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-Crb1rd8/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 Crb1rd8 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-Crb1rd8/Pjn mice but photoreceptor cell mislocalization was more extensive. External limiting membrane disruption was comparable in Tvrm266 and B6.Cg-Crb1rd8/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.

Vascular Endothelial Growth Factor A and Leptin Expression Associated with Ectopic Proliferation and Retinal Dysplasia in Zebrafish Optic Pathway Tumors.

In the central nervous system injury induces cellular reprogramming and progenitor proliferation, but the molecular mechanisms that limit regeneration and prevent tumorigenesis are not completely understood. We previously described a zebrafish optic pathway tumor model in which transgenic Tg(flk1:RFP)is18/+ adults develop nonmalignant retinal tumors. Key pathways driving injury-induced glial reprogramming and regeneration contributed to tumor formation. In this study, we examine a time course of proliferation and present new analyses of the Tg(flk1:RFP)is18/+ dysplastic retina and tumor transcriptomes. Retinal dysplasia was first detected in 3-month-old adults, but was not limited to a specific stem cell or progenitor niche. Pathway analyses suggested a decrease in cellular respiration and increased expression of components of Hif1-α, VEGF, mTOR, NFκß, and multiple interleukin pathways are associated with early retinal dysplasia. Hif-α targets VEGFA (vegfab) and Leptin (lepb) were both highly upregulated in dysplastic retina; however, each showed distinct expression patterns in neurons and glia, respectively. Phospho-S6 immunolabeling indicated that mTOR signaling is activated in multiple cell populations in wild-type retina and in the dysplastic retina and advanced tumor. Our results suggest that multiple pathways may contribute to the continuous proliferation of retinal progenitors and tumor growth in this optic pathway tumor model. Further investigation of these signaling pathways may yield insight into potential mechanisms to control the proliferative response during regeneration in the nervous system.

Requirement of Smad4 from Ocular Surface Ectoderm for Retinal Development.

Microphthalmia is characterized by abnormally small eyes and usually retinal dysplasia, accounting for up to 11% of the blindness in children. Right now there is no effective treatment for the disease, and the underlying mechanisms, especially how retinal dysplasia develops from microphthalmia and whether it depends on the signals from lens ectoderm are still unclear. Mutations in genes of the TGF-ß superfamily have been noted in patients with microphthalmia. Using conditional knockout mice, here we address the question that whether ocular surface ectoderm-derived Smad4 modulates retinal development. We found that loss of Smad4 specifically on surface lens ectoderm leads to microphthalmia and dysplasia of retina. Retinal dysplasia in the knockout mice is caused by the delayed or failed differentiation and apoptosis of retinal cells. Microarray analyses revealed that members of Hedgehog and Wnt signaling pathways are affected in the knockout retinas, suggesting that ocular surface ectoderm-derived Smad4 can regulate Hedgehog and Wnt signaling in the retina. Our studies suggest that defective of ocular surface ectoderm may affect retinal development.

Unilateral sporadic retinal dysplasia: results of histopathologic, immunohistochemical, chromosomal, genetic, and VEGF-A analyses.

PURPOSE: To describe new findings in a case of unilateral retinal dysplasia. METHODS: Histopathologic evaluation of an enucleated globe and analysis with immunohistochemical probes, karyotyping, and genetic analysis for the Norrie gene, and aqueous assay for vascular endothelial growth factor A (VEGF-A). RESULTS: Histopathological examination of the globe revealed retinal dysplasia with pseudorosette formation, abnormal or absent retinal nuclear lamination, a paucity of disorganized retinal microvasculature, retinal infoldings, advanced gliosis, persistent hyperplastic vitreous, exuberant neovascularization of the vitreous, and iris neovascularization (identical to the findings observed in bilateral Norrie disease). Immunohistochemistry disclosed GFAP-positive and GLUT-1-positive gliosis and retinal and persistent hyperplastic vitreous microvessels that were CD34-positive and GLUT-1-negative. Ki-67-positive retinal cells were polarized toward the subretinal space and absent in the retinal invaginations and pseudorosettes. A normal karyotype was found, and DNA sequencing revealed no known mutation in the region of the Norrie gene (NDP) in sputum or retinal DNA. Aqueous obtained immediately after enucleation contained an exceptionally high concentration of VEGF-A (4.5 ng/mL). CONCLUSIONS: Despite the failure to find an abnormal NDP allele, other unexplored NDP regions, an undetected defect restricted to retinal tissues, or an autosomal mutation coupled with disrupted signaling pathways may be responsible for the condition. High aqueous VEGF-A suggests that this cytokine may play a role in pathogenesis in conjunction with other pathways.

