Development of conditional knockout phenotypes.

<p><b>A.</b> H&E-stained sagittal sections of retinas from mice representing each indicated genotype (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069721#pone-0069721-t001" target="_blank">Table 1</a>), at the indicated ages. Irregularities appear in the ONL of <i>R-DCKO</i> mice at P10, and in <i>Cbp CH</i> retinas at P14. Scale bar  = 50 μm. <b>B.</b> Immunofluorescence staining for p300 (green) shows progressive loss of p300 from ONL cells of <i>R-DCKO</i> retinas between P7 and P14. Scale bar  = 25 μm. <b>C.</b> TUNEL staining for cell death was performed on three retinas of each genotype at P10, P14, and P32. TUNEL positive cells were only increased relative to age-matched <i>Cre negative</i> controls in <i>Cbp CH</i> retinas at P32 (arrow). <b>D.</b> P32 <i>Cbp CH</i> retina showing TUNEL positive cells (black arrowheads). These are frequently seen near ONL irregularities. <b>OS</b>, outer segments. Scale bar  = 50 μm. <b>E.</b> P32 <i>R-DCKO</i> retina containing one TUNEL+ cell (black arrowhead). <i>R-DCKO</i> retinas do not show increased cell death relative to <i>Cre-negative</i> littermates at any age examined.</p

Compound heterozygotes show age-dependent phenotypes.

<p><b>A</b>–<b>D.</b> Cross-sections of 8-week-old retinas stained with H&E show disrupted morphology similar to that seen at 4 weeks in <i>Cbp CH</i> and <i>R-DCKO</i> mice. Scale bar  = 50 μm. <b>E</b>–<b>G.</b> ERG testing shows persistence of the functional impairment in <i>R-DCKO</i> retinas (panels F and G). Dark-adapted b-wave deficits in some <i>p300 CH</i> mice tested at this time are reflected in the slightly decreased average and broad error bars for this genotype (Panel F orange line). Two-way repeated measures ANOVA indicated significance at p<0.0001 for dark-adapted a-waves (Panel E), b-waves (Panel F), and light-adapted b-waves (Panel G). <b>H</b>–<b>K.</b> Cross-sections of 12-week-old retinas stained with H&E. Morphologic abnormalities in <i>Cbp CH</i> retinas (panel J) have resolved, although whorls and rosettes are still seen in <i>R-DCKO</i> retinas (panel K). Scale bar  = 50 μm. <b>L</b>–<b>N.</b> ERG testing at 12 weeks revealed decreases in function in both <i>Cbp CH</i> and <i>P300 CH</i>, and <i>R-DCKO</i> retinas have lost cone responses in addition to rod function. Two-way repeated measures ANOVA indicated significance at p<0.0001 for dark-adapted a-waves (Panel L), b-waves (Panel M), and light-adapted b-waves (Panel N). Asterisks (*) indicate p<0.001 vs. <i>Cre negative</i> controls in post-hoc tests.</p

Genotypes of mice used in this study.

<p>Genotypes of mice used in this study.</p

<i>Ep300/Cbp</i> conditional knockout in cones also disrupts cone structure, gene expression and function.

<p><i>Cone opsin</i>-driven <i>Cre (CCre)</i> was used to knock out <i>Ep300/Cbp</i> in cone photoreceptors; morphology, cone gene expression/distribution, and ERG function were assessed at 6–7 weeks of age. <b>A</b>–<b>D.</b> Compared to <i>Cre negative</i> controls (Panel A; inset shows two presumptive cones), H&E staining of <i>CCre</i> conditional knockout retinas reveals no major abnormalities (panels B–D), but cells with large nuclei can be seen scattered throughout the outer retina in <i>C-DCKO</i> mice (Panel D arrowheads and high-magnification inset). <b>E.</b> Cone arrestin (red) and p300 (green) expression are decreased in these cells (arrowheads). <b>F.</b> S-opsin expression (red) is also decreased in these cells (arrowheads), which lack outer segments. <b>G.</b> Peanut agglutinin labelling (red) identifies the displaced, abnormal cells in the outer retina (arrowheads) as cones. Blue in Panels E–G is DAPI labelling of nuclei. <b>H.</b> Cone α-transducin (green) is decreased and mislocalized to the cell bodies. Draq5 (red) marks nuclei. <b>I.</b> ERGs performed on 6 week old <i>CCre</i> mice confirmed decreases in cone-driven responses in <i>C-DCKO</i> retinas (red lines). Two-way repeated measures ANOVA indicated significance at p<0.0001 for dark-adapted and light-adapted b-waves. Asterisks (*) indicate significance differences (p<0.001) from <i>Cre negative</i> controls in post-hoc tests.</p

Expression of photoreceptor genes is decreased in <i>R-DCKO</i> retinas.

