1. GCL A
NBL
ED18.5
PND3
PND6 B
GCL
Figure 1: Spatio-temporal expression of cadherin-11 in the murine retina.
A. Cadherin-11 is prominently expressed in the differentiating layer at ED18.5, by migrating cells at 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.
INL
ONL
PND60

2. A B
Merge 40x oil GS
CDH11
Merge 100x oil
Cralbp
CDH11
Chx-10
40x oil
100x oil
160 kDa
Merge 40x oil
CDH11 C
Figure 2: Co-expression of cadherin-11 and retinal cell types of the INL.
A, C. Cadherin-11 expression co-localizes with Müller glia cell bodies (Cralpb, 100x magnification) and Müller cell processes (GS, 40x magnification) and horizontal cells (160 kDa, 40x magnification), B,D but not with amacrine (HPC-1, 40x magnification) or bipolar (Chx-10, 40x and 100x magnificataion) cell types. D
Merge 40x oil
CDH11
HPC-1

3. E18.5 P3 P6
P15
P60 A B
Figure 3: Retinal histology of developing retina in CDH11+/+, CDH11+/- and CDH11-/- littermates.
Hematoxylin and Eosin (H+E) staining of 5 µm sections all 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 C

4. A B C CDH11+/+ CDH11-/- CDH11+/+ CDH11-/- CDH11+/+ CDH11-/- ED18.5
PND3
PND6
Chx- 10
160
kDa
HPC
BrdU B
CDH11+/+
CDH11-/-
NCDH
CDH11+/+
CDH11-/- C
ED18.5
ED18.5
Figure 4: Immunohistochemical analysis of CDH11+/+ vs. CDH11-/- reitnae.
All INL cell types were assayed to detect disruptions in retinal phenotype of CDH11+/+ vs. CDH11-/-. Selections sections were all 5 um think and cut through the optic nerve and lens. A. Retinal cell type markers: Bipolar & progenitor (Chx-10), horizontal (160 kDa), amacrine (HPC-1) and muller glia (Cralbp) showed no gross change at developmental timepoints ED18.5, PND3 and PND6. B, C. As well, expression of S-phase cells via BrdU incorporation and expression of NCDH (cadherin-2) also revealed no change during the 3 developmental time points tested.
PND3
PND3
PND6
PND6

5. Image J quantification
Figure 5: Image J analysis

6. Total # of T-Antigen positive cells in retina/eye
TAg+/- ;CDH11+/+
TAg+/- ;CDH11+/-
TAg+/-;CDH11-/- A B
TAg+/- ;
CDH11+/+
TAg+/- ;
CDH11+/-
TAg+/-;
CDH11-/- C 10
K-W Test:
p=0.0078 9
Total # of T-Antigen positive cells in retina/eye 8
Mouse 1 81 57 43 7
Figure 6 (PND8): T-antigen positive cells (cells of origin of RB) are significantly lower in Tag+/-;CDH11-/- vs. Tag+/-;CDH11+/+.
A. Representative sections of the three genotypes - TAg and H+E stained. 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 were made of T-antigen positive cells of the retina through the entire eye (approximately 7-8 sections, 300 µm apart were counted and tabulated with one eye per mouse analyzed). Numbers reflect the cells of origin of retinoblastoma are far less when each allele of CDH11 is lost, suggesting CDH11 affects the development of this susceptible retinal cell. C. To account for the size of the retina of each genotype, a ratio of T-antigen counts 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. 6
Mouse 2
132 52 19
Tag+ve cells / retina [cells/pixelsx10^-4] 5
Mouse 3 71 72 29 4
Mouse 4 69 83 40 3
Mouse 5 92 34 46 2 1
Avg: 89 66 33
TAg+/-
;CDH11+/+
TAg+/-
;CDH11+/-
TAg+/-
;CDH11-/-
Genotype

7. TAg+/- ;CDH11+/+ TAg+/- ;CDH11+/- TAg+/-;CDH11-/-B
Percent tumors in retinas of mice at PND28
K-W Test:
p=0.0167 9
Figure 7(PND28): Less multifocal tumors develop when CDH11 is lost.
A. A distinct phenotype is observed from representative sections of T-antigen and H+E stained sections amongst the three genotypes. T-antigen staining shows far less multifocal tumors present in CDH11-/- vs. CDH11+/+ mice, as well, H+E staining shows first signs of potential rosette formations in CDH11+/+ describing perhaps more developed tumors in CDH11+/+ vs. CDH11-/- genotype. B. Mice never regain normal phenotype from PND8 as multifocal tumors that emerge are significantly less (p=0.0167) in CDH11-/- vs. CDH11+/+. Tumor volume is calculated using image J software by measuring tumor area (T-antigen stained region) per retinal area (manually traced) of the entire eye. 8 7 6
% Tumor/retina [pixles] 5 4 3 2 1
TAg+/- ;CDH11+/+
TAg+/- ;CDH11+/-
TAg+/-;CDH11-/-
Genotype

8. TAg+/- ;CDH11+/+ TAg+/- ;CDH11+/- TAg+/-;CDH11-/-B
Percent tumors in retinas of mice at PND84
K-W Test
p=0.0602 30
Figure 8 (PND84): Tumor volume of CDH11-/- catches up to CDH11+/+ despite less multifocal tumors to start.
A. Representative H+E and T-antigen stained sections show large tumors originating from the INL of the retina. Tumors show disorganized cells, rosette formations and disrupted laminated layers due to tumor growth. No gross phenotypical difference is observed amongst the varying genotypes. B. Tumor volume of CDH11+/+ genotype is 13% vs. 13.5% and 10% in CDH11+/- and CDH11-/- respectively, describing no statistical difference (p=0.0602) amongst the genotypes. This is highly suggestive of faster growing, perhaps more aggressive tumors in CDH11-/- and CDH11+/- genotypes. Tumor volume is calculated using image J software by measuring tumor area (T-antigen stained region) per retinal area (manually traced) of the entire eye. 25 20
% Tumor per retina 15 10 5
TAg+/- ;CDH11+/+
TAg+/- ;CDH11+/-
TAg+/-;CDH11-/-
Genotype

9. 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

10. Staining for Cralbp, Muller cell marker
6 litters analyzed
Birth:P3
CDH11+/+
CDH11-/- P6
NBL
P6 cralph, CDH11+/+ is C240, -/- is C242

11. CDH11 is gradually lost in tumors of Tag-RB mice
Anti-SV40 lg(Tag)
Anti-CDH11
4 wks
Tag-RB 129/C57Bl-6
4 wks
Tag-RB 129/C57Bl-6
5 wks
Tag-RB 129/C57Bl-6
P28: TH21
5 mth
Tag-RB
C57Bl-6