Genetic Variations Strongly Influence Phenotypic Outcome in the Mouse Retina

Variation in genetic background can significantly influence the phenotypic outcome of both disease and non-disease associated traits. Additionally, differences in temporal and strain specific gene expression can also contribute to phenotypes in the mammalian retina. This is the first report of microarray based cross-strain analysis of gene expression in the retina investigating genetic background effects. Microarray analyses were performed on retinas from the following mouse strains: C57BL6/J, AKR/J, CAST/EiJ, and NOD.NON-H2-nb1 at embryonic day 18.5 (E18.5) and postnatal day 30.5 (P30.5). Over 3000 differentially expressed genes were identified between strains and developmental stages. Differential gene expression was confirmed by qRT-PCR, Western blot, and immunohistochemistry. Three major gene networks were identified that function to regulate retinal or photoreceptor development, visual perception, cellular transport, and signal transduction. Many of the genes in these networks are implicated in retinal diseases such as bradyopsia, night-blindness, and cone-rod dystrophy. Our analysis revealed strain specific variations in cone photoreceptor cell patterning and retinal function. This study highlights the substantial impact of genetic background on both development and function of the retina and the level of gene expression differences tolerated for normal retinal function. These strain specific genetic variations may also be present in other tissues. In addition, this study will provide valuable insight for the development of more accurate models for human retinal diseases

Strain specific alleles and differential expression of <i>Nr1d1</i>.

<p>(A) C57BL/6J and AKR/J chromatograms of polymorphisms identified in the ligand-binding domain of <i>Nr1d1.</i> (B) ClustalW2 sequence alignment of amino acid sequences from C57BL/6J, AKR/J, rat, chimpanzee and human. Stars indicate identity in all sequences, while dots indicate conserved amino acids. (C) C57BL/6J and AKR/J chromatograms of polymorphisms identified in the <i>Nr1d1</i> 5′UTR region. (D) ClustalW2 sequence alignment across species reveals the consensus is in accordance with AKR/J sequence. Stars indicate nucleotide conservation in all species. (E) <i>Nr1d1</i> relative expression in P30.5 AKR/J and C57BL/6J retinas (mean ± SD of mean, n = 3, p = 0.0024). (F) <i>Nr1d1</i> relative expression in P30.5 C57BL/6J, CAST/EiJ and NOD.NOH-H2^nb1 retinas (p<0.05).</p

Gene delivery of <i>Nr1d1</i> suppresses pan-retinal spotting, retinal dysplasia and function in <i>Nr2e3</i>^rd7/rd7 mice.

<p>(A–F) Fundus photographs of control and <i>rd7</i> injected retinas: (A) B6 (uninjected), (B) <i>rd7</i> (uninjected), (C) <i>GFP</i> injected, (D) <i>GFP.Nr2e3^B6</i> injected, (E) <i>GFP.Nr1d1^AKR/J</i> injected, (F) <i>GFP</i>.<i>Nr1d1</i>^B6 injected. (G–J) DAPI staining (blue) shows rescue of defects in retinal morphology 30 days after electroporation into <i>rd7</i> neonatal retinas. (G) GFP control, (H) <i>Nr2e3^B6</i> injected, (I) GFP control, (J) <i>Nr1d1</i>^AKR/J injected. L: left, R: right, GCL: ganglion cell layer, INL: inner nuclear layer, ONL: outer nuclear layer. Scale bar = 50 µm. (K, L) Representative scotopic (K) and photopic (L) electroretinograms from animals 4 month after injection with <i>GFP</i> (blue) or <i>GFP.Nr1d1^AKR/J</i> (red).</p

Expression of phototransduction genes <i>Opn1sw</i> and <i>Gnat2</i> is rescued in <i>rd7</i> retinas upon <i>Nr1d1</i> delivery.

<p>Quantitative real time PCR shows that <i>Nr1d1</i> delivery results in down-regulation of the phototransduction genes <i>Opn1sw</i> and <i>Gnat2</i> in <i>rd7</i> retinas (mean ± SD of mean, n = 3, p<0.05), to near normal levels.</p