Adenosine triphosphate-induced photoreceptor death and retinal remodeling in rats.

Many common causes of blindness involve the death of retinal photoreceptors, followed by progressive inner retinal cell remodeling. For an inducible model of retinal degeneration to be useful, it must recapitulate these changes. Intravitreal administration of adenosine triphosphate (ATP) has recently been found to induce acute photoreceptor death. The aim of this study was to characterize the chronic effects of ATP on retinal integrity. Five-week-old, dark agouti rats were administered 50 mM ATP into the vitreous of one eye and saline into the other. Vision was assessed using the electroretinogram and optokinetic response and retinal morphology investigated via histology. ATP caused significant loss of visual function within 1 day and loss of 50% of the photoreceptors within 1 week. At 3 months, 80% of photoreceptor nuclei were lost, and total photoreceptor loss occurred by 6 months. The degeneration and remodeling were similar to those found in heritable retinal dystrophies and age-related macular degeneration and included inner retinal neuronal loss, migration, and formation of new synapses; Müller cell gliosis, migration, and scarring; blood vessel loss; and retinal pigment epithelium migration. In addition, extreme degeneration and remodeling events, such as neuronal and glial migration outside the neural retina and proliferative changes in glial cells, were observed. These extreme changes were also observed in the 2-year-old P23H rhodopsin transgenic rat model of retinitis pigmentosa. This ATP-induced model of retinal degeneration may provide a valuable tool for developing pharmaceutical therapies or for testing electronic implants aimed at restoring vision.

Focal adhesion kinase (FAK) expression and activation during lens development.

PURPOSE: Regulation of lens development involves an intricate interplay between growth factor (e.g. FGF and TGFbeta) and extracellular matrix (ECM) signaling pathways. Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that plays key roles in transmitting ECM signals by integrins. In this study, we delineated patterns of FAK expression and tyrosine phosphorylation (Y397) in the developing lens and investigated its regulation by FGF2. We also examined FAK expression and activation during disrupted fiber differentiation in mice expressing a dominant-negative TGFbeta receptor. METHODS: FAK expression and activation (phosphorylation on Y397) was studied in embryonic and postnatal rodent lenses by in situ hybridization, immunofluorescence, and western blotting. Rat lens explants were used to investigate the effects of FGF2 on FAK expression and activation. Immunofluorescence and western blotting were used to examine FAK expression and phosphorylation in transgenic mice that express a dominant-negative TGFbeta receptor. RESULTS: FAK is widely expressed and phosphorylated during embryonic stages of lens morphogenesis and differentiation. However, in postnatal lenses its expression and activation becomes restricted to the posterior germinative zone and the transitional zone at the lens equator. While both NH2- and COOH-terminal antibodies revealed cytoplasmic and membrane-associated staining in lens cells, the NH2-terminal antibody also showed FAK was present in fiber cell nuclei. In vitro, FAK expression and phosphorylation on Y397 were increased by concentrations of FGF2 that initiate lens epithelial cell migration (10 ng/ml) and differentiation (50 ng/ml) but not proliferation (5 ng/ml). Moreover, reactivity for Y397 phosphorylated FAK is prominent in the nuclei of differentiating fibers both in vivo and in vitro. Disruption of TGFbeta-like signals by ectopic expression of a dominant-negative TGFbeta receptor (TbetaRII(D/N)) results in abnormal lens fiber differentiation in transgenic mice. While FAK expression is initiated normally in the posterior germinative zone of TbetaRII(D/N) transgenic lenses, as fiber differentiation proceeds, FAK becomes localized to a perinuclear compartment, decreases its association with the cytoskeleton and is poorly phosphorylated on Y(397). CONCLUSIONS: FAK is widely expressed and activated during early lens morphogenesis. During secondary lens fiber differentiation, FAK is expressed and phosphorylated on Y397 as epithelial cells exit the cell cycle, initiate migration at the equator, and undergo differentiation in the transitional zone. During terminal fiber differentiation an NH2-terminal fragment of FAK, including Y397, is translocated to the nucleus. The expression, activation, and nuclear localization of FAK are regulated, at least partly, by FGF2. FAK activity and subcellular localization are also modulated by TGFbeta-like signals. In fiber cells of TbetaRII(D/N) transgenic lenses, FAK is abnormally retained in a perinuclear compartment, loses its association with the cytoskeleton, and is poorly phosphorylated. These data suggest that integrin signaling via FAK plays important roles during lens differentiation, mediated by FGFs and TGFbeta-superfamily signals.

