumor volume was similar in all three genotypes.
A) Representative H&E and T-antigen stained sections showed large tumors originating from the INL of the retina. Tumors were composed of disorganized cells, rosette formations and disrupted laminated layers due to tumor growth. No gross phenotypical difference was observed between genotypes by H&E. B) Retinal area and tumor area of selected sections were tabulated and extrapolated to the entire retina. Total tumor volume in mice per genotype was not statistically different (p=0.42), but total retinal areas tended 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, showing no statistical difference (p=0.0935) in tumor volume with genotype. This suggested 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.
P84-PCNA
P84-Caspase-3
Percent Tumor Area per Retinal Area at PND84
K-W: p=0.93 5 1 2 3
% Total Tumor Area
per Retina [pixels]
Cdh11+/+;TAg+/-
Cdh11+/-;TAg+/-
Cdh11-/-;TAg+/- C

7. Boxplot of Tumor Volume at PND84 from a Single PND8 Cell
Tumor Volume at PND84 from a PND8 Single TAg Cell
1200
1000
K-W: p=0.008
800
Tumor Volume per TAg cell
600
400
200
Cdh11+/+;TAg+/-
Cdh11+/-;TAg+/-
Cdh11-/-;TAg+/-
Figure 7: Tumors grew faster in mice with Cdh11 allelic loss.
A) Tumor growth from a single tumor initiating cell at PND8 to PND84 in all mice of each genotype is illustrated. Tumor growth (large grey blocks) was approximately 3-fold faster in mice with Cdh11 loss compared to mice with both copies of Cdh11 (p=0.008). B) Statistical representation of tumor growth from PND8 to PND84 (median values of tumor growth per genotype).
Cdh11+/+;TAg+/-
Cdh11+/-;TAg+/-
Cdh11-/-;TAg+/-
1000
800
600
400
200
K-W: p=0.008 B
Fold increase in tumor growth
Boxplot of Tumor Volume at PND84 from a Single PND8 Cell

8. Chx-10
160kDa
HPC-1
Cdh11+/+ A
Cdh11-/- B C
Cralbp
Brd-U
Cadherin-2
Supplementary Figure 1: No gross differences were revealed 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 for bipolar & progenitor (Chx-10), horizontal (160 kDa), amacrine (HPC-1) and Müller glia (CRALBP) showed no significant change at developmental time points A) ED18.5, B) PND3 and C) PND6. As well, no changes were seen in numbers 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 displayed multifocal tumors (clusters) which stained positive for SV40 T-antigen (green). Some of these multifocal tumors lost cadherin-11 expression (red) while some retained expression (magnified picture), suggesting a gradual loss of cadherin-11 expression in tumors at this stage. B) At 5 weeks, regions of tumors that were positive for SV40 T-antigen were completely negative for cadherin-11 and regions of no tumor retained cadherin-11 expression (arrow). C) By 5 months of age, entire tumors showed 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 between genotypes (Student’s t-test p-values: p=0.3 for Hes-5 counts and p=0.35 for Caspase-3; error bars indicate 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.