Inner retinal cell development is impaired in Smoky Joe chickens.

Many different components of the retina can be affected by inherited degenerative diseases causing blindness. Currently, 5 different mutant strains of chicken have already been studied as potential models for inherited retinal degeneration; however, the potential for the blind strain of White Leghorns, called Smoky Joe (SJ), as a model remains unknown. Ocular symptoms observed within homozygous SJ birds show the birds have varying levels of blindness at hatch and by 8 wk posthatch are completely blind, but details about the development of the blindness are unclear (Salter et al., 1997). The objective of this study was to characterize the retinal development of blind and sighted SJ chicks during embryogenesis, and to monitor the numbers of the retinal cells with cell-type-specific markers. Blind SJ chicks showed less retinal cells throughout embryogenesis compared with sighted SJ chicks (P < 0.0001). Based on the histological analysis, it was determined that amacrine cells within the inner nuclear layer were the most affected cell type, showing lower numbers in the blind SJ compared with the sighted; amacrine cell development was also delayed in the blind birds, beginning 2 d later than in sighted SJ birds. Photoreceptors were also scarcely detected within the blind SJ and potentially may be an additional target of developmental impairment. Further analysis on posthatch SJ will aid in determining degenerative characteristics of a fully developed retina and its cells.

Profiling retinal biochemistry in the MPDZ mutant retinal dysplasia and degeneration chick: a model of human RP and LCA.

PURPOSE: Raman microscopy, a rapid nondestructive technique that profiles the composition of biological samples, was used to characterize retinal biochemistry in the retinal dysplasia and degeneration (rdd) and wild-type (wt) chick retina during retinogenesis and at hatching. METHODS: Embryonic day (E)13 and posthatch day (P)1 rdd and wt retinal cross-sections (n = 3 of each line at each age) were profiled using 633 helium-neon laser excitation. The biochemical composition was determined using computational analysis of the Raman spectra. In parallel histology, TUNEL and glial fibrillary acidic protein (GFAP) immunostaining were used to visualize retinal dysfunction. RESULTS: Principal component (PC) analysis of the Raman spectra identified 50 major biochemical profiles, but only PCs that made significant contributions to variation within rdd and wt retina were mapped. These significant PCs were shown to arise from DNA, various fatty acids, melanin, and a number of proteins. Distinct patterns of GFAP immunostaining and a larger population of TUNEL-positive nuclei were observed in the rdd versus wt retina. CONCLUSIONS: This study has demonstrated that Raman microscopy can discriminate between major retinal biomolecules, thus providing an unbiased account of how their composition varies due to the impact of the MPDZ null mutation in the rdd chick relative to expression in the normal wt retina.

Retina-specific activation of a sustained hypoxia-like response leads to severe retinal degeneration and loss of vision.

Loss of vision and blindness in human patients is often caused by the degeneration of neuronal cells in the retina. In mouse models, photoreceptors can be protected from death by hypoxic preconditioning. Preconditioning in low oxygen stabilizes and activates hypoxia inducible transcription factors (HIFs), which play a major role in the hypoxic response of tissues including the retina. We show that a tissue-specific knockdown of von Hippel-Lindau protein (VHL) activated HIF transcription factors in normoxic conditions in the retina. Sustained activation of HIF1 and HIF2 was accompanied by persisting embryonic vasculatures in the posterior eye and the iris. Embryonic vessels persisted into adulthood and led to a severely abnormal mature vessel system with vessels penetrating the photoreceptor layer in adult mice. The sustained hypoxia-like response also activated the leukemia inhibitory factor (LIF)-controlled endogenous molecular cell survival pathway. However, this was not sufficient to protect the retina against massive cell death in all retinal layers of adult mice. Caspases 1, 3 and 8 were upregulated during the degeneration as were several VHL target genes connected to the extracellular matrix. Misregulation of these genes may influence retinal structure and may therefore facilitate growth of vessels into the photoreceptor layer. Thus, an early and sustained activation of a hypoxia-like response in retinal cells leads to abnormal vasculature and severe retinal degeneration in the adult mouse retina.

Chapter 8. Evolution and development in the cavefish Astyanax.