Mutations of DNAI1 in primary ciliary dyskinesia: evidence of founder effect in a common mutation.

RATIONALE: Primary ciliary dyskinesia (PCD) is a rare, usually autosomal recessive, genetic disorder characterized by ciliary dysfunction, sino-pulmonary disease, and situs inversus. Disease-causing mutations have been reported in DNAI1 and DNAH5 encoding outer dynein arm (ODA) proteins of cilia. OBJECTIVES: We analyzed DNAI1 to identify disease-causing mutations in PCD and to determine if the previously reported IVS1+2_3insT (219+3insT) mutation represents a "founder" or "hot spot" mutation. METHODS: Patients with PCD from 179 unrelated families were studied. Exclusion mapping showed no linkage to DNAI1 for 13 families; the entire coding region was sequenced in a patient from the remaining 166 families. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on nasal epithelial RNA in 14 families. RESULTS: Mutations in DNAI1 including 12 novel mutations were identified in 16 of 179 (9%) families; 14 harbored biallelic mutations. Deep intronic splice mutations were not identified by reverse transcriptase-polymerase chain reaction. The prevalence of mutations in families with defined ODA defect was 13%; no mutations were found in patients without a defined ODA defect. The previously reported IVS1+2_3insT mutation accounted for 57% (17/30) of mutant alleles, and marker analysis indicates a common founder for this mutation. Seven mutations occurred in three exons (13, 16, and 17); taken together with previous reports, these three exons are emerging as mutation clusters harboring 29% (12/42) of mutant alleles. CONCLUSIONS: A total of 10% of patients with PCD are estimated to harbor mutations in DNAI1; most occur as a common founder IVS1+2_3insT or in exons 13, 16, and 17. This information is useful for establishing a clinical molecular genetic test for PCD.

WNT/Frizzled signaling in eye development and disease.

The canonical Wnt/Fzd signaling pathway is highly conserved among various species. Increasing evidence is accumulating for non-canonical Wnt signaling pathways, analogous to those discovered in Drosophila, being operative in vertebrates. Similarly, the networks of genes involved in eye development show significant conservation during evolution. The amenability of Drosophila for genetic manipulation and analysis of ocular phenotypes has delivered a great deal of information about the roles of the Wnt/Fzd signaling pathways at various stages of ocular development and growth, particularly in regulating the formation and size of the eye field, cell proliferation, polarity and differentiation. In addition to the numerous recent studies that have identified the expression of various components of these signaling pathways in the developing vertebrate eye, functional studies have revealed significant parallels in the way that Wnt/Fz signals regulate the formation of the vertebrate eye field and also the proliferation and differentiation of cells, particularly in the lens and retina. Significant advances have also recently been made in identifying mutations in these signaling pathways that underlie or contribute to various ocular diseases such as exudative vitreoretinopathy, retinal degenerations, cataract, ocular tumors and various congenital ocular malformations. Combined with the mechanistic studies in vertebrate and invertebrate models, these studies point to important functional roles for Wnt/Fzd pathways in the human eye. Further investigation of how these pathways function during eye development and growth may yield important insights into novel therapeutic approaches to treat or prevent diseases that cause blindness.

Laminin-binding integrins in rat lens morphogenesis and their regulation during fibre differentiation.

Mammalian lens development involves cell-cell and cell-ECM interactions. As integrins are a major family of cell adhesion molecules, we examined the expression patterns of several integrin subunits (alpha3A, alpha3B, alpha6A, alpha6B, beta1 and beta4) during rat lens development. RT-PCR, in situ hybridisation, immunofluorescence and immunoblotting were used to investigate expression of integrin subunits during lens development and differentiation. RT-PCR showed expression of alpha3A, alpha6A, alpha6B and beta1A but not alpha3B or beta4 subunits in postnatal rat lenses. Each subunit displayed distinct spatio-temporal expression patterns. beta1 integrin was expressed in both epithelium and fibres. alpha3A subunit expression was restricted to the epithelium; expression ceased abruptly at the lens equator. Expression of the alpha6A subunit increased during fibre differentiation, whereas alpha6B expression was predominantly associated with epithelial cells during lens development. In lens epithelial explants, FGF induced some of the changes in integrin expression that are characteristic of fibre differentiation in vivo. One notable exception was the inability of FGF to reproduce the distinctive down-regulation of the alpha3 isoform that is associated with initiation of elongation in vivo. Interestingly, vitreous treatment was able to reproduce this shift in alpha3 expression indicating that another factor(s), in addition to FGF, may be required for full and complete transition from an epithelial cell to a fibre cell. Integrin subunit expression therefore appears to be highly regulated during lens development and fibre differentiation with evidence of major changes in alpha3 and alpha6 isoform expression. These results indicate that integrins may play important roles in development and growth of the lens. How specific integrin subunits influence the behaviour of cells in different developmental compartments of the lens remains to be determined.

