1. Figure 1 A B GCL NBL ED18.5 PND3 PND6 GCL
Figure 1: Expression of Cadherin-11 in Developing Murine Retina.
A) Cadherin-11 was expressed in the differentiating layer at (embryonic day) ED18.5, by migrating cells at (post natal day) PND3 and again in an area where differentiating cells are present PND6. B) In adults, (PND60) cadherin-11 expression was restricted to cell types of the INL, with high expression by Müller glia processes that span the entire retina. These studies were performed using a different and more specific antibody than previously used in Marchong et al, 2004.
INL
ONL
PND60
Figure 1

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. Brenda: I don’t understand what needs to be added/changed based on reviewer#2’s comment.
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 arrows) cells.
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
Figure 2

3. Complete At home: Delete first 4 & put in colour as per reviewer#2
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 papillary-optic nerve plane. At developmental time points, ED18.5, PND3, PND6, PND15 (data not shown) and PND60 (adult), no gross retinal phenotypic differences were observed between A) Cdh11+/+, B) Cdh11+/-, and C) Cdh11-/- Littermates.
Figure 3
Complete At home: Delete first 4 &
put in colour as per reviewer#2

4. Figure 4 A B C cadherin-11 TAg
Figure 4: 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 TAg (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 TAg 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.
Figure 4

5. Figure 5 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]
9000
8000
7000
Figure 5: 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 TAg staining. The single TAg-positive cells in the INL of the retina 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 TAg-positive cells per retinal area were extrapolated to the entire retina. The total number of TAg-positive cells of origin of retinoblastoma was 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 was similar (p=0.83) between the Cdh11 genotypes. C) The ratio of TAg-positive 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. 2
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
% TAg +ve cells / retina
(cells/pixels)
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0.00
Cdh11+/+;TAg+/-
Cdh11+/-;TAg+/-
Cdh11-/-;TAg+/-
Figure 5

6. Figure 6 A B Cdh11+/+;TAg+/- Cdh11+/-;TAg+/- Cdh11-/-;TAg+/-
Percent Tumor Area per Retinal Area at PND28
Figure 6: At PND28, fewer multifocal tumors developed when Cdh11 alleles were lost.
A) A distinct Cdh11 loss phenotype was observed from representative sections of TAg and H&E stains. Fewer TAg-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 (TAg stained region) as a percentage of retinal area (manually traced) for every 60th section (approximately 300 µm apart) through the eye and extrapolated to the entire retina. The Kruskal Wallis Test was used to assess significance and error bars represent standard deviations. 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+/-
Figure 6

7. Figure 7 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 7: At PND84, total tumor volume was similar in all three genotypes.
A) Representative H&E and TAg 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 differences were observed in different genotypes on H&E stained sections. B) Retinal area and tumor area of every 60th section were tabulated and extrapolated to the entire retina. Total tumor volume per genotype was not statistically different (p=0.42), but total retinal areas tended to be larger when Cdh11 was 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, since there were fewer tumor-originating cells and consequently fewer multifocal tumors initially (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.
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
Figure 7

8. Boxplot of Tumor Volume at PND84 from a Single PND8 Cell
Cdh11+/+;TAg+/-
Cdh11+/-;TAg+/-
Cdh11-/-;TAg+/-
1000
800
600
400
200
K-W: p=0.008
Fold increase in tumor growth
Boxplot of Tumor Volume at PND84 from a Single PND8 Cell
Figure 8A: Tumors grew faster in mice with Cdh11 allelic loss. Single tumor initiating cells at PND8 and advanced tumor at PND84 were best represented by average TAg-positive cells per genotype at PND8 and total tumor volume in pixels at PND84, respectively. Due to technical reasons, the same mouse cannot be evaluated at both time points to calculate actual tumor volume per mouse. Thus, to study tumor volume from a single cell at PND8 to advanced tumor at PND84, we estimated a baseline number of tumor initiating cells per retina by averaging the total number of TAg-positive cells over the 5 mice per genotype. The ratio of tumor volume in pixels at PND84 to the average number of TAg-positive cells for the three genotypes at PND8, estimates the tumor volume (large grey blocks) and describes a significant increase in volume (3 fold, p=0.008) in mice with Cdh11 loss compared to mice with both copies of Cdh11. Since we observed a near significant trend to larger overall retinal size in the absence of Cdh11 alleles at PND84 (Figure 6B), we also compared the ratio of total tumor volume per retina at PND84 to total TAg-positive cells per retina for each genotype (data not  shown). After controlling for the trend to larger retinas, loss of Cdh11 alleles showed significantly larger tumor volumes.  B) Cdh11 facilitates cell death in TAg tumors.
Every 60th section of PND84 eyes was counted for cells positive for activated caspase-3, extrapolated to the entire retina and represented as a ratio to tumor volume per eye. The number of dying cells in tumors of mice with normal Cdh11 alleles show wide variation, but are significantly more abundant (p<0.037) than in mice with mutant Cdh11 alleles. (Make as one figure with the staining and data on PCNA?) B
Figure 8

9. Supplementary Figure 1 Cdh11+/+ A Cdh11-/- B C
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.
Supplementary Figure 1

10. Figure 2: At PND84, proliferation, as determined by PNCA expressing cells, were similar in both genotypes. I present this as supplementary data b/c I think we need to should show some sort of proliferation assay especially since we conclude that it is not thru prolif, rather it is thru apoptosis!
In this small study, two animals per genotype were analyzed for the proliferation marker PCNA. We show that on average the %PCNA cells with respect to tumor area is similar (p<0.113) in mice with normal Cdh11 alleles and mice with mutant Cdh11 alleles.
Supplementary Figure 2

11. Hes5 and Caspase-3 Positive cells in retinas of mice80
p = 0.3
p = 0.35 70 60
CDH11+/+;TAg+/- 50
CDH11-/-;TAg+/-
Number of Positively Stained Cells 40 30 20 10
Hes-5
Activated
Caspase-3
Supplementary Figure 3: Quantitation of Hes-5 and Activated Caspase-3 Positive Cells in Retinae of Cdh11+/+;TAg+/- and Cdh11-/-;TAg+/- mice at PND8. is this really necessary? Should we omit it? I don’t think it adds much to the ppr.
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).
Supplementary Figure 3