Localization of the <i>loxP</i> sites and Crb2 probe in the Crb2 targeting construct.

<p>In the Crb2 targeting construct the <i>loxP</i> recombination sites are located in intron 9 and within exon 13 in the 3′ non-coding region. Upon Cre-mediated recombination the exons 10, 11, 12 and part of exon 13 are removed. The 60 bp probe for the Crb2 gene is located downstream of the 3’ <i>loxP</i>, before the polyadenylation signal. Two sets of primers were used to detect changes in Crb2 expression, they were located in exon 7 (primer 534/535) and in exon 11/12 (primer 536/537). In blue are represented the coding exons of the gene, in black the 3’ untranslated region. </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

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

Loss of CRB2 results in gliosis and microglia activation in the adult retina.

<p>Immunohistochemistry pictures from 2M mouse retinae. Sections were stained with antibodies against: Glial fibrillary acidic protein (GFAP) and Glutamine synthetase (GS) (<b>A</b>, <b>B</b>), SOX9 and GS (<b>C</b>, <b>D</b>), CD45 (<b>E</b>, <b>F</b>), CD11b (<b>G</b>, <b>H</b>). The mutant retinas showed activated Müller glia cells, detected by an increase in the GFAP staining in the outer nuclear layer (arrowhead) mainly in areas of cellular mislocalization (<b>B</b>). At foci, Müller glia cell nuclei (SOX9-positive) were detected in the outer nuclear layer (arrowheads) (<b>D</b>). Staining by anti-CD45 and anti-CD11b showed an increase in ectopic activated microglia cells in the outer nuclear layer and adjacent to ectopic photoreceptor nuclei located in the subretinal space (arrowheads; F and H). No morphological changes were observed in the control retinae. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars: 20 µm. Scale bar in the inset: 10 µm.</p

Loss of CRB2 affects lamination of photoreceptor cells.

<p>Immunohistochemistry pictures from 10 days old mouse retinas. Sections were stained with antibodies against: recoverin (<b>A</b>, <b>B</b>), rhodopsin and cone arrestin (<b>C</b>, <b>D</b>), M-opsin (<b>E</b>, <b>F</b>), peanut agglutinin (PNA) (<b>G</b>, <b>H</b>). In the mutant retinas several photoreceptor nuclei localized in the subretinal space were positive for recoverin (<b>B</b>) and rhodopsin (<b>D</b>). Some cone arrestin (<b>D</b>), M-opsin (<b>F</b>) positive nuclei were also misplaced in the subretinal place, showing that also cone photoreceptors lamination was affected. Outer segment from the cone photoreceptor cells, stained with PNA, were present in both retinas (<b>G</b>, <b>H</b>). However, in the mutant retinas these segments were located between photoreceptor nuclei and not in contact with the retinal pigmented epithelium (<b>H</b>). GCL, ganglion cell layer; INL, inner nuclear layer; OLM, outer limiting membrane; ONL, outer nuclear layer. Scale bars: 25 µm.</p

Tree-chart of correlative gene expression patterns from gene cluster analysis.

<p>Tree-chart of correlative gene expression patterns from gene cluster analysis.</p

Loss of CRB2 results in retinal disorganization in mice on C57BL/6J genetic background.

<p>Toluidine stained light microscopic pictures, of retina sections, from the control (<b>A</b>, <b>C</b> and <b>E</b>) and from the <i>Crb2</i>Chx10 cKO on C57BL/6J genetic background (<b>C</b>, <b>D</b> and <b>F</b>), at different ages, P10 - (<b>A</b>, <b>B</b>), 1M - (C, D), 3M - (<b>E</b>, <b>F</b>). At P10 (<b>B</b>), several photoreceptor nuclei were localized ectopically in the subretinal space. At 1M (<b>D</b>) protrusion of photoreceptor cell nuclei in the subretinal space and gaps in the outer limiting membrane were observed. At 3M (<b>F</b>) we observed thinner outer nuclear layers, with rows of photoreceptors cells protruding into the subretinal space through the outer limiting membrane and protrusions of inner nuclear layer cells into the outer nuclear layer. No abnormalities were observed in the control. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bar: 50 µm.</p

Crb2 and signalling pathways.

<p>Transcript levels measured by quantitative real time PCR at P3 (A) and P10 (B) in 5-6 control and Crb2 cKO retinas showed only changes in Hey1 (<i>P</i> = 0.041). P3: <i>Notch1 </i><i>P</i> = 0.144, <i>Kaiso </i><i>P</i> = 0.440, β-catenin <i>P</i> = 0.082, c-myc <i>P</i> = 0.711, cyclin D1 <i>P</i> = 0.226, Cyr61 <i>P</i> = 0.4687, <i>Shh </i><i>P</i> = 0.411. P10: <i>Smo </i><i>P</i> = 0.182, <i>gli1 </i><i>P</i> = 0.262, <i>P120-catenin </i><i>P</i> = 0.228, β-catenin <i>P</i> = 0.837, Hey2 <i>P</i> = 0.472, <i>elF4E </i><i>P</i> = 0.236, RPS6 <i>P</i> = 0.196, <i>PIK3K1 </i><i>P</i> = 0.375. Data are presented as mean ± SEM *<i>P</i><0.05; **<i>P</i><0.01.</p

Lack of CRB2 leads to the disruption of the apical CRB protein complex.

<p>Immunohistochemistry pictures from (P10) retina sections stained for subapical region markers: CRB2 (<b>A</b>, <b>B</b>), CRB1 (<b>C</b>, <b>D</b>), PALS1 (<b>E</b>, <b>F</b>), PAR3 (<b>G</b>, <b>H</b>), MUPP1 (<b>I</b>, <b>J</b>) and for adherens junction markers: β-Catenin (<b>K</b>, <b>L</b>), Catenin pp120 (P120) (<b>M</b>, <b>N</b>), N-Cadherin (<b>O</b>, <b>P</b>), Zona occludens-1 (ZO-1) (<b>Q</b>, <b>R</b>), Nectin1 (<b>S</b>, <b>T</b>). CRB2 was absent in the knockout retina (<b>B</b>), in contrast to control (<b>A</b>). CRB1, PALS1 and MUPP1 staining showed disruption of the CRB complex at the subapical region at sites of cellular mislocalization (<b>D</b>, <b>F</b> and <b>J</b>). PAR3 was also lost at sites of disruption (<b>H</b>). Staining using adherens junctions markers β-Catenin (<b>L</b>), P120 (<b>M</b>), N-cadherin (<b>P</b>), ZO-1 (<b>R</b>) and Nectin1 (<b>T</b>) showed disruption of the adherens junctions. Moreover, ectopic photoreceptor nuclei protruded into the subretinal space (<b>J</b> and <b>T</b>). No morphological changes were observed in the control retinas. OLM, outer limiting membrane; ONL, outer nuclear layer. Scale bars: 20 μm.</p