1. A B GCL NBL ED18.5 PND3 PND6 GCL INL
Figure 1: Spatio-temporal expression of cadherin-11 in the 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 throughout the retina
ONL
PND60

2. A
GCL C
INL
ONL
Chx-10
40x oil
CDH11
40x oil GS
CDH11
Merge 40x oil
INL
INL
Chx-10
100x oil
CDH11
Cralbp
CDH11
Merge 100x oil D B
INL
160 kDa
CDH11
Merge 40x oil
ONL
HPC-1
CDH11
Figure 2: Co-expression of cadherin-11 and retinal cell types of the inner nuclear layer
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 (syntaxin, 40x magnification) cell types

3. E18.5 P3 P6
P15
P60
GCL A
INL
ONL
GCL B
INL
ONL
GCL C
Figure 3: Histology of developing retina in 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, E18.5, P3, P6, P15 and P60 (adult), no gross retinal phenotype was observed between Cdh11+/+ (A), Cdh11+/- (B) and Cdh11-/- (C) littermates
INL
ONL

4. Cdh11+/+ Cdh11-/- Cdh11+/+ Cdh11-/- Cdh11+/+ Cdh11-/-A B C
Cdh11+/+
Cdh11-/-
Cdh11+/+
Cdh11-/-
Cdh11+/+
Cdh11-/-
Chx-10
160kDa
HPC-1
Cralbp
Brud-U
Figure 4: No gross differences are revealed in differentiation of retinal cell types, proliferation or expression of cadherin-2 retinae of Cdh11+/+ vs. Cdh11-/- mice
All INL cell types were assayed to detect disruptions in retinal phenotype of Cdh11+/+ vs. Cdh11-/-. 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 Brd-U incorporation or cadherin-2
Cadherin-2

5. Manually select retina Program measures retinal area in pixels
Cornea
Retinal
pigment
Epithelium
(folded over)
Lens
Figure 5: Using ImageJ software to quantify tumor volume in retinas of mice
Retina
T-antigen
stained
tumor
Manually select retina
Program measures retinal
area in pixels
Change picture type to 8-bit
Threshold tumor region
and program measures area of
threshold region within retina
in pixels
Excel : Calculate % tumor/retina

6. Total # of T-Antigen positive cells in retina/eye
TAg+/-;Cdh11+/+
TAg+/-;Cdh11+/-
TAg+/-;Cdh11-/- C 10
Ratio of T-Antigen positive cells to retinal area B
K-W Test:
p=0.0118 9 8
Figure 5 (PND8): Cdh11 allelic loss significantly reduces T-antigen cell number (cells of origin of retinoblastoma)
A) Representative sections of TAg+/-;Cdh11+/+, TAg+/-;Cdh11+/-, and TAg+/-;Cdh11-/- 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 retina 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.
K-W Test:
p=0.0078
TAg+/-;
Cdh11+/+
TAg+/- ;
Cdh11+/-
TAg+/-;
Cdh11-/- 7 6
Total # of T-Antigen positive cells in retina/eye
TAg+ve cells / retina
[cells/pixelsx10^-4] 5
Mouse 1 81 57 43 4
Mouse 2
132 52 19 3
Mouse 3 71 72 29 2
Mouse 4 69 83 1 40
Mouse 5 92 34 46
TAg+/-;Cdh11+/+
TAg+/-;Cdh11+/-
TAg+/-;Cdh11-/-
Avg: 89 66 33

7. Percent tumor in retinae of mice at PND28
TAg+/-;Cdh11+/+
TAg+/-;Cdh11+/-
TAg+/-;Cdh11-/- B
Percent tumor in retinae of mice at PND28 9
K-W Test:
p=0.0167
Figure 6 (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+/+ 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. 8 7 6 5 4
% Tumor/retina [pixles] 3 2 1
TAg+/-;Cdh11+/+
TAg+/-;Cdh11+/-
TAg+/-;Cdh11-/-

8. Percent tumors in retinae of mice at PND84 30 B K-W Test p=0.0935 25
TAg+/-;Cdh11+/+
TAg+/-;Cdh11+/-
TAg+/-;Cdh11-/-
Percent tumors in retinae of mice at PND84 30 B
K-W Test
p=0.0935 25
Figure 7 (PND84): Tumor volume in mice with Cdh11 null alleles catches up to mice with normal Cdh11, despite less multifocal tumors to start
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) amongst the three genotypes, 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 7. 20
% Tumor per retina 15 10 5
TAg+/-;Cdh11+/+
TAg+/-;Cdh11+/-
TAg+/-;Cdh11-/-

9. A C B
CDH11
CDH11
CDH11
CDH11
Figure 8: Loss of cadherin-11 protein expression is seen gradually 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) (top panel) while some do not (bottom panel), 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 TAg 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. Add pictures Genotypes TAg+/- ;CDH11+/+ TAg+/- ;CDH11-/-
Cell Type Marker
Hes-5
Caspase-3
Average of 5 mice: 45 51 56
Hes5 and Caspase-3 Positive cells in retinas of mice 80 70 60
Tag+/-; CDH11+/+
Tag+/-; CDH11-/- 50
Number of Positively Stained Cells 40 30 20
Add pictures 10
Hes 5
Caspase-3

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
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
Table 1: Antibody List