Loss of CRB2 results in gliosis and microglia activation.

<p>Immunohistochemistry pictures from P10 mouse retinae. Sections were stained with antibodies against: SOX9 and Glutamine synthetase (GS) (<b>A</b>, <b>B</b>), GFAP (<b>C</b>, <b>D</b>), CD45 (<b>E</b>, <b>F</b>), CD11b (<b>G</b>, <b>H</b>). The location of nuclei of Müller glia cells, stained with SOX9 was not altered in the mutant retinas (<b>B</b>), however disruption at the apical end feet of the Müller glia cells were observed at sites with photoreceptor protrusions (<b>B</b>). The mutant retinas showed activated Müller glia cells, detected by a moderate increase in the GFAP staining in the outer nuclear layer (arrowhead) (<b>D</b>). An increase in activated microglia cells in the outer nuclear layer, stained with anti-CD45 and anti-CD11b, was detected (<b>F</b> and <b>H</b>). No morphological changes were observed in the control retinae. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; OLM, outer limiting membrane; ONL, outer nuclear layer; OPL, outer plexiform layer. Scale bars: 20 µm.</p

Strategy to select RPE signature genes.

<p>In the first step of this strategy we laser dissect the RPE (and its adjacent layers, the CH and PR) for specific tissue collection. In the second step we statistically correct for possible contamination by adjacent layers.</p

This diagram depicts the tight junction gene expression of mouse and human RPE, divided in four categories: high expression (>90^th percentile), moderate (50-90^th percentile), low (10-50^th percentile) and very low (<10^th percentile).

<p>On the x-axis the four categories are displayed and on the y-axis the amount of genes found in a category is depicted. Light blue circles contain genes expressed in mouse RPE, Dark blue circles genes expressed in human RPE. Genes inside the overlapping parts of the circles are expressed in the RPE in both species in that category.</p

Confirmation of microarray results by sqRT-PCR.

<p>Beta-actin (<i>Bact</i>), a housekeeping gene, was used to normalize gene expression in mouse CH, RPE and PR. The light blue bars indicate expression levels in CH, the blue bars expression levels in the RPE and the dark blue bars indicate expression levels in PR. Similar to the microarray data the expression level is highest in the RPE and lowest in the PR. The sqRT-PCR results confirm our findings; however <i>Tshr</i> and <i>Slc16a8</i> show expression lower in RPE compared to choroid. Overall, the sqRT-PCR confirmation rate in this, and in all our previous studies (combined), using exactly the same methodology and platform to investigate neuroepithelia from human donor eyes and brains was 87% [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141597#pone.0141597.ref012" target="_blank">12</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141597#pone.0141597.ref014" target="_blank">14</a>].</p

Transcript levels of the Crumbs family members.

<p>Transcript levels of Crb2, Crb1 and Crb3 measured by quantitative real time PCR at P3 and P10 (<b>A</b>), in 5-6 control and Crb2 cKO retinas. At P3, we detected a decrease of approximately 70% in the Crb2 transcript using primers located between the recombined area (<i>P</i> = 0.004). At P10 we did not found differences in the same transcript (<i>P</i> = 0.381). No differences were found in the levels of CRB1 (P3: <i>P</i> = 0.116; P10: <i>P</i> = 0.277) and CRB3 (P3: <i>P</i> = 0.912; P10: <i>P</i> = 0.869) transcripts. Data are presented as mean ± SEM **<i>P</i><0.01. Evaluation of the recombination efficiency of a Chx10<i>Cre</i> mouse line (<b>B</b>). The mT/mG reporter mouse line expresses membrane-targeted red fluorescent protein. After Chx10Cre-mediated recombination, the mT sequence is excised allowing expression of membrane-targeted enhanced green fluorescent protein (mG). Confocal laser scanning microscope pictures of (1M) retina sections from mT/mG (B1) and mT/mG::Chx10Cre (B2). While in the mT/mG only mT signal could be detected, in mT/mG*Chx10Cre retinas expression of mT and mG was found, suggesting mutant adjacent to wild type cells. GCL, ganglion cell layer; INL, inner nuclear membrane; ONL, outer nuclear layer. Scale bars: 20 μm.</p

Retinas lacking CRB2 show ectopic synapses.

<p>Immunohistochemistry pictures from P10 mouse retinae. Retina sections were stained with antibodies against: PKCα and PSD-95 (<b>A</b>, <b>B</b>), Calretinin (<b>C</b>, <b>D</b>) and PAX6 (<b>E</b>, <b>F</b>). In the <i>Crb2</i>Chx10 cKO retinae the outer plexiform layer, stained by an PSD-95 antibody, is thinner and showed some disruptions (arrows), ectopic synapses were also detected in the outer nuclear layer (arrowheads) (<b>B</b>). Some bipolar cells, stained with anti-PKCα (<b>B</b>), and some PAX6-positive amacrine cells (<b>F</b>) were misplaced in the outer plexiform layer of the mutant retinas. Calretinin positive amacrine cells (<b>D</b>) seemed not affected in the <i>Crb2</i>Chx10 cKO retinae. No morphological changes were observed in the control retinae. GCL, ganglion cell layer; INL, inner nuclear layer; OLM, outer limiting membrane; ONL, outer nuclear layer. Scale bars: 20 µm.</p

Strategy to determine “Interspecies RPE signature genes”.

<p>Schematic overview of our comparison strategy: our “Mouse RPE signature genes” dataset and “Human RPE signature genes” dataset, which contains (a modification of) two human RPE transcriptome datasets [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141597#pone.0141597.ref012" target="_blank">12</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141597#pone.0141597.ref015" target="_blank">15</a>]. This resulted in a new dataset, “Interspecies RPE signature genes”.</p

Most significant canonical pathways identified by Ingenuity for the “Mouse High RPE gene expression” and “Human High RPE expression gene expression” datasets.

<p>The left y-axis displays the–log of Benjamini-Hochberg corrected p-value. The right y-axis displays the ratio of the number of genes derived from our dataset, divided by the total number of genes in the pathway. The blue line indicates the threshold of the BH corrected p-value of 0.1.</p

The 22 signature genes that are specifically expressed in both RPE in mouse and in human.

<p>Derived from a comparison between our “Mouse RPE signature genes” dataset (this study) and two (modified) studies on the human RPE transcriptome [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141597#pone.0141597.ref012" target="_blank">12</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141597#pone.0141597.ref015" target="_blank">15</a>]. We show the gene symbol, genbank ID for both species and the GO annotation of each gene.</p><p>The 22 signature genes that are specifically expressed in both RPE in mouse and in human.</p

Overview of the major biological functions found in a functional annotation by Ingenuity of the “Mouse High RPE gene expression” and “Human High RPE gene expression” datasets.

<p>The p-value for these categories are indicated as a range because each category contains sub-functions that have their own p-value.</p><p>Overview of the major biological functions found in a functional annotation by Ingenuity of the “Mouse High RPE gene expression” and “Human High RPE gene expression” datasets.</p