Mapping of genetic modifiers of Nr2e3 rd7/rd7 that suppress retinal degeneration and restore blue cone cells to normal quantity

The retinal degeneration 7 (rd7) mouse, lacking expression of the Nr2e3 gene, exhibits retinal dysplasia and a slow, progressive degeneration due to an abnormal production of blue opsin-expressing cone cells. In this study we evaluated three strains of mice to identify alleles that would slow or ameliorate the retinal degeneration observed in Nr2e3 ^rd7/rd7 mice. Our studies reveal that genetic background greatly influences the expression of the Nr2e3 ^rd7/rd7 phenotype and that the inbred mouse strains CAST/EiJ, AKR/J, and NOD.NON-H2 ^nb1 carry alleles that confer resistance to Nr2e3 ^rd7/rd7 -induced retinal degeneration. B6.Cg-Nr2e3 ^rd7/rd7 mice were outcrossed to each strain and the F₁ progeny were intercrossed to produce F₂ mice. In each intercross, 20–24% of the total F₂ progeny were homozygous for the Nr2e3 ^rd7/rd7 mutation in a mixed genetic background; approximately 28–48% of the Nr2e3 ^rd7/rd7 homozygotes were suppressed for the degenerative retina phenotype in a mixed genetic background. The suppressed mice had no retinal spots and normal retinal morphology with a normal complement of blue opsin-expressing cone cells. An initial genome scan revealed a significant association of the suppressed phenotype with loci on chromosomes 8 and 19 with the CAST/EiJ background, two marginal loci on chromosomes 7 and 11 with the AKR/J background, and no significant QTL with the NOD.NON-H2 ^nb1 background. We did not observe any significant epistatic effects in this study. Our results suggest that there are several genes that are likely to act in the same or parallel pathway as NR2E3 that can rescue the Nr2e3 ^rd7/rd7 phenotype and may serve as potential therapeutic targets. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Novel vertebrate genes and putative regulatory elements identified at kidney disease and NR2E1/fierce loci

Fierce (frc) mice are deleted for nuclear receptor 2e1 (Nr2e1), and exhibit cerebral hypoplasia, blindness, and extreme aggression. To characterize the Nr2e1 locus, which may also contain the mouse kidney disease (kd) allele, we compared sequence from human, mouse, and the puffer fish Fugu rubripes. We identified a novel gene, c222389, containing conserved elements in noncoding regions. We also discovered a novel vertebrate gene conserved across its length in prokaryotes and invertebrates. Based on a dramatic upregulation in lactating breast, we named this gene lactation elevated-1 (LACE1). Two separate 100-bp elements within the first NR2E1 intron were virtually identical between the three species, despite an estimated 450 million years of divergent evolution. These elements represent strong candidates for functional NR2E1 regulatory elements in vertebrates. A high degree of conservation across NR2E1 combined with a lack of interspersed repeats suggests that an array of regulatory elements embedded within the gene is required for proper gene expression.     

The distribution and developmental regulation of NMDA receptor subunit proteins in the outer and inner retina of the rat

In order to investigate whether N-methyl-D-aspartate (NMDA) receptors with distinct pharmacological properties are differentially distributed within the retinal layers, the spatial distribution and temporal regulation of all NMDA receptor subunits was analyzed in parallel on the protein level in the rat retina during development. Immunohistochemistry was performed on retinal sections at different developmental ages between embryonic (E) days 20/21 and the adult stage using specific antibodies against NMDA subunits (NR1, NR2A-D). All NMDA subunits were expressed in the rat retina postnatally but showed different spatial patterns. In particular, and in contrast to previous in situ hybridization studies, labeling of NR2 subunits was observed in horizontal cell bodies and in the outer plexiform layer, indicating that functional NMDA receptors are expressed in this retinal cell type in the rat. Expression of NR2D was restricted to the inner retina and seemed to be involved in neurotransmission within the rod pathway. In the inner plexiform layer (IPL), distinct patterns of labeling were observed for different NMDA subunits. NR1 was found in two bands which can be related to the off- and on-signal pathways, whereas NR2A and NR2B were located in two bands within the off-sublaminae of the IPL. The antibody against NR2C was distributed throughout the whole IPL, and NR2D was expressed exclusively in the innermost part of the IPL where rod bipolar cell terminals terminate. Distinct bands of immunoreactivity in the IPL were observed only from P14 on. In conclusion, there are clear differences in the spatial distribution and temporal expression of NMDA receptor subtypes in the rodent retina. This indicates that specific retinal cells selectively express glutamate receptors composed of different subunit combinations and thus display different pharmacological and kinetic properties.     