BMP and activin receptor expression in lens development.

PURPOSE: Members of the TGFbeta super-family have been shown to play important roles in lens development, including lens placode formation and fiber differentiation, and also induce changes characteristic of some forms of cataract. Previous studies demonstrated expression of TGFbeta receptors during lens morphogenesis. However, the expression patterns of activin and BMP receptors or their signaling mediators, the Smad proteins, have not been well documented. In this study we examine the spatio-temporal expression patterns of activin receptors (ActRIIA, ActRIIB, ALK1, and ALK2), BMP receptors (BMPRII, ALK3, and ALK6), and the distribution of the phosphorylated forms of Smad1 and Smad2 during normal lens development (E12-P21) and aberrant development in transgenic mice that express dominant negative TGFbeta receptors. METHODS: RT-PCR was used to identify receptor expression in total RNA isolated from P2 and P21 rat lenses. cDNAs were cloned and used for in situ hybridization analysis of spatio-temporal expression patterns in wild type and transgenic (OVE550 and OVE591) lenses. Expression of ALK3 was also examined by immunofluorescence and immunoblotting. Antibodies for phosphorylated forms of Smad1 and Smad2 were used to examine activation of BMP and activin signaling. RESULTS: RT-PCR of RNA from postnatal lenses showed distinct expression of ActRIIA, ActRIIB, BMPRII, and ALK3 but not ALK1, ALK2, or ALK 6. In situ hybridization with specific probes for BMPRII, ActRII, and ALK3 showed ubiquitous expression in ectoderm, lens pit, optic vesicle, and peri-optic mesenchyme during early lens formation at E12. During subsequent lens differentiation, from E14 onwards, expression of these receptors became increasingly restricted to the lens epithelium and to the equatorial region, including the germinative and transitional zones, where cells proliferate and commence differentiation, respectively. Expression for both receptors declined rapidly with fiber differentiation and maturation. Immunofluorescence with specific antibodies for phospho-Smad1 and phospho-Smad2 showed distinct localization of these signaling mediators in epithelial cells of the germinative zone and in fibers undergoing early differentiation in the transitional zone. Further investigation of the expression of these receptors in lenses of transgenic mice, which ectopically express a truncated TbetaRII, showed marked up regulation and aberrant expression of ALK3, but not BMPRII or ActRII. CONCLUSIONS: These results indicate that multiple members of the TGFbeta family have the potential to signal during lens fiber differentiation and suggest there may be cross-talk between different signaling pathways.

A role for Wnt/beta-catenin signaling in lens epithelial differentiation.

The differentiation of epithelial cells and fiber cells from the anterior and posterior compartments of the lens vesicle, respectively, give the mammalian lens its distinctive polarity. While much progress has been made in understanding the molecular basis of fiber differentiation, little is known about factors that govern the differentiation of the epithelium. Members of the Wnt growth factor family appear to be key regulators of epithelial differentiation in various organ systems. Wnts are ligands for Frizzled receptors and can activate several signaling pathways, of which the best understood is the Wnt/beta-catenin pathway. The presence of LDL-related protein coreceptors (LRPs) 5 or 6 has been shown to be a requirement for Wnt signaling through the beta-catenin pathway. To access the role of this signaling pathway in the lens, we analyzed mice with a null mutation of lrp6. These mice had small eyes and aberrant lenses, characterized by an incompletely formed anterior epithelium resulting in extrusion of the lens fibers into the overlying corneal stroma. We also showed that multiple Wnts, including 5a, 5b, 7a, 7b, 8a, 8b, and Frizzled receptors 1, 2, 3, 4, and 6, were detected in the lens. Expression of these molecules was generally present throughout the lens epithelium and extended into the transitional zone, where early fiber elongation occurs. In addition to both LRP5 and LRP6, we also showed the expression of other molecules involved in Wnt signaling and its regulation, including Dishevelleds, Dickkopfs, and secreted Frizzled-related proteins. Taken together, these results indicate a role for Wnt signaling in regulating the differentiation and behavior of lens cells.