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+/-**
2230* **
3874* **
6417*
Average
2484
2244
6072
Mouse 5
2496
5179
4388
Mouse 4
2262
4752
5040
Mouse 3
1226
3432
9900
Mouse 2
2683
3762
6683
Mouse 1
Retinal Area
(pixels)
TAg +
(cells)
Retinal Area (pixels)
Cdh11-/-;TAg+/-
Cdh11+/-;TAg+/-
Cdh11+/+;TAg+/-
Figure 4: At PND8, Cdh11 Allelic Loss Significantly Reduces T-antigen Cell Number (cells of origin of retinoblastoma).
A) Representative sections of Cdh11+/+;TAg+/-, Cdh11+/-;TAg+/-, and Cdh11-/-;TAg+/-, 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 illustrates no major phenotypical difference. B, C) Manual counts of T-antigen positive cells extrapolated to the total retina revealed 2-fold and 3-fold less (p=0.01) T-antigen cells (cells of origin of retinoblastoma) when one allele of Cdh11 is lost each time. To account for retinal size, a ratio of T-antigen cell numbers to total retinal area were made. Kruskal-Wallis test was used to assess significance. Error bars represent standard deviations.
K-W Test p=0.01*, K-W Test p=0.83**
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

5. Additional Expts Done: P28-PCNA
Cdh11+/-;TAg+/-
Cdh11-/-;TAg+/-
Cdh11+/+;TAg+/- B
Percent tumor volume in retinas of mice at PND28
% Tumor/retina [pixels]
K-W Test:
Cdh11+/-;
TAg+/-
Cdh11-/-;
Cdh11+/+; 1 2 3 4 5 6 7 8 9
Figure 5: At 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+/+ 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=0.0167) 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. Kruskal Wallis test was used to assess significance. Error bars represent standard deviations.

6. Additional Expts Done: P84-PCNA P84-Caspase-3
Cdh11+/-;TAg+/-
Cdh11-/-;TAg+/-
Cdh11+/+;TAg+/- B
K-W Test p=0.07*, K-W Test p=0.42**
916621* ** * **
422592*
693914**
Average
828190
Mouse 10
770770
Mouse 9
642670
Mouse 8
958984
Mouse 7
365500
558540
201450
Mouse 6
758631
Mouse 5
866768
378864
428038
Mouse 4
707355
298656
329928
Mouse 3
284202
571644
Mouse 2
994686
360756
Mouse 1
(pixels)
Retinal Area
Tumor Area
Cdh11-/-;TAg+/-
Cdh11+/-;TAg+/-
Cdh11+/+;TAg+/-
Figure 6: At PND84, Tumor volume in all Three Genotypes Show no Significant Difference, Describing a Trend of Faster Growing Tumors in Mice with Mutant Cdh11 Alleles.
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), 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 5. Kruskal Wallis test was used to assess significance. Error bars represent standard deviations. C) Tumor growth was calculated by a two-way ANOVA which revealed no significant correlation (p=0.0972), but a strong trend of faster growing tumors in mice with mutated Cdh11 alleles.
Cdh11+/-;
TAg+/-
Cdh11-/-;
Cdh11+/+; C
% Tumor per retina
Percent Total Tumor Volume in Retinas of Mice at PND84
K-W Test
P=0.42 5 1 2 3

7. Chx-10
160kDa
HPC-1
Cdh11+/+ A
Cdh11-/- B C
Cralbp
Brd-U
Cadherin-2
Supplementary Figure 1: No gross differences are 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: 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 BrdU incorporation or cadherin-2 expression.

8. 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 SV40 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 SV40 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.

9. PCNA Clone PC10; Dako, Denmark
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
Additional Expts Done:
P8: Hes 5, Caspase 3, PCNA, Cralbp
PCNA Clone PC10; Dako, Denmark
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.

10. ? 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.

11. 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