Tyrosinase-related protein 2 promoter targets transgene expression to ocular and neural crest-derived tissues

In an effort to identify a promoter suitable for studying early ocular development, we generated transgenic mice carrying the lacZ reporter gene linked to the tyrosinase-related protein 2 (TRP2) promoter. TRP2-lacZ was expressed in early retinal pigment epithelium (RPE) and early neural crest cells in embryos. The promoter activity was robust and consistent in independent transgenic lines. The transgene was also expressed in the optic nerve and neural crest-derived neuronal cells in which the endogenous TRP2 gene is not expressed. This suggests that repressor elements may be missing in the promoter used in this study. To test whether this promoter can be used to study melanocyte development, we cross-mated TRP2-lacZ transgenic mice with mice heterozygous for the Patch (Ph) mutation. The pattern of beta-galactosidase activity in the embryos correlates well with the pigmentation phenotype in postnatal and adult Ph/+ mice. We also generated transgenic mice expressing fibroblast growth factor 9 (FGF9) directed by the TRP2 promoter and examined the effect on ocular development. Ectopic expression of FGF9 in the early embryonic RPE switched its differentiation pathway to a neuronal fate, resulting in formation of a duplicated neural retina in transgenic mice. These studies demonstrate that the TRP2 promoter is valuable for transgenic studies of ocular differentiation and development of neural crest cells.     

Serum Differentially Modifies the Transcription and Translation of NMDAR Subunits in Retinal Neurons

The N-methyl-d-Aspartate type of glutamate receptor (NMDAR) plays a major role in the vertebrate retina. Expression of NR1 splice-variants and NR2 subunits in the retina differs from that in the brain, suggesting a tissue-specific heteromeric assembly of NMDARs. We previously demonstrated that serum alters retinal glutamate receptor properties. In order to relate this effect to NMDAR subunit composition, we here studied the effect of serum on the expression of NMDAR subunits and splice-variants in chick retinal neurons in primary culture. Our results show that mRNA and protein expression of NR1 alternative splice-variants and NR2 subunits are differentially modified by glutamate contained in serum. Such alteration suggests that NMDAR structure is reversed to embryonic heteromeric composition, through the control of subunit availability. The present findings could be relevant for the understanding of the lack of effect in the retina, of drugs which have been shown to protect cortical neurons from glutamate-induced excitotoxicity in those pathological or clinical conditions in which the retina is exposed to serum. Special issue article in honor of Dr. Ricardo Tapia.     

The zebrafish orphan nuclear receptor genes nr2e1 and nr2e3 are expressed in developing eye and forebrain

Mammalian Nr2e1 (Tailless, Mtll or Tlx) and Nr2e3 (photoreceptor-specific nuclear receptor, Pnr) are highly related orphan nuclear receptors, that are expressed in eye and forebrain-derived structures. In this study, we analyzed the developmental expression patterns of zebrafish nr2e1 and nr2e3. RT-PCR analysis showed that nr2e1 and nr2e3 are both expressed during embryonic and post-embryonic development. To examine the spatial distribution of nr2e1 and nr2e3 during development whole-mount in situ hybridization was performed. At tailbud stage, initial nr2e1 expression was localized to the rostral brain rudiment anterior to pax2.1 and eng2 expression at the prospective midbrain-hindbrain boundary. During subsequent stages, nr2e1 became widely expressed in fore- and midbrain primordia, eye and olfactory placodes. At 24hpf, strong nr2e1 expression was detected in telencephalon, hypothalamus, dorsal thalamus, pretectum, midbrain tectum, and retina. At 2dpf, the initially widespread nr2e1 expression became more restricted to distinct regions within the fore- and midbrain and to the retinal ciliary margin, the germinal zone which gives rise to retina and presumptive iris. Expression of nr2e3 was exclusively found in the developing retina and epiphysis. In both structures, nr2e3 expression was found in photoreceptor cells. The developmental expression profile of zebrafish nr2e1 and nr2e3 is consistent with evolutionary conserved functions in eye and rostral brain structures.     

Distinct patterns of expression of the RB gene family in mouse and human retina

Although RB1 function is disrupted in the majority of human cancers, an undefined cell of developing human retina is uniquely sensitive to cancer induction when the RB1 tumor suppressor gene is lost. Murine retinoblastoma is initiated only when two of the RB family of genes, RB1 and p107 or p130, are inactivated. Although whole embryonic retina shows RB family gene expression by several techniques, when E14 developing retina was depleted of the earliest differentiating cells, ganglion cells, the remaining proliferating murine embryonic retinal progenitor cells clearly did not express RB1 or p130, while the longer splice form of p107 was expressed. Each retinal cell type expressed some member of the RB family at some stage of differentiation. Rod photoreceptors stained for the RB1 protein product, pRB, and p107 in only a brief window of postnatal murine development, with no detectable staining for any of the RB family proteins in adult human and mouse rod photoreceptors. Adult mouse and human Muller glia, ganglion and rare horizontal cells, and adult human, but not adult mouse, cone photoreceptors stained for pRB. The RB gene family is dynamically and variably expressed through retinal development in specific retinal cells.     

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.