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.

Retinal structural and microvascular abnormalities in retinal dysplasia imaged by OCT and OCT angiography.

OBJECTIVE: To describe the in vivo structural characteristics of multifocal and geographic retinal dysplasia visualized with advanced retinal imaging including confocal scanning laser ophthalmoscopy (cSLO), optical coherence tomography (OCT), en face OCT, and the novel vascular imaging technique OCT angiography (OCTA). DOGS STUDIED AND PROCEDURES: Two dogs were diagnosed with unilateral multifocal or geographic retinal dysplasia and underwent advanced retinal imaging under general anesthesia at the Retinal Disease Studies Facility of the University of Pennsylvania. RESULTS: In both cases, the morphological pattern of the lesions was similar including outer retinal folds that invaginated and formed tubular retinal rosettes, surrounding a central inner retinal thickening (multifocal) or plaque (geographic). The two dogs had multiple vascular anomalies in the lesions such as increased tortuosity, abnormal change of vessel diameter including aneurysms and capillary network disruption. We also identified increased autofluorescence by AF cSLO with short wavelength light source (488 nm and barrier filter at 500 nm), and several areas of photoreceptor loss associated with the lesions. CONCLUSION: The use of OCTA allowed the identification of microvascular abnormalities associated with multifocal and geographic retinal dysplasia in two dogs. To our knowledge, this is the first report where the dye-free OCTA technique is used to study vascular lesions in canine retinas.

Progenitor death drives retinal dysplasia and neuronal degeneration in a mouse model of ATRIP-Seckel syndrome.

Seckel syndrome is a type of microcephalic primordial dwarfism (MPD) that is characterized by growth retardation and neurodevelopmental defects, including reports of retinopathy. Mutations in key mediators of the replication stress response, the mutually dependent partners ATR and ATRIP, are among the known causes of Seckel syndrome. However, it remains unclear how their deficiency disrupts the development and function of the central nervous system (CNS). Here, we investigated the cellular and molecular consequences of ATRIP deficiency in different cell populations of the developing murine neural retina. We discovered that conditional inactivation of Atrip in photoreceptor neurons did not affect their survival or function. In contrast, Atrip deficiency in retinal progenitor cells (RPCs) led to severe lamination defects followed by secondary photoreceptor degeneration and loss of vision. Furthermore, we showed that RPCs lacking functional ATRIP exhibited higher levels of replicative stress and accumulated endogenous DNA damage that was accompanied by stabilization of TRP53. Notably, inactivation of Trp53 prevented apoptosis of Atrip-deficient progenitor cells and was sufficient to rescue retinal dysplasia, neurodegeneration and loss of vision. Together, these results reveal an essential role of ATRIP-mediated replication stress response in CNS development and suggest that the TRP53-mediated apoptosis of progenitor cells might contribute to retinal malformations in Seckel syndrome and other MPD disorders.This article has an associated First Person interview with the first author of the paper.

Focal/multifocal and geographic retinal dysplasia in the dog-In vivo retinal microanatomy analyses.

PURPOSE: To examine the in vivo microanatomy of retinal folds and geographic lesions in dogs with acquired or inherited retinal dysplasia. MATERIAL AND METHODS: Thirteen dogs had retinal microanatomy evaluation under general anesthesia using cSLO/sdOCT; two eyes had noninherited multifocal retinal folds, five had inherited multifocal retinal folds (drd1 or drd2), and 10 geographic retinal dysplasia. Retinas from two drd2 carrier dogs were examined by histology and immunohistochemistry (IHC) after in vivo imaging. RESULTS: Retinal folds are the common feature of acquired focal/multifocal or geographic retinal dysplasia, are indistinguishable structurally from those associated with syndromic oculoskeletal dysplasia, and represent outer nuclear layer invaginations and rosettes visible by sdOCT. In dogs heterozygous for oculoskeletal dysplasia, the folds form clusters in a perivascular distribution along superior central vessels. IHC confirmed photoreceptor identity in the retinal folds. The geographic dysplasia plaques are not focally detached, but have inner retinal disorganization and intense autofluorescence in cSLO autofluorescence mode that is mainly limited to the geographic lesion, but is not uniform and in some extends beyond the plaques. CONCLUSION: We propose that the autofluorescent characteristic of the geographic lesions is associated with an inner retinal disruption associated with perivascular or infiltrating macrophages and phagocytosis of cellular debris. As well, we suggest restructuring the examination forms to distinguish the folds that are sporadically distributed from those that have a perivascular distribution as the latter likely represent carriers for drd. In this latter group, DNA testing would be a helpful tool to provide specific breeding advice.

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.

Retinal dysplasia in American pit bull terriers--phenotypic characterization and breeding study.