The Notch signaling pathway in retinal dysplasia and retina vascular homeostasis.

The retina is one of the most essential elements of vision pathway in vertebrate. The dysplasia of retina cause congenital blindness or vision disability in individuals, and the misbalance in adult retinal vascular homeostasis leads to neovascularization-associated diseases in adults, such as diabetic retinopathy or age-related macular degeneration. Many developmental signaling pathways are involved in the process of retinal development and vascular homeostasis. Among them, Notch signaling pathway has long been studied, and Notch signaling-interfered mouse models show both neural retina dysplasia and vascular abnormality. In this review, we discuss the roles of Notch signaling in the maintenance of retinal progenitor cells, specification of retinal neurons and glial cells, and the sustaining of retina vascular homeostasis, especially from the aspects of conditional knockout mouse models. The potential of Notch signal manipulation may provide a powerful cell fate- and neovascularization-controlling tool that could have important applications in treatment of retinal diseases.

Relative quantification of white blood cell mitochondrial DNA and assessment of mitochondria by use of transmission electron microscopy in English Springer Spaniels with and without retinal dysplasia.

OBJECTIVE: To compare relative amounts of WBC mitochondrial DNA (mtDNA; assessed via real-time PCR assay) and morphology of lymphocyte mitochondria (assessed via transmission electron microscopy [TEM]) in blood samples collected from English Springer Spaniels with and without retinal dysplasia. ANIMALS: 7 and 5 client-owned English Springer Spaniels (1 to 11 years old) with and without retinal dysplasia, respectively. PROCEDURES: Blood samples were obtained from affected and unaffected dogs via venipuncture. Genomic DNA was extracted from WBCs of the 7 affected and 5 unaffected dogs, and relative quantification of the cytochrome c oxidase subunit 1 gene (COX1) was determined via analysis of real-time PCR results. White blood cells from 3 affected and 4 unaffected dogs were embedded in epoxide resin for TEM; cross sections were examined for lymphocytes, which were measured. The mitochondria within lymphocytes were quantified, and the mitochondrial surface area per lymphocyte cross section was calculated. A masked technique was used to compare mitochondrial morphology between the 2 groups. RESULTS: Compared with the smallest measured quantity of mtDNA among unaffected dogs, mtDNA amounts varied among unaffected (1.08- to 4.76-fold differences) and affected dogs (1- to 2.68-fold differences). Analysis of lymphocyte measurements and mitochondrial surface area, morphology, and quantity revealed no significant differences between affected and unaffected dogs. CONCLUSIONS AND CLINICAL RELEVANCE: No significant differences were detected in relative amounts of WBC mtDNA or the size, number, or morphology of lymphocyte mitochondria in English Springer Spaniels affected with retinal dysplasia, compared with results for unaffected control dogs.

Proteomic profiling of the retinal dysplasia and degeneration chick retina.