The teleost Astyanax mexicanus is a single species consisting of two radically different forms: a sighted pigmented surface-dwelling form (surface fish) and a blind depigmented cave-dwelling form (cavefish). The two forms of Astyanax have favorable attributes, including descent from a common ancestor, ease of laboratory culture, and the ability to perform genetic analysis, permitting their use as a model system to explore questions in evolution and development. Here, we review current research on the molecular, cellular, and developmental mechanisms underlying the loss of eyes and pigmentation in Astyanax cavefish. Although functional eyes are lacking in adults, cavefish embryos begin to develop eye primordia, which subsequently degenerate. The major cause of eye degeneration appears to be apoptotic cell death of the lens, which prevents the growth of other optic tissues, including the retina. Ultimately, the loss of the eye is the cause of craniofacial differences between cavefish and surface fish. Lens apoptosis is induced by enhanced activity of the Hedgehog signaling system along the cavefish embryonic midline. The absence of melanin pigmentation in cavefish is due to a block in the ability of undifferentiated melanoblasts to accumulate L-tyrosine, the precursor of L-DOPA and melanin, in melanosomes. Genetic analysis has shown that this defect is caused by a hypomorphic mutation in the p/oca2 gene encoding an integral melanosomal membrane protein. We discuss how current studies of eye and pigment regression have revealed some of the mechanisms in which cavefish development has been changed during evolution.

Generation of immature retinal neurons from proliferating cells in the pars plana after retinal histogenesis in mice with retinal degeneration.

PURPOSE: To study the differentiation of immature retinal neurons/retinal precursors in the ciliary epithelium after retinal histogenesis in mice with inherited or acquired retinal degeneration. METHODS: Immunoreactivity to anti-recoverin, rhodopsin, and Pax6 antibodies and binding to peanut agglutinin were analyzed histologically. The distribution and differentiation of immature retinal neurons/retinal precursors in the ciliary epithelium of mice with inherited (C3H/HeJ) and acquired (C57BL mice injected with 60 mg/kg N-methyl-N-nitrosourea) retinal degeneration were assessed. Proliferating retinal progenitors were labeled with bromodeoxyuridine (BrdU), and they were studied histologically using retinal markers. RESULTS: Many cells of rod and cone photoreceptor lineage were identified within the ciliary epithelium of the pars plana in adult mice with inherited retinal degeneration. Tracking experiments using BrdU indicated that some of recoverin-positive cells in the pars plana (approximately 3%) were generated after retinal histogenesis, and few were produced at or after postnatal day 24 (P24). The induction of acquired retinal degeneration in adult wild-type mice (P30) increased the number of BrdU-positve cells by roughly fourfold and recoverin-positive cells by approximately 17-fold in the pars plana. Moreover, some (approximately 1.5%) of the recoverin-positive cells were newly generated from dividing retinal progenitors in the adult pars plana. CONCLUSIONS: In response to retinal damage, an increased number of immature retinal neurons/retinal precursors was observed in the pars plana of mice with acquired and inherited retinal degeneration. Some of these cells differentiated from proliferating cells even after retinal histogenesis.

An essential role for frizzled 5 in mammalian ocular development.

Microphthalmia, coloboma and persistent fetal vasculature within the vitreous cavity are among the most common human congenital ocular anomalies, and each has been associated with a variety of genetic disorders. Here we show that, in the mouse, loss of frizzled 5 (Fz5) - a putative Wnt receptor expressed in the eye field, optic cup and retina - causes all of these defects with high penetrance. In the developing Fz5(-/-) eye, the sequence of defects, in order of appearance, is: increased cell death in the ventral retina, delayed and/or incomplete closure of the ventral fissure, an excess of mesenchymal cells in the vitreous cavity, an excess of retinal astrocyte precursors and mature astrocytes, and persistence of the hyaloid vasculature in association with a large number of pigment cells. Fz5(-/-) mice also exhibit a late-onset progressive retinal degeneration by approximately 6 months of age, which might be related to the expression of Fz5 in Müller glia in the adult retina. These results demonstrate a central role for frizzled signaling in mammalian eye development and are likely to be relevant to the etiology of congenital human ocular anomalies.

[Hypercapnia- and trans-arachidonic acid-induced retinal microvascular degeneration: implications in the genesis of retinopathy of prematurity]. / La rétinopathie du prématuré: Rôle de l'acide trans-arachidonique dans la dégénérescence des micro-vaisseaux de la rétine.

