Volcano plots showing the relationship between fold change (represented as mean A – mean B) and the level of significance (represented by the <i>Fs</i> permutated <i>p</i>-value).

<p>Differentially expressed probe sets (q<0.05 shown in red across all fold change levels) at and fold change greater than 2 are depicted in volcano plots in three pairwise comparisons. (A) <i>rd6/rd6</i> (<i>Mfrp^rd6</i>/<i>Mfrp^rd6</i>) P0 vs B6 (C57BL/6J) P0, (B) <i>rd6/rd6</i> P14 vs B6 P14 and (C) <i>rd6/rd6</i> P14 vs <i>rd6/rd6</i> P0.</p

qRT-PCR analysis of RPE and retinal- specific genes in homozygous <i>Mfrp^rd6</i>, <i>Tulp1^tvrm124 and Rpe65^tvrm148</i> mutants.

<p>(A) In homozygous <i>Mfrp^rd6</i> mutant mice, the transcripts in the visual cycle (<i>Rpe65, Lrat</i> and <i>Rgr</i>), phototransduction pathway (<i>Rgs9</i>, <i>GuCa1b</i>, <i>Pde6a</i>) and structural components of rods and cones (<i>Fscn2</i> and <i>RpGrip1</i>) were significantly decreased relative to the wild-type control (B6/J), validating the microarray results. (B) qRT-PCR analysis in <i>Tulp1^tvrm124/Tulp1^tvrm124</i> mutants at P14 revealed no significant change in any of the transcripts tested. (C) In <i>Rpe65^tvrm148/Rpe65^tvrm148</i> mutants, there was only a significant increase in <i>RpGrip1</i> from transcripts tested, relative to wild-type (B6/J) controls. The data are expressed as relative fold change (RFC) after normalizing to the wild-type control. RFC was calculated using ΔΔC_T method after internal calibration to β-Actin control. Each value represents RFC ± S.E.M. * <i>P</i><0.05 and ** <i>P</i><0.001 relative to controls. N = 3–6 per group.</p

Primers for qRT-PCR.

<p>Primers for qRT-PCR.</p

Ingenuity pathway analysis identified Visual Cycle and Phototransduction pathways to be downregulated genes in homozygous <i>Mfrp^rd6</i> mutant mice.

<p>(A) Genes in the visual cycle (RPE) and phototransduction pathway (rod photoreceptors) are represented in this panel. The asterisk represents the visual cycle genes (<i>Rpe65, Lrat and Rgr</i>), phototransduction pathway genes (<i>Rgs9</i>, <i>Guca1b</i>, <i>Pde6a</i>) and genes encoding structural components of the rod-cells (<i>Rpgrip1</i> and <i>Fscn2</i>) that were validated by qRT-PCR. (B) Genes in the visual cycle (RPE) and phototransduction pathway (cone photoreceptors) are represented in this panel. The asterisk represents the visual cycle genes (<i>Rpe65</i>, <i>Lrat</i> and <i>Rgr</i>), Müller glia cell expressed gene (<i>Rgr</i>), phototransduction pathway genes (<i>Rgs9</i> and <i>Guca1b</i>), and genes encoding structural components of the cone cells (<i>RpGrip1</i> and <i>Fscn2</i>) that were validated by qRT-PCR. The molecules associated with the symbols are as depicted in the inset. The solid and dashed lines represent direct or indirect interactions, respectively, between the genes. The arrow indicates interaction between genes. A =  Activation, B = Binding, E = Expression (includes metabolism/synthesis for chemicals), I (Inhibition), PP (Protein-Protein binding), P (Phosphorylation/Dephosphorylation), RB (Regulation of binding), MB (Group/complex Membership).</p

Cellular localization of <i>Prss56 and Glul</i> in B6 (C57BL/6J) and <i>Mfrp^rd6/Mfrp^rd6</i> mice.

<p>(A) By <i>in situ</i> hybridization, in B6 (C57BL/6J) controls at P14, we observed <i>Prss56</i> transcript in only very few cells of the inner nuclear layer (INL) of the retina (top panel), whereas in <i>Mfrp^rd6/Mfrp^rd6</i> mutants, an intense staining of <i>Prss56</i> transcript was observed in INL of the retina (bottom panel). (B) By 2-plex <i>in situ</i> hybridization, in B6 controls, we observed co-localization of <i>Prss56</i> (red) and <i>Glul</i> (pseudo colored green) transcripts in only a few cell body of the (INL) of the retina (top panel), whereas in <i>Mfrp^rd6/Mfrp^rd6</i>, strong co-localization of <i>Prss56</i> and <i>Glul</i> transcripts in the cell body of the INL of the retina was observed (bottom panel). (C) Glutamine synthetase (GS) staining of Müller cells. In both C57BL/6J (B6) and <i>Mfrp^rd6/Mfrp^rd6</i> mice, Müller cells marked with glutamine synthetase showed a similar localization pattern (inset, top and bottom panels) as observed for <i>Prss56 in situ</i> hybridization staining, suggesting that Müller cells in the INL of retina express <i>Prss56</i> transcripts at P14.</p

Transcripts that are upregulated in <i>Mfrp^rd6</i>^/<i>Mfrp^rd6</i> mutant relative to B6 control at P14.