<p><b>A.</b> Summarized microarray findings for <i>R-DCKO</i> vs. <i>Cre neg</i> retinas. Each gene was categorized by the cell process in which it functioned, and results for each category are represented as a percentage of all the down- or up-regulated genes (see Supplemental Tables 2 & 3 for details). <b>B.</b> Schematic distribution of the 62 down-regulated photoreceptor or phototransduction-related genes in <i>R-DCKO</i> microarrays (red), compared with the 247 retinal disease loci listed in RetNet (<a href="https://sph.uth.edu/RetNet/home.htm" target="_blank">https://sph.uth.edu/RetNet/home.htm</a>; green) and a list of 230 genes down-regulated in <i>Crx^−/−</i> retinas compiled from published sources <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069721#pone.0069721-Blackshaw1" target="_blank">[51]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069721#pone.0069721-Livesey1" target="_blank">[53]</a>. Numbers in overlapping areas indicate the numbers of genes affected in both/all three conditions. All overlapping genes are listed in Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069721#pone.0069721.s009" target="_blank">Tables S4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069721#pone.0069721.s010" target="_blank">S5</a>. <b>C</b>–<b>F.</b> Expression of the indicated rod gene (<b>C. </b><b><i>Nrl</i></b><b>; D. </b><b><i>Crx;</i></b><b> E.</b> <b><i>Rhodopsin (Rho);</i></b><b> F. </b><b><i>Rod Transducin (Gnat1)</i></b>) was assessed by quantitative RT-PCR (<b>qRT-PCR</b>) at P14, and is expressed as percent of the level of <i>Cre-negative</i> littermate controls (<b><i>% Cre neg</i></b>). Protein localization was verified by immunohistochemistry (<b>IHC</b>) at P30. Scale bar  = 20 µm for all images. Levels of acetylated histone H3 <b>(AcH3)</b> or H4 <b>(AcH4)</b> on the respective promoter was determined by quantitative chromatin immunoprecipitation (<b>qChIP</b>) at P14, and is expressed as the value from the immunoprecipitated sample divided by the value from the untreated “input” sample, multiplied by 100 (“IP/input”).</p

Differential expression of mutant CRX protein/RNA in <i>K-IN</i> mouse retinas.

<p><b>A–G</b>. Paraffin embedded sagittal sections of P10 mouse retinas were stained with the mouse monoclonal CRX M02 antibody (Abnova) and imaged by fluorescent microscopy. ONL-outer nuclear layer, INL-inner nuclear layer, GCL-ganglion cell layer. <b>H</b>. SDS-PAGE and Western blot analyses of CRX proteins made by the indicated mouse strains at P10, using the rabbit polyclonal CRX 119b-1 (α-CRX) antibody <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004111#pgen.1004111-Peng1" target="_blank">[7]</a> and mouse monoclonal anti-β-ACTIN (α-BACT, Sigma-Aldrich). Positive bands correlating with the ∼37 kD full-length CRX and ∼27 kD truncated CRX^[E168d2] are visible. Lanes are numbered for reference (below). <b>I</b>. CRX protein levels were quantified by measuring the intensities of the CRX^[E168d2] and full-length bands normalized to the β-ACTIN control using LI-COR Odyssey Image Studio software. The results are presented as fold changes (FC) relative to full-length CRX level in <i>WT</i> retina. (*p≤0.05) <b>J</b>. <i>Crx</i> mRNA levels were determined by quantitative real-time PCR using allele specific PCR primer pairs. Separate primer pairs were used to amplify <i>WT Crx</i> alone and total <i>Crx</i> (<i>WT</i>+<i>mutant</i>) in <i>E168d2</i> and <i>R90W</i> mice (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004111#s4" target="_blank">Materials and Methods</a>). The results are presented as FC relative to <i>WT</i> retina. (*p≤0.05).</p

Heterozygous <i>E168d2/</i>+, <i>E168d2neo/</i>+ and <i>R90W/</i>+ mice have graded deficits in retinal function.

<p><b>A–I</b>. Retinal function of <i>E168d2/+</i>, <i>E168d2neo/+</i> and <i>R90W/+</i> and <i>+/−</i> mice was assessed by electroretinography at 1 mo (<b>A–C</b>), 3 mo (<b>D–F</b>) and 6 mo (<b>G–I</b>). Average peak amplitude responses for dark-adapted A-waves and B-waves and light-adapted B-waves are plotted against the log of the flash intensity (Log [cd*s/m²]). Genotype*flash intensity interactions for peak amplitude (by two-way ANOVA) were significant (p<0.05) at all ages for each wave form tested. <i>E168d2/+</i> mice show severe deficits in all wave responses at each age compared to responses from either <i>WT</i> or <i>E168d2neo/+</i> mice (green vs. black and red line, respectively). Peak responses in <i>E168d2neo/+</i> mice are higher than <i>E168d2/+</i> (red vs. green line), but remain significantly decreased compared to <i>WT</i> (red vs. black line) with exceptions for 6 mo dark-adapted B-waves (<b>H</b>). <i>R90W/+</i> and <i>+/−</i> mice have mostly normal retinal function (blue or orange vs. black line) but <i>R90W/+</i> have subtle significant deficits in light-adapted B-waves at 6 mo (<b>I</b>, blue vs. black line). (Relative to <i>WT</i>: *p<0.05; relative to <i>E168d2neo/+</i>: *‘p<0.05, brackets indicate all enclosed data points are significant). Error bars: SEM.</p

Homozygous <i>E168d2</i>, <i>R90W</i> and −/− mice show graded changes in retinal gene expression.