PURPOSE: In our previous paper we undertook proteomic analysis of the normal developing chick retina to identify proteins that were differentially expressed during retinal development. In the present paper we use the same proteomic approach to analyze the development and onset of degeneration in the retinal dysplasia and degeneration (rdd) chick. The pathology displayed by the rdd chick resembles that observed in some of the more severe forms of human retinitis pigmentosa. METHODS: Two-dimensional gel electrophoresis (pH 4-7), gel image analysis, and mass spectrometry were used to profile the developing and degenerating retina of the rdd and wild-type (wt) chick retina. RESULTS: Several proteins were identified by mass spectrometry that displayed differential expression between normal and rdd retina between embryonic day 12 (E12) and post-hatch day 1 (P1). Secernin 1 displayed the most significant variation in expression between rdd and wt retina; this may be due to differential phosphorylation in the rdd retina. Secernin 1 has dipeptidase activity and has been demonstrated to play a role in exocytosis; it has been shown to be overexpressed in certain types of cancer and has also been suggested as a potential neurotoxicologically relevant target. Its role in the retina and in particular its differential expression in the degenerate rdd retina remains unknown and will require further investigation. Other proteins that were differentially expressed in the rdd retina included valosin-containing protein, beta-synuclein, stathmin 1, nucleoside diphosphate kinase, histidine triad nucleotide-binding protein, and 40S ribosomal protein S12. These proteins are reported to be involved in several cellular processes, including the ubiquitin proteasome pathway, neuroprotection, metastatic suppression, transcriptional and translational regulation, and regulation of microtubule dynamics. CONCLUSIONS: This proteomic study is the first such investigation of the rdd retina and represents a unique data set that has revealed several proteins that are differentially expressed during retinal degeneration in the rdd chick. Secernin 1 showed the most significant differences in expression during this degeneration period. Further investigation of the proteins identified may provide insight into the complex events underlying retinal degeneration in this animal model.

Defective formation of the inner limiting membrane in laminin beta2- and gamma3-null mice produces retinal dysplasia.

PURPOSE: Retinal basement membranes (BMs) serve as attachment sites for retinal pigment epithelial cells on Bruch's membrane and Müller cells (MCs) on the inner limiting membrane (ILM), providing polarity cues to adherent cells. The beta2 and gamma3 chains of laminin are key components of retinal BMs throughout development, suggesting that they play key roles in retinal histogenesis. This study was conducted to analyze how the absence of both beta2- and gamma3-containing laminins affects retinal development. Methods. The function of the beta2- and gamma3-containing laminins was tested by producing a compound deletion of both the beta2 and the gamma3 laminin genes in the mouse and assaying the effect on postnatal retinal development by using anatomic and electrophysiological techniques. Results. Despite the widespread expression of beta2 and gamma3 laminin chains in wild-type (WT) retinal BMs, the development of only one, the ILM, was disrupted. The postnatal consequence of the ILM disruption was an alteration of MC attachment and a resultant disruption in MC apical-basal polarity, which culminated in retinal dysplasia. Of importance, although their density was altered, retinal cell fates were unaffected. The laminin mutants have a markedly decreased visual function, resulting in part from photoreceptor dysgenesis. Conclusions. These data suggest that beta2 and gamma3 laminin isoforms are critical for the formation and stability of the ILM. These data also suggest that attachment of the MC to the ILM provides important polarity cues to the MC and for postnatal retinal histogenesis.

Role of INK4a locus in normal eye development and cataract genesis.

The murine INK4a locus encodes the critical tumor suppressor proteins, p16(INK4a) and p19(ARF). Mice lacking both p16(INK4a) and p19(ARF) (INK4a-/-) in their FVB/NJ genetic backgrounds developed cataracts and microophthalmia. Histopathologically, INK4a-/- mice showed defects in the developmental regression of the hyaloid vascular system (HVS), retinal dysplasia, and cataracts with numerous vacuolations, closely resembling human persistent hyperplastic primary vitreous (PHPV). Ocular defects, such as retinal fold and abnormal migration of lens fiber cells, were observed as early as embryonic day (E) 15.5, thereby resulting in the abnormal differentiation of the lens. We also found that ectopic expression of p16(INK4a) resulted in the induction of gammaF-crystallin, suggesting an important role of INK4a locus during mouse eye development, and also providing insights into the potential genetic basis of human cataract genesis.