<p>The number in the column heading (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110299#pone-0110299-t002" target="_blank">Table 2</a>) represents the mouse identity used in the microarray analysis.</p><p>Transcripts that are upregulated in <i>Mfrp^rd6</i>^/<i>Mfrp^rd6</i> mutant relative to B6 control at P14.</p

RPE65 protein expression in RPE cells from B6 (C57BL/6J) and homozygous <i>Mfrp^rd6</i> mice.

<p>(A) Western blot analysis of RPE65 protein extracted from RPE cells of B6 (C57BL/6J) and homozygous <i>Mfrp^rd6</i> mice. There was a 2.0-fold decrease in RPE65 protein in <i>Mfrp^rd6/Mfrp^rd6</i> (Lane 2) relative to the B6 control (Lane 1), whereas in <i>Rpe65^tvrm148/Rpe65^tvrm148</i> mutant, RPE65 protein was undetected (Lane 3). β-Actin loading confirms equal protein loading in all lanes (1–3). (B) Quantitation of RPE65 protein in RPE cells of B6 (C57BL/6J) and <i>Mfrp^rd6/Mfrp^rd6</i> mice. Student's T test was used to calculate statistical significance (* <i>P</i><0.05 relative to B6 control).</p

Independent qRT-PCR validation of genes that were differentially expressed in <i>Mfrp^rd6/Mfrp^rd6</i> mutants by array analysis.

<p>The upregulated gene <i>Prss56</i> was evaluated at three different time points. (A) At P7, there was a 3.5-fold increase in <i>Prss56</i> transcript and it increased to 14-fold by P14, followed by a further 70-fold increase in <i>Prss56</i> transcript in <i>Mfrp^rd6/Mfrp^rd6</i> mutants at P21. (B) At P14, when compared to the <i>Mfrp^rd6/Mfrp^rd6</i> mutant, in <i>Tulp1^tvrm124/Tulp1^tvrm124</i> mutants, there was no significant change in <i>Prss56</i> transcript, whereas in <i>Rpe65^tvrm148/Rpe65^tvrm148</i> mutants, there was a significant decrease. (C) Wildtype levels of <i>Prss56</i> transcript revealed a significant decrease between P7 and P21 timpoints, whereas the decrease from P7 to P14 was not statistically significant. Data are expressed as relative fold change (RFC) in the <i>Prss56</i> transcript after normalizing to the wild-type control (B6). RFC was calculated by the ΔΔC_T method using β-actin as an internal calibrator. Each value represents RFC ± S.E.M. * <i>P</i><0.05 and ** <i>P</i><0.001 relative to controls. N = 3 per group.</p

Mouse models of human ocular disease for translational research

<div><p>Mouse models provide a valuable tool for exploring pathogenic mechanisms underlying inherited human disease. Here, we describe seven mouse models identified through the Translational Vision Research Models (TVRM) program, each carrying a new allele of a gene previously linked to retinal developmental and/or degenerative disease. The mutations include four alleles of three genes linked to human nonsyndromic ocular diseases (<i>Aipl1</i>^{<i>tvrm119</i>}, <i>Aipl1</i>^{<i>tvrm127</i>}, <i>Rpgrip1</i>^{<i>tvrm111</i>}, <i>Rho</i>^{<i>Tvrm334</i>}) and three alleles of genes associated with human syndromic diseases that exhibit ocular phentoypes (<i>Alms1</i>^{<i>tvrm102</i>}, <i>Clcn2</i>^{<i>nmf289</i>}, <i>Fkrp</i>^{<i>tvrm53</i>}). Phenotypic characterization of each model is provided in the context of existing literature, in some cases refining our current understanding of specific disease attributes. These murine models, on fixed genetic backgrounds, are available for distribution upon request and may be useful for understanding the function of the gene in the retina, the pathological mechanisms induced by its disruption, and for testing experimental approaches to treat the corresponding human ocular diseases.</p></div

Marker analysis of homozygous <i>Fkrp</i>^{<i>tvrm53</i>} mice at 1–2 months of age.

<p>(A) Localization of FKRP in B6J control retina (n = 3). FKRP (<i>red</i>) colocalizes with β-dystrophin to synaptic processes in the OPL. (B) Co-staining of FKRP (<i>red</i>) with CTBP2 (<i>green</i>) shows FKRP surrounds the synaptic ribbons. (C) IHC of rhodopsin (<i>red</i>) and pan-laminin (<i>green</i>) in B6J control (n = 6) and homozygous <i>Fkrp</i>^{<i>tvrm53</i>} mice (n = 7). (D) IHC of pan-laminin in ocular cryosections of samples as in C. (E) IHC of collagen IV (COL IV, <i>red</i>) and β-dystroglycan (β-DYS, <i>green</i>) in cryosections (n = 5 and 6 for B6J and mutant mice, respectively). (F) Glial fibrillary acidic protein (GFAP) staining of retinal cryosections indicated Müller cell activation in homozygous <i>Fkrp</i>^{<i>tvrm53</i>} mice (n = 4) compared to B6J controls (n = 3). Retinal layers are labeled as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183837#pone.0183837.g001" target="_blank">Fig 1B</a>. <i>Asterisks</i>, abnormal vessels in (D, E). <i>Bars</i>, (A) 20 μm, (B) 5 μm, (C-D) 50 μm. (E-F) 100 μm.</p