<p><b>A–B</b>. Venn diagram showing overlap of genes that are differentially expressed at P10, as identified by Illumina gene expression mouseRef6 microarray. The number of genes in each group is indicated. <i>E168d2neo/d2neo</i>, <i>R90Wneo/Wneo</i> and <i>−/−</i> mice show a high degree of overlap in differentially expressed genes. <b>C</b>. Percentage of differentially expressed genes for each genotype that are directly bound by WT CRX protein in <i>WT</i> and <i>Nrl KO</i> retinas (based on the published ChIP-Seq datasets <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004111#pgen.1004111-Corbo1" target="_blank">[21]</a>). For all mutant genotypes, differentially expressed genes are enriched for direct CRX targets. <b>D</b>. Heat map of commonly downregulated genes in <i>E168d2neo/d2neo</i>, <i>R90Wneo/Wneo</i> and <i>−/−</i> mice show graded changes in gene expression of commonly downregulated genes. <b>E</b>. Cellular processes associated with commonly downregulated genes, based on gene ontology provided by Mouse Genome Informatics, showing a widespread effect of <i>Crx</i> mutations on visual and cellular pathways. <b>F–I</b>. P14 paraffin embedded sagittal retinal sections of <i>WT</i>, <i>E168d2neo/d2neo</i>, <i>R90Wneo/Wneo and −/−</i> mice were stained with Rhodopsin (RHO, green) and DAPI (blue), and imaged by wide field fluorescence at 40×. Note that RHO is absent in <i>E168d2neo/d2neo</i>, while mislocalized to ONL in <i>R90Wneo/Wneo</i> and <i>−/−</i>. <b>J–M</b>. Validation of microarray results by qRT-PCR analyses on selected CRX target genes, <i>Rho</i>, <i>Arr3</i>, <i>Opn1mw</i> and <i>Opn1sw</i> in retinas of P10 homozygous mice from the indicated strains, shown as FC relative to <i>WT</i>. (*p<0.05; Error bars: STDEV).</p

Graded changes in CRX target gene expression in heterozygous <i>E168d2/</i>+, <i>E168d2neo/</i>+ and <i>R90W/</i>+ mice.

<p><b>A–D</b>. Paraffin embedded sagittal retinal sections of 1 mo <i>WT</i> and the indicated heterozygous mutant mice were stained with mouse monoclonal anti-Rhodopsin RetP-1 antibody (Chemicon) (RHO, red) and DAPI nuclear conterstaining (blue), and imaged by widefield fluorescence at 40×. <i>E168d2/+</i> shows reduced rod OS length and mislocalized RHO in ONL. <b>E–H</b>. qRT-PCR analysis of four CRX target genes, <i>Rho</i>, <i>Arr3</i>, <i>Opn1mw</i>, <i>Opn1sw</i> in the indicated heterozygous mice at P10 and P21 (*p<0.05; bracketed *FDR p<0.09; Error bars: SEM). Note at P10, the expression of <i>Opn1mw</i> and <i>Opn1sw</i> CRX target genes are reduced in all mutant models. However, at P21, expression recovers in <i>R90W/+</i> and <i>+/−</i> mice, while remaining reduced in <i>E168d2/+</i> and <i>E168d2neo/+</i> mice.</p

Heterozygous <i>E168d2/</i>+, <i>E168d2neo/</i>+ and <i>R90W/</i>+ mice display distinct changes in cone density and M/S opsin gradient formation.

<p><b>A</b>. Diagram showing regions of flat-mounted retina selected for cone density image analyses. <b>B–D</b>. Cone density of 1 mo and 1 yr old mice was determined by counting PNA+ cells on flat-mounted retinas in the dorsal (D), central (C), nasal/temporal (N/T) and ventral (V) regions. <b>B</b>. Total cone density over all regions (*p<0.05). <b>C–D</b>. Cone density in each region in1 mo (<b>C</b>, *p<0.05) and 1 yr old (<b>D</b>) mice. ND-not determined. Error bars: SEM. Note that genotype*retinal region interaction (by two-way ANOVA) was significant at 1 mo (p = 0.04) but not 1 yr (p = 0.11). <b>E–L</b>. Flat-mounted retinas from 1 mo mice of the indicated genotype were stained for OPN1SW (SOP, green), red/green opsin (MOP, red) and the pan cone marker peanut agglutinin (PNA, blue), showing sample images from the dorsal (<b>E–H</b>) and ventral (<b>I–L</b>) regions. Image scale bars: 25 µM. Unlike <i>WT</i> samples (<b>E&I</b>), <i>E168d2/+</i>, (<b>F&J</b>) and <i>E168d2neo/+</i> (<b>G&K</b>) samples show a small number of PNA+ cones that did not express either cone opsin (white arrows). <b>M–P</b>. Fraction of cones in each region expressing SOP, MOP, both SOP/MOP or no opsin (*p<0.05).</p