Quantification of BrdU positive RPE cells.

<p>BrdU positive RPE cells and total RPE cell numbers were counted in the middle of the treated area of retinal cryosections obtained from subretinally injected mice 5 days after vector administration at a titre of 10⁷ T.U./ml. Following injection of LNT.shZO-1 or LNT.ZONAB BrdU positive cells increased to 2.4% and 2.0% of total RPE cell number, respectively, whereas very few BrdU positive cells were identified in LNT.shGFP (0.05%) treated eyes. (*<b><i>P</i></b><0.001 compared with LNT.shGFP control. Student's <i>t</i>-test. n = 4 [20 measurements from 4 eyes per treatment group]).</p

Downregulation of ZO-1 or overexpresion of ZONAB induces RPE pyknosis and breaks.

<p>Two features were assessed: RPE pyknosis and RPE breaks 10 days after subretinal injection of vectors at 10⁸ T.U./ml. Pyknotic RPE cells were defined as hyperpigmented cells that were not within a continuous monolayer. Percentage of retinal sections in which RPE pyknosis and breaks were observed is plotted. LNT.shZO-1 and LNT.ZONAB treated eyes contained many pyknotic RPE cells. RPE breaks or cell loss occurred adjacent to pyknotic RPE areas in LNT.shZO-1 or LNT.ZONAB treated eyes, respectively. (n = 4, 20 measurements from 4 eyes per treatment group).</p

Lentivirally-mediated modulation of ZO-1 and ZONAB expression.

<p>Retinal cryosections were obtained from eyes 5 days after the subretinal injections of LNT.shGFP (<b>A,B</b>), LNT.ZONAB (<b>C,D</b>) or LNT.shZO-1 (<b>E,F</b>) at 10⁷ T.U./ml. Immunostaining was performed using antibodies against ZONAB (left panel) and ZO-1 (right panel). Following injection of LNT.shGFP, ZONAB can only be detected at low levels in the RPE (<b>A</b>) and ZO-1 was observed in the RPE as apical dots depending on the section (<b>B</b>). Subretinal injection of LNT.ZONAB resulted in an elevation of ZONAB levels in RPE cells (<b>C</b>) and did not affect ZO-1 levels (<b>D</b>). Depletion of ZO-1 expression resulted in a slight increase in ZONAB expression (<b>E</b>) and decreased of ZO-1 expression (<b>F</b>) compared with control eyes (<b>B</b>). Nuclei were counterstained with DAPI. White arrows, RPE monolayer. Size bar, 20 µm. n = 4 per treatment group.</p

Manipulation of ZO-1 and ZONAB expression increases RPE cell proliferation.

<p>Retinal cryosections were obtained from eyes 5 days after the subretinal injections of LNT.shGFP (<b>A,B</b>), LNT.ZONAB (<b>C,D</b>) or LNT.shZO-1 (<b>E,F</b>) at 10⁷ T.U./ml. BrdU was injected intraperitoneally following the subretinal vector administration. Immunostaining was performed using antibodies against RPE65 (left panel) and BrdU (right panel). In eyes injected with LNT.shGFP, high levels of RPE65 were evident in RPE cells (<b>A</b>) and there was no evidence of proliferation judged by the absence of BrdU staining (<b>B</b>). Overexpression of ZONAB did not change RPE65 levels (<b>C</b>) but increased the number of BrdU positive cells, indicative of proliferation (<b>D</b>, white arrowhead). Delivery of LNT.shZO-1 did not affect RPE65 expression (<b>E</b>) but increased BrdU positive cells suggesting RPE proliferation (<b>F,</b> white arrowhead). Nuclei were counterstained with DAPI. Size bar, 20 µm. n = 4 per treatment group.</p

Downregulation of ZO-1 or overexpresion of ZONAB induce changes in fluorescein angiograms.

<p>Late-phase fluorescein angiograms of RNAi-treated eyes were obtained at 10, 20 and 30 days after subretinal injection of vectors (10⁸ T.U./ml). No abnormal changes in fluorescence were observed in LNT.shGFP injected eyes. Marked hyperfluorescence, indicating RPE cell loss, was seen in either LNT.shZO-1 or LNT.ZONAB treated eyes. Increasing intensity of the hyperfluorescence between timepoints suggests that RPE cell loss progressed over time. n = 4 per treatment group.</p

Ultrastructure of the RPE after 10 days following subretinal injection of vectors at 10⁸ T.U./ml.

<p>In LNT.shGFP treated eyes (<b>A</b>), the RPE monolayer lies on Bruch's membrane, has apical microvilli towards the photoreceptor outer segments the cells are interconnected by tight junctions (<b>A,</b> black arrow. <b>B</b>, white arrows). In eyes either with depleted levels of ZO-1 (<b>C</b>) or overexpressing ZONAB (<b>D</b>), the RPE monolayer was highly disorganised with a marked loss of the epithelial monolayer characteristics, areas of RPE cells located on top of each other (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015730#pone-0015730-g006" target="_blank">Figure 6</a>: RPE pyknosis) and accumulation of extracellular debris was also seen (<b>C,</b> asterisk). RPE cells appeared flattened and elongated with absent microvilli (black arrows indicate the flat apical side of the cell), reduced basal infoldings, and mesenchymal-like morphology. In addition, numerous vacuoles were present within the cells (<b>D,</b> asterisk). In areas adjacent to RPE breaks (<b>E</b>), the RPE retained some of its epithelial characteristics, such as, microvilli present on the apical membrane and intracellular basal infoldings. However, melanin vesicle maturation was defective (asterisks). R, RPE cell nuclei. OS, photoreceptor outer segments. Mv, microvilli. BM, Bruch's membrane. BI, basal infoldings. Ph, phagosome. Size bar, 1 µm (except in <b>B</b>, 200 nm). n = 4 per treatment group.</p

Downregulation of ZO-1 or overexpression of ZONAB affects retinal morphology.

<p>Retinal semithin sections were obtained after 10 days of subretinal injection of LNT.shGFP (<b>A,D</b>), LNT.ZONAB (<b>B,E</b>) or LNT.shZO-1 (<b>C,F</b>) at 10⁷ T.U./ml (left panel, ×40 magnification) and 10⁸ T.U./ml (right panel, ×20 magnification). Areas in red rectangles are shown in higher magnification. The LNT.shGFP treated eyes (<b>A,D</b>) exhibit normal retinal architecture showing that there are no adverse effects induced either by the lentiviral vector itself or by the expression of shRNA. Following injection of either LNT.ZONAB (<b>B,E</b>) or LNT.shZO-1 (<b>C,F</b>) signs of RPE puknosis and multilayerisation (black arrows) as well as retinal folding and rosette formation in areas corresponding to those with severe RPE abnormalities (white arrows) were observed. GCL, ganglion cell layer. IPL, inner plexiform layer. INL, inner nuclear layer. OPL, outer plexiform layer. ONL, outer nuclear layer. IS, inner segments. OS, outer segments. RPE, retinal pigment epithelium. CH, choroid. Size bar, 20 µm. n = 4 per treatment group.</p