Axon fasciculation defects and retinal dysplasias in mice lacking the immunoglobulin superfamily adhesion molecule BEN/ALCAM/SC1.

The immunoglobulin superfamily adhesion molecule BEN (other names include ALCAM, SC1, DM-GRASP, neurolin, and CD166) has been implicated in the control of numerous developmental and pathological processes, including the guidance of retinal and motor axons to their targets. To test hypotheses about BEN function, we disrupted its gene via homologous recombination and analyzed the resulting mutant mice. Mice lacking BEN are viable and fertile, and display no external morphological defects. Despite grossly normal trajectories, both motor and retinal ganglion cell axons fasciculated poorly and were occasionally misdirected. In addition, BEN mutant retinae exhibited evaginated or invaginated regions with photoreceptor ectopias that resembled the "retinal folds" observed in some human retinopathies. Together, these results demonstrate that BEN promotes fasciculation of multiple axonal populations and uncover an unexpected function for BEN in retinal histogenesis.

Overproduction and partial purification of the Norrie disease gene product, norrin, from a recombinant baculovirus.

Abnormal vascularization of the peripheral retina and retinal detachment are common clinical characteristics of Norrie disease (ND), familial exudative vitreoretinopathy, Coats' disease, and retinopathy of prematurity. Although little is known about the molecular basis of these diseases, studies have shown that all of these diseases are associated with mutations in the ND gene. In spite of this, little is known about norrin, its molecular mechanism of action, and its functional relationship with the development of abnormal retinal vasculature. To obtain a large quantity of norrin for structural and functional studies, we have overproduced it in insect cells. For this purpose, a cDNA fragment (869 bp) was isolated from a human retinal cDNA library by amplification and was cloned into an expression vector. The purified plasmid was co-transfected with wild-type linearized Bac-N-Blue DNA into S. frugiperda Sf21 insect cells. The recombinant virus plaques were purified and clones were selected based on the level of recombinant protein expressed in Sf21 cells infected with a purified recombinant virus. From these, a high-titer stock was generated and subsequently used to prepare a fused protein on a large scale. The protein was partially purified by the process of immobilized metal affinity chromatography and the use of ion exchange chromatography

Loss of heterozygosity for the NF2 gene in retinal and optic nerve lesions of patients with neurofibromatosis 2.

Individuals affected with the neurofibromatosis 2 (NF2) cancer predisposition syndrome develop specific ocular lesions. To determine whether these lesions result from altered NF2 gene expression, microdissection and PCR were used to investigate 40 ocular lesions from seven eyes of four NF2 patients for LOH, with markers that flank the NF2 gene on chromosome 22q. NF2 protein (merlin) expression was also evaluated in these lesions, using immunohistochemistry. Retinal hamartoma was observed in all seven eyes, including one with combined pigment epithelial and retinal hamartoma (CPERH). Retinal tufts were present in four eyes (three patients), retinal dysplasia in two eyes (two patients), optic nerve neurofibroma in one eye, iris naevoid hyperplasia in two eyes (two patients) and pseudophakia in all eyes. Markers were informative in three patients (six eyes from three unrelated families). One patient was non-informative due to prolonged decalcification. All retinal and optic nerve, but not iris lesions, demonstrated consistent LOH for the NF2 gene. Merlin was not expressed in the retina, optic nerve, or iris lesions. These results suggest that inactivation of the NF2 gene is associated with the formation of a variety of retinal and optic nerve lesions in NF2 patients.

Progressive photoreceptor degeneration, outer segment dysplasia, and rhodopsin mislocalization in mice with targeted disruption of the retinitis pigmentosa-1 (Rp1) gene.

