Gene expression analysis of GeneChip hybridizations using IPA: List of cellular functional pathways which show major changes in genetic regulation, as a result of the <i>rds</i> defect.

<p>This list was generated with input of all genes which are differentially regulated at one or more time points.</p

Activated microglia are present in mature <i>rds</i> retina.

<p>(A) Immunofluorescent micrographs of age-matched <i>rds</i> and WT murine ocular cryosections are shown; DAPI-stained nuclei appear blue. Labeling of microglia by anti-Iba1 and anti-CD11b antibodies (red, <i>left and middle panels</i>) shows distributions of resting filamentous (arrowheads) and activated ameboid (arrows) microglia. GFAP labeling (red, <i>right panels</i>) shows distributions of activated Müller glial cells and reflects retinal stress. (B) Quantitative analysis of activated microglial migration in <i>rds</i> (filled bars) and WT (unfilled bars) retinas. Iba1 labeled cells were tabulated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031371#s4" target="_blank">Methods</a> (averages are reported s.e.m.; <i>n = 3</i>). The results show migration of activated microglia into nuclear layers of the P21 <i>rds</i> retina.</p

Quantitative PCR analyses for validation of gene expression changes in the <i>rds</i> model.

<p>For each gene, the maximal fold change value (<i>rds</i>:Nrl-eGFP vs. Nrl-eGFP) between P6 and P21 was plotted. Error bars indicate the standard error of the mean (s.e.m.; <i>n = 3</i>). Genes were classified into functional pathways, using Ingenuity Pathways Analysis software. Expression of most genes changed less than two-fold, and dysregulation in a variety of functional pathways was seen.</p

Transient upregulation may reflect homeostatic mechanisms.

<p>Differential regulation of numerous genes was observed at P14; however, expression returned to baseline levels by P21. This temporal pattern included genes mainly representing cellular organization and lipid metabolism functional pathways.</p

Strategy and sample generation for gene expression profiling of structurally abnormal rod photoreceptors.

<p>(A) Photoreceptors in the <i>rds</i> mouse fail to elaborate OSs. OS disk membrane biosynthesis begins at ∼P10, OSs are well established by P14, and reach full length by ∼P21. (B) <i>Left panels</i>: immunofluorescence analyses of retinal cryosections; DAPI-stained nuclei appear blue. eGFP labeled photoreceptors (green) were observed in WT, Nrl-eGFP and <i>rds</i>:Nrl-eGFP retinas; however, OSs labeled with anti-P/rds antibody PabMPCT (red), were only detected in WT and Nrl-eGFP retinas. <i>Right panels</i>: fluorescence-activated cell sorting was utilized to enrich rod photoreceptors from trypsin-dissociated mouse retinas. Typical histograms, generated using a BD Biosciences FACSVantage SE cell sorter, illustrate GFP-containing rod photoreceptors detected in dissociated (P9) retinas from Nrl-eGFP and <i>rds</i>:Nrl-eGFP, but not WT mice. Collected rod cell fractions, typically ∼500,000 cells per retina, are indicated (brackets).</p

Neurotrophic factors are upregulated in the <i>rds</i> retina.

<p>Maximal Fold change values from qPCR study show strong upregulation of <i>CNTF</i> and <i>GDNF</i>, which are known to have a neuroprotective role for photoreceptors. Error bars indicate standard error of the mean (<i>n = 3</i>).</p

Human mRNA rescues zebrafish <i>bbs9</i>-spMO phenotype.

<p>h<i>W</i> and h<i>M</i> represent wild type and mutant human mRNA, respectively. The arrows indicate eye phenotype. (<b>A</b>) The uninjected control (top) and <i>bbs9</i>-spMO alone injected (bottom) zebrafish at 72 hpf. (<b>B</b>) Rescue of <i>bbs9</i>-spMO eye phenotype by hW 100 pg (top), but not by lower dose of 50 pg (bottom). (<b>C</b>) The <i>bbs9</i>-spMO phenotype is not rescued by h<i>M</i> as the eye defect remains in the morphants. (<b>D</b>) The quantification of embryos' eye size at 72 hpf in rescue experiment using human mRNAs co-injected with <i>bbs9</i>-spMO. X-axis shows category of embryos scored. Y-axis shows the eye size in pixels. Data are presented as mean ± SEM. Statistically significant and non-significant observations are indicated with p value and n.s., respectively.</p

Exon 5-targeted <i>bbs9</i> splice morpholino affects eye development independent of p53 pathway.