OBJECTIVE: The purpose of this study was to investigate the inheritance and phenotype of retinal dysplasia (RD) in the American pit bull terrier. ANIMALS STUDIED: A breeding colony established from a single female pure-bred American pit bull terrier dog with RD. PROCEDURES: A female pure-bred American pit bull terrier with RD was donated to the Veterinary Hospital of Federal University of Paraná, Curitiba, Brazil. A breeding colony was established and the phenotype and inheritance of the condition investigated. Regular ophthalmic examinations and fundus photography were performed on three generations of offspring from the founder animal. Some animals were additionally studied by optical coherence tomography. Ocular histopathology was performed on some animals from the colony. RESULTS: Fifty-seven offspring were produced in two generations from the affected founder female. Thirty-two were diagnosed with RD and showed a spectrum of severity of lesions including multifocal, and or geographic lesions and some developed retinal detachment. Histologic examination demonstrated retinal folds, rosettes, and areas of retinal detachment. The affected dogs were shorter in stature than the unaffected littermates. Breeding studies suggested the trait has an autosomal dominant mode of inheritance. DNA testing showed that the affected dogs were negative for the known gene mutations for canine dwarfism with RD. CONCLUSION: This is a report of a novel inherited form of RD that affects American pit bull terriers.

Spectral domain optical coherence tomography imaging of the posterior segment of the eye in the retinal dysplasia and degeneration chicken, an animal model of inherited retinal degeneration.

OBJECTIVE: To investigate qualitative and quantitative differences in the structure of the posterior segment of the eye in 1-day post-hatch and 12-month-old retinal dysplasia and degeneration (rdd) and wild-type chickens. ANIMAL STUDIED: Retinal dysplasia and degeneration and wild-type chickens. PROCEDURE: Using a commercially available spectral domain optical coherence tomography (OCT) system, 15° horizontal line scans were performed in both eyes of 24 live birds. Qualitative differences in retinal lamination and choroidal structure were investigated, and retinal and choroidal thickness were measured. RESULTS: Progressive retinal thinning with loss of outer retinal lamination and changes in the appearance of the choroid were seen in the rdd birds. Mean total retinal thickness was 202 µm (SD 7.8) and 251 µm (SD 8.8) in the rdd and wild-type chicks and 154 µm (SD 18) and 280 µm (SD 10.8) in the adult birds. Much of the difference was the result of loss of outer retinal lamination and thickness in the rdd birds. Mean choroidal thickness was 76 µm (SD 19.6) and 112 µm (SD 36.9) in the rdd and wild-type chicks and 85 µm (SD 23.7) and 228 µm (SD 44.1) in the rdd and wild-type adult birds, respectively. CONCLUSIONS: Differences in retinal and choroidal structure and thickness between rdd and wild-type birds were evident on spectral domain OCT imaging at 1-day post-hatch and more marked at 1 year. Spectral domain OCT may provide a reliable end point for therapeutic intervention in this animal model of inherited retinal degeneration.

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.

Ectopic proliferation contributes to retinal dysplasia in the juvenile zebrafish patched2 mutant eye.

PURPOSE: Patched is a well-studied tumor suppressor and negative regulator of the Hedgehog (Hh) pathway. Earlier work in this laboratory has shown that embryonic zebrafish patched2 (ptc2) mutant retinas possess an expanded ciliary marginal zone (CMZ) and phenotypes similar to those in human patients with basal cell naevus syndrome (BCNS), a congenital disorder linked to mutations in the human PTCH gene. This study extends the analysis of retinal structure and homeostasis in ptc2-/- mutants to juvenile stages, to determine whether Patched 2 function is essential in the postembryonic eye. METHODS: Histologic, immunohistochemical, and molecular analyses were used to characterize retinal defects in the 6-week-old juvenile ptc2-/- retina. RESULTS: Juvenile ptc2-/- mutants exhibited peripheral retinal dysplasias that included the presence of ectopic neuronal clusters in the inner nuclear layer (INL) and regions of disrupted retinal lamination. Retinal dysplasias were locally associated with ectopic proliferation. BrdU/EdU labeling and immunohistochemistry assays demonstrated that a population of ectopically proliferating cells gave rise to the ectopic neuronal clusters in the INL of ptc2-/- mutants and that this contributed to retinal dysplasia in the mutant eye. CONCLUSIONS: These results demonstrate a direct link between overproliferation and retinal dysplasia in the ptc2-/- juvenile retina and establish ectopic proliferation as the likely cellular underpinning of retinal dysplasia in juvenile ptc2-/- mutants.

Mitochondrial transcription factor A (Tfam) gene sequencing and mitochondrial evaluation in inherited retinal dysplasia in miniature schnauzer dogs.