Retinitis pigmentosa (RP), a common group of human retinopathic diseases, is characterized by late-onset night blindness, loss of peripheral vision, and diminished or absent electroretinogram (ERG) responses. Mutations in the photoreceptor-specific gene RP1 account for 5-10% of cases of autosomal dominant RP. We generated a mouse model of the RP1 form of RP by targeted disruption of the mouse ortholog (Rp1) of human RP1. In Rp1(-/-) mice, the number of rod photoreceptors decreased progressively over a period of 1 year, whereas that of cone photoreceptors did not change for at least 10 months. Light and electron microscopic analysis revealed that outer segments of Rp1(-/-) rods and cones were morphologically abnormal and became progressively shorter in length. Before photoreceptor cell death, rhodopsin was mislocalized in inner segments and cell bodies of Rp1(-/-) rods. Rod ERG amplitudes of Rp1(-/-) mice were significantly smaller than those of Rp1(+/+) mice over a period of 12 months, whereas those of Rp1(+/-) mice were intermediate. The decreases in cone ERG amplitudes were slower and less severe than those in rods. These findings demonstrate that Rp1 is required for normal morphogenesis of photoreceptor outer segments and also may play a role in rhodopsin transport to the outer segments. The phenotype of Rp1 mutant mice resembles the human RP1 disease. Thus, these mice provide a useful model for studies of RP1 function, disease pathology, and therapeutic interventions.

Control of Müller glial cell proliferation and activation following retinal injury.

Müller glial cells are the major support cell for neurons in the vertebrate retina. Following neuronal damage, Müller cells undergo reactive gliosis, which is characterized by proliferation and changes in gene expression. We have found that downregulation of the tumor supressor protein p27Kip1 and re-entry into the cell cycle occurs within the first 24 hours after retinal injury. Shortly thereafter, Müller glial cells upregulate genes typical of gliosis and then downregulate cyclin D3, in concert with an exit from mitosis. Mice lacking p27Kip1 showed a constitutive form of reactive gliosis, which leads to retinal dysplasia and vascular abnormalities reminiscent of diabetic retinopathy. We conclude that p27Kip1 regulates Müller glial cell proliferation during reactive gliosis.

Time-specific action of N-methyl-N-nitrosourea in the occurrence of retinal dysplasia and retinal degeneration in neonatal mice.

The morphologic response of neonatal mouse retina to the alkylating agent N-methyl-N-nitrosourea (MNU) was examined at different periods of retinal development. A dose of 60 mg/kg N-methyl-N-nitrosourea was injected intraperitoneally to neonatal C57BL mice at 0, 3, 5, 8, 11, 14, 17, and 20 days of age and to C3H mice at 0 days of age, and the retinas were examined sequentially. In the C57BL mice, MNU evoked a time-dependent occurrence of retinal dysplasia and retinal degeneration. With MNU treatment at day 0 and day 3 (the stage of retinal cell proliferation), retinal dysplasia characterized by the progressive disorganization of neuroblasts, which led to the formation of rosettes, was found in the outer neuroblastic/nuclear layer above the normal pigment epithelial cells during days 8-20, but decreased at day 50. The rosettes were surrounded by photoreceptor segments and Müller cell processes, and by photoreceptor nuclei. The MNU response was related to retinal differentiation; following MNU treatment at day 5 or 8 (the stage of retinal cell differentiation) the cells were much less sensitive (i.e. no retinal response was found). However, with MNU treatment at days 11, 14, 17, and 20 (after cellular differentiation), retinal degeneration characterized by selective photoreceptor apoptosis was seen. These results suggest that there is a critical period for the time of MNU administration in the development of mouse retinal lesions. In C3H (rd/rd) mice, MNU treatment at day 0 resulted in retinal degeneration with only slight rosette formation at the peripheral retina.

Lectin-histochemical study of O-linked glycoconjugates in dysplastic retina of Norrie disease.

