Mapping of genetic modifiers of Eya1 bor/bor in CAST/EiJ and BALB/cJ that suppress cochlear aplasia and associated deafness

Mice homozygous for the hypomorphic allele Eya1 ( bor ) exhibit cochlear aplasia, with associated deafness, and renal hypoplasia, similar to Branchio-Oto-Renal syndrome (BOR) in humans. Although much is known about the genetics of the disease, little is known about the factors that modify its phenotypic expression. We have recently detailed two modifier loci (Mead1 and Mead2) in a C3HeB/FeJ-Eya1 ( bor/+ ) x C57BL/6 J intercross that suppress the ear-related phenotypes in our hypomorphic mutants. In this study we report corroborating evidence for our initial finding with the identification of two modifier loci mapping to the same region in CAST/EiJ and BALB/cJ. Furthermore, we describe an additional locus (Mead3) on chromosome 19 in CAST/EiJ, within which the previously cloned suppressor Nxf1 resides. The suppression effect on cochlear coiling was studied on congenic line(s) for each protective allele. The penetrance and suppressor strength of these alleles vary by strain and locus. Eya1 ( bor/bor ) hypomorphs, when homozygous for each of the three protective alleles (CAST/EiJ, C57BL/6 J, or BALB/cJ) at the Mead1 or Mead2 locus, exhibit completely penetrant suppression of cochlear agenesis. At the Mead1 locus, the C57BL/6 J and BALB/cJ alleles have comparable strengths. At the Mead2 locus, the C57BL/6 J and CAST/EiJ alleles have comparable strengths. In contrast, mice with genotype Eya1 ( bor/bor )Mead3(CAST/CAST) exhibit incomplete penetrance (50%) and a wide range of cochlear coiling (1/4-1(1/2) turns). The identification of these additional modifier alleles could provide crucial clues for evaluating the candidate genes.     

A novel mutation in Prph2 , a gene regulated by Nr2e3 , causes retinal degeneration and outer-segment defects similar to Nr2e3 rd7/rd7 retinas

The nmf193 mutant was generated by a large-scale ENU mutagenesis screen and originally described as having a dominantly inherited phenotype characterized by fundus abnormalities. We determined that nmf193 mice exhibit outer-segment defects and progressive retinal degeneration. Clinical examination revealed retinal spotting apparent at 6 weeks of age. Histologic analysis of homozygous mutant mice at 6 weeks indicated an absence of outer segments (OS) and a 50% reduction of photoreceptor cells which progressed to complete loss of photoreceptors by 10 months. Mice heterozygous for the nmf193 mutation had a less severe phenotype of shortened outer segments at 2 months with progressive loss of photoreceptor cells to 50% by 10 months. A positional cloning approach using a DNA pooling strategy was performed to identify the causative mutation in nmf193 mice. The nmf193 mutation was linked to chromosome 17 and fine mapped to an interval containing the peripherin/rds (Prph2) gene. Mutation analysis identified a single base change in Prph2 that causes aberrant splicing between exons 1 and 2. Interestingly, a comparative histologic analysis demonstrated that Prph2 ^nmf193/+ mutants have similar photoreceptor degeneration to that of Nr2e3 ^rd7/rd7 . We show that Prph2 mRNA and protein levels are reduced in the Nr2e3 ^rd7/rd7 mutant compared to control littermates. Chromatin immunoprecipitation analysis shows that Prph2 is a direct target of NR2E3. In addition, the downregulation of Prph2 gene expression is similar in both the Nr2e3 ^rd7/rd7 and Prph2 ^nmf193/+ mutants, suggesting that the reduction of Prph2 may contribute to the degenerative pathology seen in Nr2e3 ^rd7/rd7 .     