<p>(<b>A</b>) At 24 hpf, the <i>p53</i>-atgMO (1.5 ng) alone injection did not elicit a phenotype. The <i>bbs9</i>-spMO (1 ng) injection alone caused developmental defects in the eye, brain and tail of morphants. However, co-injection of <i>p53</i>-atgMO reduced the defects seen by the <i>bbs9</i>-spMO injection alone, though mild eye defect remained the tail becomes normal (bottom panel). (<b>B</b>) Higher magnification of morphants' head region. Top, middle and bottom rows are 24-, 48- and 72-hpf, respectively. Left and right column of panels are <i>p53</i>-atgMO without and with <i>bbs9</i>-spMO, respectively. At 48 hpf the effect of <i>bbs9</i>-spMO injection on eye size visible (compare the arrows). The <i>bbs9</i>-spMO injection also resulted in hydrocephalous (compare the arrow heads). The defects seen at 48 hpf are weaker at 72 hpf. (<b>C</b>) The gel photograph of RT-PCR showing exon-skipping by <i>bbs9</i>-spMO. mRNA isolated from individual embryos was used for RT-PCR. U, C (4 and 6 ng) and B (1, 4, 6 ng) represent un-injected, control, and <i>bbs9</i>-spMO, respectively. Splice blocking gave an additional smaller (marked e5skip) band along with the original WT band. The bottom panel shows β-actin control for respective samples. (<b>D</b>) Quantification of the effect of morpholino(s) injection on eye size. X-axis shows the morpholinos used and time (hpf) of scoring. Y-axis shows eye size in pixels (mean ± SEM).</p

Zebrafish <i>bbs9</i> gene: structural comparison and expression pattern.

<p>(<b>A</b>) A comparison of <i>BBS9</i> exon:intron structure between human (<i>H. sapiens</i>, top blue), mouse (<i>M. musculus</i>, middle black) and zebrafish (<i>D. rerio</i>, bottom gray/black). The filled and open boxes indicate coding exons and UTRs, respectively. The blue and black boxes represent validated exons. The gray boxes represent exons present in provisional sequence XM_002664792.1. Exons 2 to 8 are highly conserved across species (boxed area within hatched square). The yellow arrow points to yellow mark on exon 5, which represents the missense mutation G→A (p.G141R) in human BBS9 protein. Under the zebrafish <i>bbs9</i> transcript, the red line represents <i>bbs9</i>-spMO targeting site at intron4:exon5 boundary. (<b>B</b>) The protein sequence alignment (clustalW) between human (NP_940820.1), mouse (NP_848502.1) and predicted zebrafish BBS9 (904 amino acids). Exon 5 is highlighted (yellow), and the position of missense mutation (p.G141R) in human is highlighted by a black rectangle. The bar coding on top of the sequences represents degree of conservation (red and blue represent maximum and minimum conservation, respectively). (<b>C, D</b>) <i>In situ</i> hybridization analysis at 11 hpf and 15 hpf. Left and right panels represent the sense and anti-sense probes generated from <i>bbs9</i> cDNA. (<b>E</b>) <i>In situ</i> hybridization analysis at 48 hpf. Expression of <i>bbs9</i> in the eye, brain and somites gives a strong signal with the anti-sense probe compared to the background signal from the sense probe. Compare the strong signal in the head regions (arrows). Left and right panels represent lateral and dorsal views, respectively.</p

GA lesion growth rates for each individual in the combined study.

<p>The measured area of GA was square-root transformed. From the transformed area the growth rate was calculated per year in [mm/year]. Growth rates from each individual were then obtained by calculating the mean of all growth rates of the individual. If both eyes were affected, the mean of both eyes were calculated resulting in a single growth variable per individual. These individual growth rates were further transformed by the natural logarithm (ln) and were stratified either by (A) the genotype at ARMS2_rs10490924 or (B) the genotype at C3_rs2230199 or (C) the presence or absence of bilateral GA.</p