High oxygen tension is a major factor in the genesis of retinopathy of prematurity (ROP). However, clinical and experimental evidence also suggest a significant role for high levels of carbon dioxide (CO(2)). Hypercapnia is a facilitator of nitration in vitro, and nitrative stress is known to have an important role in microvascular degeneration leading to ischemia in conditions such as ROP. We hereby present evidence that prolonged exposure to CO(2) impairs developmental retinal neovascularisation through a mechanism involving increased endothelial nitric oxide synthase and induction of a nitrative stress; effects of hypercapnia are independent of its hyperaemic effects. Moreover, in a model of oxygen-induced retinopathy, we demonstrate that an in vivo nitrative stress associated with retinal vasoobliteration results in nitration of cis-arachidonic acids into trans-arachidonic acids (TAAs). TAAs act in turn as mediators of nitrative stress by causing microvascular degeneration by inducing expression of the anti-angiogenic factor thrombospondin-1. These recent findings establish a previously unexplored means by which hypercapnia hinders efficient neovascularisation and provide new insight into the molecular mechanisms of nitrative stress on microvascular injury involving TAA, therefore opening new therapeutic avenues in the management of nitrative stress disorders such as in ischemic retinopathies (of prematurity and of diabetes) and encephalopathies.

The effect of in ovo ethanol exposure on retina and optic nerve in a chick embryo model system.

Ocular anomalies seen in children with fetal alcohol syndrome (FAS) suggest that ocular structures are sensitive to alcohol exposure during their development. This study was designed to investigate the effect of in ovo ethanol (EtOH) exposure on retinal development and myelinization of optic nerve fibers at an ultra structural level in a chick embryo model system. Prior to incubation, fertilized chicken eggs were injected once with 100 microl of either 0.9% NaCl (vehicle control), or EtOH solutions at different doses (10, 30, or 50%, v:v in 0.9% NaCl) into their air sacs and incubated at 37.5 degrees C and saturation humidity. On day 20 embryos were analyzed in terms of their viability and growth and the optic cups including the optic nerves were dissected out. Specimens were processed for electron microscopy (EM). Results showed that, EtOH significantly decreased the viability of chick embryos (P < 0.045), and caused significant prenatal growth retardation (P < 0.004) in a dose-dependant manner. Light microscopy of semi thin sections revealed that prenatal exposure to EtOH resulted in both retinal degeneration and optic nerve hypoplasia (P < 0.001) in a dose-dependant manner. EM revealed that a dose-dependant decrease in the number of myelinated nerve fibers was profound in groups exposed to EtOH (P < 0.001). Furthermore, the myelin coats observed were thinner than those seen in control embryos. In groups exposed to EtOH myelin sheets were unorganized and contained vacuolar structures in between them. The tissue in between the cells and optic nerve fibers, on the other hand, lost its intact appearance with vacuolar and vesicular structures in between them. In addition, the optic nerve fibers contained granular accumulations in EtOH exposed groups. A dose dependent degeneration was also observed in retinas of EtOH exposed groups. The effect of EtOH was profound in pigment epithelium (PE), inner plexiform layer (IPL), and ganglion cell layer (GC). Mitochondrial deficiencies, and alterations in melanin granule number and distribution dominated the defects seen in PE. On the other hand, EM findings of all the affected layers were suggestive of induced cell death in EtOH exposed groups. Thus, this study suggests retinal development with the emphasis on melanin pigmentation in PE and optic nerve myelinization as potential targets of prenatal EtOH exposure and discusses potential mechanisms of EtOH action on these tissues.

Mouse eye gene microarrays for investigating ocular development and disease.

Microarray technology can facilitate simultaneous expression analysis of thousands of genes and assist in delineating cellular pathways involved in development or disease pathogenesis. Since public databases and commercial cDNA microarrays have an under-representation of eye-expressed genes, we generated over 3000 expressed sequence tags from three unamplified mouse eye/retina cDNA libraries. These eye-expressed genes were used to produce cDNA microarrays. Methodology for printing of slides, hybridization, scanning and data analysis has been optimized. The I-gene microarrays will be useful for establishing expression profiles of the mouse eye/retina and provide a resource for defining molecular pathways involved in development, aging and disease.

Zebrafish mutagenesis yields eye morphological mutants with retinal and lens defects.

A chemical mutagenesis to identify zebrafish eye morphological mutants was performed by screening F(3) larvae at 5 and 7 days post-fertilization (dpf) for changes in eye or pupil size. Based on histological analysis, four different phenotypic classes were obtained. The two Class I and three Class II mutants are all characterized by small eyes and exhibit defects in early retinal development or unregulated cell death, respectively. The single Class III mutant has reduced ocular pigmentation. The three Class IV mutants display defects in the ocular lens, including one mutant line with normal sized eyes and pupils that develops lens opacity at 7 dpf.

sine oculis is a homeobox gene required for Drosophila visual system development.