Knockout of Nr2e3 prevents rod photoreceptor differentiation and leads to selective L-/M-cone photoreceptor degeneration in zebrafish

Mutations in the photoreceptor cell-specific nuclear receptor gene Nr2e3 increased the number of S-cone photoreceptors in human and murine retinas and led to retinal degeneration that involved photoreceptor and non-photoreceptor cells. The mechanisms underlying these complex phenotypes remain unclear. In the hope of understanding the precise role of Nr2e3 in photoreceptor cell fate determination and differentiation, we generated a line of Nr2e3 knockout zebrafish using CRISPR technology. In these Nr2e3-null animals, rod precursors undergo terminal mitoses but fail to differentiate as rods. Rod-specific genes are not expressed and the outer segment (OS) fails to form. Formation and differentiation of cone photoreceptors is normal. Specifically, there is no increase in the number of UV-cone or S-cone photoreceptors. Laminated retinal structure is maintained. After normal development, L-/M-cones selectively degenerate, with progressive shortening of OS that starts at age 1 month. The amount of cone phototransduction proteins is concomitantly reduced, whereas UV- and S-cones have normal OS lengths even at age 10 months. In vitro studies show Nr2e3 synergizes with Crx and Nrl to enhance rhodopsin gene expression. Nr2e3 does not affect cone opsin expression. Our results extend the knowledge of Nr2e3's roles and have specific implications for the interpretation of the phenotypes observed in human and murine retinas. Furthermore, our model may offer new opportunities in finding treatments for enhanced S-cone syndrome (ESCS) and other retinal degenerative diseases.     

Identification of Arhgef12 and Prkci as genetic modifiers of retinal dysplasia in the Crb1rd8 mouse model

Mutations in the apicobasal polarity gene CRB1 lead to diverse retinal diseases, such as Leber congenital amaurosis, cone-rod dystrophy, retinitis pigmentosa (with and without Coats-like vasculopathy), foveal retinoschisis, macular dystrophy, and pigmented paravenous chorioretinal atrophy. Limited correlation between disease phenotypes and CRB1 alleles, and evidence that patients sharing the same alleles often present with different disease features, suggest that genetic modifiers contribute to clinical variation. Similarly, the retinal phenotype of mice bearing the Crb1 retinal degeneration 8 (rd8) allele varies with genetic background. Here, we initiated a sensitized chemical mutagenesis screen in B6.Cg-Crb1^rd8/Pjn, a strain with a mild clinical presentation, to identify genetic modifiers that cause a more severe disease phenotype. Two models from this screen, Tvrm266 and Tvrm323, exhibited increased retinal dysplasia. Genetic mapping with high-throughput exome and candidate-gene sequencing identified causative mutations in Arhgef12 and Prkci, respectively. Epistasis analysis of both strains indicated that the increased dysplastic phenotype required homozygosity of the Crb1^rd8 allele. Retinal dysplastic lesions in Tvrm266 mice were smaller and caused less photoreceptor degeneration than those in Tvrm323 mice, which developed an early, large diffuse lesion phenotype. At one month of age, Müller glia and microglia mislocalization at dysplastic lesions in both modifier strains was similar to that in B6.Cg-Crb1^rd8/Pjn mice but photoreceptor cell mislocalization was more extensive. External limiting membrane disruption was comparable in Tvrm266 and B6.Cg-Crb1^rd8/Pjn mice but milder in Tvrm323 mice. Immunohistological analysis of mice at postnatal day 0 indicated a normal distribution of mitotic cells in Tvrm266 and Tvrm323 mice, suggesting normal early development. Aberrant electroretinography responses were observed in both models but functional decline was significant only in Tvrm323 mice. These results identify Arhgef12 and Prkci as modifier genes that differentially shape Crb1-associated retinal disease, which may be relevant to understanding clinical variability and underlying disease mechanisms in humans.     