Mitochondrial transcription factor A (Tfam) has been implicated in the pathogenesis of retinal dysplasia in miniature schnauzer dogs and it has been proposed that affected dogs have altered mitochondrial numbers, size, and morphology. To test these hypotheses the Tfam gene of affected and normal miniature schnauzer dogs with retinal dysplasia was sequenced and lymphocyte mitochondria were quantified, measured, and the morphology was compared in normal and affected dogs using transmission electron microscopy. For Tfam sequencing, retina, retinal pigment epithelium (RPE), and whole blood samples were collected. Total RNA was isolated from the retina and RPE and reverse transcribed to make cDNA. Genomic DNA was extracted from white blood cell pellets obtained from the whole blood samples. The Tfam coding sequence, 5' promoter region, intron1 and the 3' non-coding sequence of normal and affected dogs were amplified using polymerase chain reaction (PCR), cloned and sequenced. For electron microscopy, lymphocytes from affected and normal dogs were photographed and the mitochondria within each cross-section were identified, quantified, and the mitochondrial area (µm²) per lymphocyte cross-section was calculated. Lastly, using a masked technique, mitochondrial morphology was compared between the 2 groups. Sequencing of the miniature schnauzer Tfam gene revealed no functional sequence variation between affected and normal dogs. Lymphocyte and mitochondrial area, mitochondrial quantification, and morphology assessment also revealed no significant difference between the 2 groups. Further investigation into other candidate genes or factors causing retinal dysplasia in the miniature schnauzer is warranted.

A de novo deletion of 20q11.2-q12 in a boy presenting with abnormal hands and feet, retinal dysplasia, and intractable feeding difficulty.

Proximal interstitial deletions involving 20q11-q12 are very rare. Only two cases have been reported. We describe another patient with 20q11.21-q12 deletion. We precisely mapped the 6.5-Mb deletion and successfully determined the deletion landmarks at the nucleotide level. Common clinical features among the three cases include developmental delay, intractable feeding difficulties with gastroesophageal reflux, and facial dysmorphism including triangular face, hypertelorism, and hypoplastic alae nasi, indicating that the 20q11.2-q12 deletion can be a clinically recognizable syndrome. This is also supported by the fact that the three deletions overlap significantly. In addition, unique features such as arthrogryposis/fetal akinesia (hypokinesia) deformation and retinal dysplasia are recognized in the patient reported herein.

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.

Mutation discovered in a feline model of human congenital retinal blinding disease.

PURPOSE: To elucidate the gene defect in a pedigree of cats segregating for autosomal dominant rod-cone dysplasia (Rdy), a retinopathy characterized extensively from a clinical perspective. Disease expression in Rdy cats is comparable to that in young patients with congenital blindness (Leber congenital amaurosis [LCA] or retinitis pigmentosa [RP]). METHODS: A pedigree segregating for Rdy was generated and phenotyped by clinical ophthalmic examination methods including ophthalmoscopy and full-field flash electroretinography. Short tandem repeat loci tightly linked to candidate genes for autosomal dominant retinitis pigmentosa in humans were genotyped in the pedigree. RESULTS: Significant linkage was established to the candidate gene CRX (LOD = 5.56, = 0) on cat chromosome E2. A single base pair deletion was identified in exon 4 (n.546delC) in affected individuals but not in unaffected littermates. This mutation generates a frame shift in the transcript, introducing a premature stop codon truncating the putative CRX peptide, which would eliminate the critical transcriptional activation region. Clinical observations corroborate previously reported clinical reports about Rdy. Results show that the cone photoreceptor system was more severely affected than the rods in the early disease process. CONCLUSIONS: A putative mutation causative of the Rdy phenotype has been described as a single base pair deletion in exon 4 of the CRX gene, thus identifying the first animal model for CRX-linked disease that closely resembles the human disease. As such, it will provide valuable insights into the mechanisms underlying these diseases and their variable presentation, as well as providing a suitable model for testing therapies for these diseases.

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.

Ocular toxicity caused by Paclitaxel in neonatal sprague-dawley rats.

BACKGROUND: The toxic effects of paclitaxel (PTX) on neonatal eyes have not been evaluated. MATERIALS AND METHODS: PTX was dissolved in solvent containing polyethoxylated castor oil and intraperitoneally administered to male and female Sprague-Dawley rats at a dose of 0, 2, 4 and 8 mg/kg at 0 day of age, 4 mg/kg at 14 days of age, or 8 mg/kg at 12-18 weeks of age. Eyes were histologically examined 1 and/or 7 days after PTX. RESULTS: Male and female rats that received 4 mg/kg or more of PTX at 0 days of age developed cataracts and retinal dysplasias, while the rats that received other dosing regimens did not develop ocular lesions. Epithelial cells in the lens were apoptotic on day 1, and lens fibers were degenerative at day 7, indicating the development of cataracts. Scattered foci of apoptosis in the neuroblastic layer of the retina on day 1, and rosettes were seen on day 7, suggestive of retinal dysplasia. CONCLUSION: Neonatal rats that received a threshold dose of PTX (4 mg/kg) at a critical period (0 days of age) developed cataracts and retinal dysplasia; however, the 2 mg/kg dose at 0 days of age and the 4 or 8 mg/kg dose at 14 days of age or older caused no ocular damage. Thus, the determination of the dose and timing of PTX treatment administered during the early developmental stage requires great care to avoid ocular toxicity.