The somda (sine oculis-medusa) mutant is the result of a P element insertion at position 43C on the second chromosome. somda causes aberrant development of the larval photoreceptor (Bolwig's) organ and the optic lobe primordium in the embryo. Later in development, adult photoreceptors fail to project axons into the optic ganglion. Consequently optic lobe development is aborted and photoreceptor cells show age-dependent retinal degeneration. The so gene was isolated and characterized. The gene encodes a homeodomain protein expressed in the optic lobe primordium and Bolwig's organ of embryos, in the developing adult visual system of larvae, and in photoreceptor cells and optic lobes of adults. In addition, the SO product is found at invagination sites during embryonic development: at the stomadeal invagination, the cephalic furrow, and at segmental boundaries. The mutant somda allele causes severe reduction of SO embryonic expression but maintains adult visual system expression. Ubiquitous expression of the SO gene product in 4-8-hr embryos rescues all somda mutant abnormalities, including the adult phenotypes. Thus, all deficits in adult visual system development and function results from failure to properly express the so gene during embryonic development. This analysis shows that the homeodomain containing SO gene product is involved in the specification of the larval and adult visual system development during embryogenesis.

Effects of the antioxidant butylated hydroxytoluene (BHT) on retinal degeneration induced transplacentally by a single low dosage of N-methyl-N-nitrosourea (MNU).

A 1 mg/kg dose of the DNA alkylating agent, N-methyl-N-nitrosourea (MNU), when administered on day 16 of gestation provokes a progressive retinal degeneration in CD-1 albino mice reared under standard fluorescent lighting conditions (12 hr light: 12 hr dark); this degeneration begins at about 4 weeks post-natally and worsens with age. It is accelerated by constant fluorescent light exposure but is retarded greatly by constant darkness, suggesting the importance of secondary insults in the post-natal period for development of the degenerative disease. To determine whether the secondary photochemical damage might be specifically blocked, MNU-exposed and control animals in the present study were fed an antioxidant-enriched diet of Purina mouse chow supplemented with 0.75% butylated hydroxytoluene (BHT). A second group of MNU-exposed and control animals were fed a non-BHT supplemented standard Purina mouse chow diet. Systematic measurements of the number of rows of photoreceptor cell nuclei, the thickness of the inner/outer segment layer, and the thickness of the whole retina were made, to quantify and degenerative changes in animals 2, 4, 6, and 8 weeks of age. By 8 weeks, retinas of BHT-fed, MNU-exposed animals were significantly thicker and had more rows of photoreceptor cell nuclei than regular-diet, MNU-exposed animals. Moreover, the retinas of BHT-fed animals, both for MNU-exposed and controls, demonstrated sporadic morphologic changes in the form of circular configurations composed of ganglion cells, arcades of nuclear and plexiform layers, and, in one control animal, a hyperplastic nodule. These experiments suggested that MNU-induced retinal degeneration may be retarded by a BHT-enriched diet; however, continuous high doses of this compound pre- and postnatally may induce other retinal abnormalities.

Glutamate-induced cellular injury in isolated chick embryo retina: Müller cell localization of initial effects.

The neurotoxic and gliotoxic effects of glutamate and several glutamate analogues were studied in isolated chick embryo retinas. To facilitate examination of initial pathological events, a short-term incubation system was developed and used for light microscopic and autoradiographic investigation. Low-dose, short-term glutamate treatment of 12-day retinas resulted in a glial-specific lesion in the Müller cells, characterized by extensive cellular edema; at higher concentrations and/or longer treatment times, neurotoxic as well as gliotoxic effects were seen. The early glial damage was identical in appearance to that seen after incubation with DL-alpha-aminoadipate and other reported gliotoxins. No evidence of a similar glial-specific action was seen after administration of kainic acid, although extensive neuronal degeneration did result. Incubation of retinas with tritiated glutamate (3H-glu) revealed a selective uptake of the label by Müller cells. Autoradiographic grains were localized over Müller foot processes at the inner limiting membrane, and by 30 minutes labeled the entire glial system. Prior treatment with neurotoxic levels of glutamate did not alter the autoradiographic localization to glial cells. Possible glial-neuronal interactions and their effect on cytotoxic patterns are discussed.