Localization of peripherin/rds in the disk membranes of cone and rod photoreceptors: relationship to disk membrane morphogenesis and retinal degeneration

The outer segments of vertebrate rod photoreceptor cells consist of an ordered stack of membrane disks, which, except for a few nascent disks at the base of the outer segment, is surrounded by a separate plasma membrane. Previous studies indicate that the protein, peripherin or peripherin/rds, is localized along the rim of mature disks of rod outer segments. A mutation in the gene for this protein has been reported to be responsible for retinal degeneration in the rds mouse. In the present study, we have shown by immunogold labeling of rat and ground squirrel retinas that peripherin/rds is present in the disk rims of cone outer segments as well as rod outer segments. Additionally, in the basal regions of rod and cone outer segments, where disk morphogenesis occurs, we have found that the distribution of peripherin/rds is restricted to a region that is adjacent to the cilium. Extension of its distribution from the cilium coincides with the formation of the disk rim. These results support the model of disk membrane morphogenesis that predicts rim formation to be a second stage of growth, after the first stage in which the ciliary plasma membrane evaginates to form open nascent disks. The results also indicate how the proteins of the outer segment plasma membrane and the disk membranes are sorted into their separate domains: different sets of proteins may be incorporated into membrane outgrowths during different growth stages of disk morphogenesis. Finally, the presence of peripherin/rds protein in both cone and rod outer segment disks, together with the phenotype of the rds mouse, which is characterized by the failure of both rod and cone outer segment formation, suggest that the same rds gene is expressed in both types of photoreceptor cells.     

Differential roles of ERK1/2 and JNK in retinal development and degeneration

Programmed cell death is well established as a key factor in the development of the vertebrate nervous system of which the retina is a unique sensory component. However, it is of utmost importance for the survival of post-mitotic tissues such as the retina that the execution of the cell death program is kept under stringent control once development is complete. This is exemplified by the many retinal dystrophies where aberrant apoptosis results in loss of distinct cell layers in the mature retina and often culminates in blindness. In this study, we report that the extracellular signal-regulated kinase (ERK1/2) pathway plays a key role in the regulation of apoptosis during retinal development. We show that as the retina matures, the emphasis shifts towards survival and ERK1/2 is activated resulting in phosphorylation of the potent BH3-only protein Bim(EL) and a dramatic decline in Bim(EL) expression via proteasomal degradation. We find that activation of ERK1/2 also occurs in response to injury in retinal explants. However, this is a transient response and appears to be overcome by Jun N-terminal kinase activation resulting in induction of Bim(EL) mRNA and photoreceptor apoptosis. Our findings provide new insights into the intracellular pathways responsible for regulating apoptosis during neuronal development and degeneration.     

Differential modulation of retinal degeneration by Ccl2 and Cx3cr1 chemokine signalling

Microglia and macrophages are recruited to sites of retinal degeneration where local cytokines and chemokines determine protective or neurotoxic microglia responses. Defining the role of Ccl2-Ccr2 and Cx3cl1-Cx3cr1 signalling for retinal pathology is of particular interest because of its potential role in age-related macular degeneration (AMD). Ccl2, Ccr2, and Cx3cr1 signalling defects impair macrophage trafficking, but have, in several conflicting studies, been reported to show different degrees of age-related retinal degeneration. Ccl2/Cx3cr1 double knockout (CCDKO) mice show an early onset retinal degeneration and have been suggested as a model for AMD. In order to understand phenotypic discrepancies in different chemokine knockout lines and to study how defects in Ccl2 and/or Cx3cr1 signalling contribute to the described early onset retinal degeneration, we defined primary and secondary pathological events in CCDKO mice. To control for genetic background variability, we compared the original phenotype with that of single Ccl2, Cx3cr1 and Ccl2/Cx3cr1 double knockout mice obtained from backcrosses of CCDKO with C57Bl/6 mice. We found that the primary pathological event in CCDKO mice develops in the inferior outer nuclear layer independently of light around postnatal day P14. RPE and vascular lesions develop secondarily with increasing penetrance with age and are clinically similar to retinal telangiectasia not to choroidal neovascularisation. Furthermore, we provide evidence that a third autosomal recessive gene causes the degeneration in CCDKO mice and in all affected re-derived lines and subsequently demonstrated co-segregation of the naturally occurring RD8 mutation in the Crb1 gene. By comparing CCDKO mice with re-derived CCl2(-/-)/Crb1(Rd8/RD8), Cx3cr1(-/-)/Crb1(Rd8/RD8) and CCl2(-/-)/Cx3cr1(-/-)/Crb1(Rd8/RD8) mice, we observed a differential modulation of the retinal phenotype by genetic background and both chemokine signalling pathways. These findings indicate that CCDKO mice are not a model of AMD, but a model for an inherited retinal degeneration that is differentially modulated by Ccl2-Ccr2 and Cx3cl1-Cx3cr1 chemokine signalling.     