The carbohydrate chains of O-linked glycoconjugates in dysplastic retina from a Japanese female infant with Norrie disease were examined by lectin histochemistry. The avidin-biotinylated peroxidase method was used. The retina was highly dysplastic and composed of undifferentiated embryonic tissues containing a number of rosettes of varying sizes. The lumina of the rosettes were stained by peanut agglutinin, which recognizes the Gal beta 1,3GalNAc sequence of O-linked glycans. However, the lumina were not labeled by wheat germ agglutinin, which reacts with sialic acid and/or N-acetylglucosamine. These observations suggest that the O-linked glycoconjugates in the lumina of rosettes were not sialylated in the present case. Their lack of terminal sialic acids may be related to the rosette formation.

Glial-, neuronal- and photoreceptor-specific cell markers in rosettes of retinoblastoma and retinal dysplasia.

Previous studies have shown that a rosette formation represents an attempt to form embryonic retinal tissue, primarily rods and cones. To test the theories as to the origin and characteristics of retinoblastoma cells, we compared the characteristics of tumor rosettes with those of dysplastic rosettes seen in retinal dysplasia using the glial, neuronal and photoreceptor markers. Forty-four retinoblastoma and one retinal dysplasia specimens were analyzed by indirect immunohistochemistry, using specific antibodies against glial fibrillary acidic protein, S-100 protein, myelin basic protein, neuron-specific enolase, neurofilament, retinal S-antigen and retinal pigment epithelial antigen. In human retinoblastoma, all the glial, neuronal, retinal pigment epithelial, and photoreceptor cell markers, except for the neurofilament, were present in parts of rosette-forming tumor cells. However, their localization was different for each antigen and it was not clear whether each tumor cell possesses several antigens. These immuno-positive tumor cells were cytologically indistinguishable from other rosette-forming cells at the light microscopic level. In retinal dysplasia, neuron specific enolase and retinal S-antigen were diffusely expressed in the dysplastic rosettes, however, other antigen were not seen in those rosettes. The staining pattern by immunocytochemistry is totally different in tumor rosettes from dysplastic ones. We found varying localizations of different immunoreactivities within tumor rosettes. These results led us to suggest that tumor cells in the rosettes of retinoblastoma may have the ability to differentiate into neural and glial cells. To prove the theory that retinoblastoma cells may have originated from a primitive neuroectodermal cell capable of multipotentiality, further investigation is needed.

Cyclic GMP, calcium and photoreceptor sensitivity in mice heterozygous for the rod dysplasia gene designated "rd".

The rise in photoreceptor cGMP, induced by less than 1.0 nM extracellular calcium, is delayed in retinas of mice heterozygous for the rod dysplasia gene (+/rd). The calcium ionophore A23187 reduces the delay, suggesting that +/rd outer segments contain more calcium than normal. In turn, this might explain the increased photosensitivity of the +/rd retina. During the response to low calcium there is no correlation in +/rd retinas between the total concentration of cGMP and the photoresponse amplitude and its time to peak. The observations imply that either free cGMP is abnormally independent of the bound pool in the +/rd photoreceptor outer segment or that factors other than cGMP and its phosphodiesterase are modulating the rising phase of the response. The time-to-peak of PIII in a +/rd retina, incubated in a standard medium and stimulated with dim light, is abnormally delayed. Reduction of extracellular calcium induces an abnormal delay as well in responses to higher light levels. In addition to this, a second delay manifests slowly in both the normal and the +/rd retina. More studies are needed to explain these observations.

Intramembranous particle distribution and filipin binding in dysplastic canine retina.

We examined dysplastic canine retina for changes in intramembranous particle (IMP) density and filipin binding to sterols. Differences in IMP density were identified in incipiently dysplastic fetal retina and also in the degree of filipin binding near the onset of the dysplastic process. The data suggest that there are temporal differences in IMPs and filipin-sterol complexes that may be related to the formation of retinal folds and disorganized dysplastic retina proliferation.