1. A B PND3 ED18.5 PND6 PND60 GCL NBL INL ONL
Expression of Cadherin-11 in Developing 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 that span the eitire 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
Co-expression of Cadherin-11 and Retinal Cell Types in Adult Retina.
A, B) Cadherin-11 expression co-localizes with Muller glia cell bodies (Cralbp, 100x magnification), Muller 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.

3. ED18.5
PND3
PND6
PND15
PND60 B C A
GCL
INL
ONL
Retinal Histology of Cdh11+/+, Cdh11+/-, and Cdh11-/- Littermates.
Hematoxylin and Eosin (H&E) staining of 5 um 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. Chx-10
160kDa
HPC-1
Cdh11+/+ A
Cdh11-/- B C
Cralbp
Brd-U
Cadherin-2
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 Muller 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 expression.

5. A B Technique for Quantifying Tumor Volume in Retinae of Mice
Cornea
Retinal
pigment
epithelium
(folded over)
Technique for Quantifying Tumor Volume in Retinae of Mice
A) One section every 60th section (green lines), such that 5-6 sections were analyzed per eye make to better estimate tumor volume. B) These selected sections were immuno-stained for T-antigen (representative of tumor area) using DAB for visualization in bright field. Using Image J software, retinae of mice were manually traced (yellow) and measured as retinal area in pixels. The image was then converted to an 8-bit grey scale, and using a manually selected threshold tool, the tumor area (DAB stained) within the selected retina was highlighted and measured by the program in pixels. Total retina and tumor areas of all selected sections per retina in one eye per animal were estimated calculating for percent tumor volume, (tumor area in pixels/retina area in pixels) * 100.
Lens
Retina
T-antigen
stained
tumor

6. cadherin-11
TAg A B C
Gradual Loss of Cadherin-11 Expressionn 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.

7. Total # of T-Antigen positive cells in retina/eye
Cdh11+/-;
TAg+/-
Cdh11-/-;
Cdh11+/+;
Cdh11+/-;TAg+/-
Cdh11-/-;TAg+/-
Cdh11+/+; TAg+/- A 1 2 3 4 5 6 7 8 9 10
TAg+ve cells / retina
(cells/pixelsx10^-4) B
Total # of T-Antigen positive cells in retina/eye
Mouse 1
Mouse 3
Mouse 4
Mouse 5
Avg:
Mouse 2 81
132 71 69 92 89 57 52 72 83 34 66 43 19 29 40 46 33
K-W Test:
p=0.0118 C
p=0.0078
Ratio of Tag-positive cells to retinal area
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 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 retinal 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. Kruskal-Wallis test was used to assess significance. Error bars represent standard deviations.

8. 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
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 Muller 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

9. A B Percent tumors in retinas of mice at PND28 Cdh11+/-;TAg+/-1 2 3 4 5 6 7 8 9
% Tumor/retina [pixles]
K-W Test:
p=0.0167
Cdh11+/-;TAg+/-
Cdh11-/-;TAg+/-
Cdh11+/+;TAg+/-
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 um apart) through the eye. Kruskal Wallis test was used to assess significance. Error bars represent standard deviations.

10. A B Cdh11+/+;TAg+/- Cdh11+/-;TAg+/- Cdh11-/-;TAg+/-
Percent tumors in retinas of mice at PND84 B 30
At PND84, Tumor volume in all Three Genotypes Show no Significant Difference.
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 . Kruskal Wallis test was used to assess significance. Error bars represent standard deviations. 25
K-W Test
p=0.0935 20
% Tumor per retina 15 10 5
Cdh11+/+;TAg+/-
Cdh11+/-;TAg+/-
Cdh11-/-;TAg+/-

11. Tumor growth from PND28 - PND84
TAg+/-;Cdh11+/+
TAg+/-;Cdh11+/-
TAg+/-;Cdh11-/- 1 2 3 4 5 6 7 8 9 10
Fold increase
tumor growth
Tumor growth from PND28 - PND84
Increase in Tumor Growth from PND28-84.
Tumor growth was calculated by dividing percent tumor growth at PND84 by percent tumor growth at PND28 to get a fold increase in tumor growth. This figure clearly depicts faster growing tumors in mice with mutated Cdh11 alleles.

12. 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# , Lot# 06247
1:2000
Brd-U (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: 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
Antibody List: This table is informative of all antibodies, corresponding company names and dilutions used in this study.

13. . PND8 PND28 PND84 TAg+/-; Cdh11+/+ Cdh11-/- tumor foci
Retinoblastoma
Tumor
faster
progression
tumor .
tumor foci
cells of origin of RB
lose
cadherin-11