Peripheral, central and total RPE cell cultures have different proliferation profiles.

<p>Peripheral, central and total RPE cells were cultured for 9 days then received a single 4-hour BrdU pulse prior to fixation. Representative microphotographs of BrdU-labelled cultures of (A) peripheral RPE cells and (B) central RPE cells. White arrows indicate pigmented BrdU⁺ cells. (C) A graph indicating the number of BrdU⁺ cells in cultures harvested from each region (Peripheral: circles. Central: squares. Total: triangles). Mann-Whitney Test; *p = 0.02, **p = 0.04. No statistical significance was found between central and total RPE BrdU⁺ cells counts. Error bars = SEM. Peripheral and central RPE cells were cultured as above for 3 days, fixed and immuno-stained with Ki67. Representative microphotographs of Ki67⁺ cells of (D) peripheral cells and (E) central cells. White arrows indicate Ki67⁺ cells. The number of Ki67⁺ cells counted from each region is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018921#pone-0018921-g002" target="_blank">Figure 2F</a> where cell densities in the two cultures were similar (Periphery: circle points. Central; square points). Mann-Whitney Test; *p = 0.05. Error bars = SD. In A–C, when labelled cell numbers are normalised against the total cell number for the two regions the differences remain significantly different (Mann-Whitney Test; BrdU⁺/total cell number - Periphery against Centre; p = 0.037 and Ki67⁺/total cell number - Periphery against Centre; p = 0.0237). In vivo protein expression of active (phosphorylated) beta-catenin in peripheral and central RPE (G). N = 3 eyes. Scale bars = 5 µm.</p

Gene and protein expression profile in peripheral and central RPE tissue of E-Cadherin.

<p>Gene expression of E-Cadherin, in RPE tissue obtained from the periphery and centre of adult mouse RPE in vivo (A) and in vitro (B). Graph shows relative expression levels from independent samples normalized to ACTB (n is at least 4 from each region). Protein expression of E-Cadherin (FITC) on adult mouse RPE flatmounts (C) and a graph showing FITC/pixel intensity from each RPE region (D). Central cells consistently expressed greater levels of E-Cadherin than those in the periphery. Inserts show individual RPE cells in higher magnification. N = 3 eyes. Scale bar = 5 µm. Mann-Whitney Test, * P<0.05, **p<0.002. Error bars = SEM.</p

Central RPE cells do not inhibit peripheral RPE cells from proliferating via diffusible signals.

<p>RPE cultures were setup from peripheral and central RPE and were allowed to proliferate at low density for one week, before peripheral RPE cultures were introduced to central RPE medium for two days before a 4-hour BrdU pulse. (A) The graph indicates the number of BrdU⁺ cells per region per total cell number. Periphery (circle points), central (square points) and control periphery (triangle points). Cell number per culture; 5,000 cells/well. Mann-Whitney Test; *p = 0.03. **p = 0.02. Peripheral RPE versus control RPE periphery was not found to be statistically significant (p = 0.68). Error bars = SD. (B) RPE cultures were setup from peripheral and central RPE and allowed to proliferate at high density for one week, before peripheral RPE cultures were introduced to central RPE medium for two days before a 4-hour BrdU pulse. The graph indicates the number of BrdU⁺ cells per region. Periphery (circle points), central (square points) and control periphery (triangle points). Cell number per culture; 10,000 cells/well. Error bars = SD.</p

Quantitative Real-Time PCR analysis of gene expression in peripheral and central RPE tissue and cell cultures.

<p>Gene expression of p27^kip1 (Ai.), Cyclin D1 (Bi.), and mTOR (Ci.), in RPE tissue obtained from the periphery and centre of adult mouse RPE. Gene expression of p27^kip1 (Aii.), Cyclin D1 (Bii.), and mTOR (Cii.) in cultured cells obtained from the periphery and centre of adult mouse RPE. Graphs show relative gene expression levels from independent samples normalized to ACTB (at least 6 independent samples obtained from each region); Mann-Whitney Test, * P<0.05 in each case.</p

Protein expression profile in peripheral and central RPE tissue of F-actin and ZO-1 junctional markers.

<p>Phalloidin (F-actin) dye (FITC) expression on adult mouse RPE flatmounts (A) and a graph showing FITC/pixel intensity from each RPE region (B). Greater levels of F-actin were present on junctions centrally than in the periphery. Protein expression profile of ZO-1 in peripheral and central RPE (C). Inserts show individual RPE cells in higher magnification. ZO-1 is distributed less regularly in the periphery that in the centre. N = 3 eyes. Scale bar = 5 µm. Mann-Whitney Test, **p<0.002. Error bars = SEM.</p

Peripheral RPE cells proliferate in vivo and in vitro.

<p>The adult mouse RPE was investigated to confirm it contains a peripheral region where cell division occurs. Animals were given daily BrdU injections for 5 consecutive days and were sacrificed 1, 2, 3 and 4 weeks after the last injection day. (A) Photomicrograph of the RPE flatmount indicating, in the circle, the peripheral region in which BrdU⁺ RPE cells were found. The dotted white line illustrates the peripheral and central RPE zones. (B) Higher magnification showing cells positive for BrdU (in red). (C) RPE cells were cultured for 9 days and on final day they received a single 4-hour BrdU pulse prior to fixation. The majority of cells expressed Otx2 (RPE-specific marker, red), with a number of cells also co-expressing BrdU (green). Scale bar = 5 µm. N (number of eyes examined) = 3.</p

Schematic graph of the adult mouse RPE and characteristics of peripheral and central RPE cells.

<p>Peripheral and Central RPE cells express different levels of genes that play crucial role in cell proliferation. The levels of protein and gene expression for key cell adhesion molecules is also different between these two RPE populations, indicating distinct cell-cell contact behaviour.</p

Pathway analysis of transcription factors via DAVID Functional Annotation.

<p>Transcription factors expressed in young CEC-DM, old CEC-DM and CEC culture (RPM>10) that are more than two-fold higher in young CEC-DM versus stroma, were analyzed in DAVID. Pathways that are over represented are ranked in descending order of percentage of genes representing this category.</p

Genes not expressed in the corneal stroma.

<p>Gene list is filtered for RPM>10 for CEC Young, CEC Old and CEC Culture, and RPM<1 for corneal stroma. Genes are ranked in descending order of RPM values of CEC Young. All values are rounded off to 2 decimal places.</p

High-throughput QPCR analysis of genes lowly or not expressed in corneal stroma.

<p>BM: bone marrow; IVS: interventricular septum; SG: salivary gland; SM: skeletal muscle; SI: small intestine; SC: spinal cord; U/C: uterus/cervix; FB: fetal brain; FL: fetal liver; SF: stromal fibroblast; CC: CEC culture; CEC-DM-y: CEC-DM young; CEC-DM-o: CEC-DM-old.</p