Drosophila Lin-7 is a component of the Crumbs complex in epithelia and photoreceptor cells and prevents light-induced retinal degeneration

The Drosophila Crumbs protein complex is required to maintain epithelial cell polarity in the embryo, to ensure proper morphogenesis of photoreceptor cells and to prevent light-dependent retinal degeneration. In Drosophila, the core components of the complex are the transmembrane protein Crumbs, the membrane-associated guanylate kinase (MAGUK) Stardust and the scaffolding protein DPATJ. The composition of the complex and some of its functions are conserved in mammalian epithelial and photoreceptor cells. Here, we report that Drosophila Lin-7, a scaffolding protein with one Lin-2/Lin-7 (L27) domain and one PSD-95/Dlg/ZO-1 (PDZ) domain, is associated with the Crumbs complex in the subapical region of embryonic and follicle epithelia and at the stalk membrane of adult photoreceptor cells. DLin-7 loss-of-function mutants are viable and fertile. While DLin-7 localization depends on Crumbs, neither Crumbs, Stardust nor DPATJ require DLin-7 for proper accumulation in the subapical region. Unlike other components of the Crumbs complex, DLin-7 is also enriched in the first optic ganglion, the lamina, where it co-localizes with Discs large, another member of the MAGUK family. In contrast to crumbs mutant photoreceptor cells, those mutant for DLin-7 do not display any morphogenetic abnormalities. Similar to crumbs mutant eyes, however, DLin-7 mutant photoreceptors undergo progressive, light-dependent degeneration. These results support the previous conclusions that the function of the Crumbs complex in cell survival is independent from its function in photoreceptor morphogenesis.     

Comparison and characterization of retinal pericytes and retinal pigment epithelial cells on subcellular IP3-sensitive Ca2+ pools

Abstract. A comparative study of inositol 1,4,5-trisphosphate (IP₃)-induced Ca²⁺ mobilization in bovine retinal capillary pericytes (BRCP) and bovine retinal pigment epithelial cells (BRPE) was carried out. Both cells were permeabilized with saponin. The two cell types had similar basal levels of [Ca²⁺]i (130 nM for BRCP, 132 nM for BRPE) and responded to IP, in a dose-dependent manner. However, when stimulated by various concentrations of IP₃ (1–10 μM), the increase in [Ca²⁺]i of BRCP was always two- to threefold higher than that in BRPE. Subcellular-fractionation studies showed that a single population of IP₃ binding site with a high affinity and high specificity of IP₃ mainly localized to plasma membrane in these two cell types. Although the dissociation constant of specific [³²P]-IP₃ binding sites (Kd 1.9–2.8 nM) was similar, the profile of maximal binding capacity (Bmax) of each fraction was markedly different. In comparison, plasma membrane fractions of BRCP were with Bmax of 165 fmol/mg protein versus 90 fmol/mg protein for BRPE membranes. The ATP-dependent Ca²uptake and IP₃-dependent Ca²⁺ release were observed in the both plasma membrane fractions. With quantitative correlation, the membrane fraction (2 mg) of BRCP released 0.2 nmol Ca²⁺ whereas BRPE only released 0.07 nmol Ca²⁺ with the same dose of IP₃ (5 μM). The selectively higher density of IP, binding sites in coupling to the larger Ca²⁺-release in the membrane of BRCP suggests that the quantity of Ca²⁺ mobilized is determined by the spatially preferential distribution of membrane-associated IP₃ binding sites. These findings may provide an explanation for the differences observed between BRCP and BRPE in IP,-induced DNA replication.