The aqueous humor outflow pathway of zebrafish.

PURPOSE: The structures of the ocular anterior segment responsible for aqueous humor secretion and absorption have been well characterized in mammals. However, the underlying molecular and cellular mechanisms that regulate aqueous humor flow have remained elusive. Experimental analysis in Danio rerio, the zebrafish, is providing mechanistic insights into many cellular processes relevant to normal human physiology and disease. To facilitate studies on the molecular and cellular mechanisms of aqueous humor dynamics using this species, the authors have characterized the anatomy of aqueous secretion and outflow in adult zebrafish eyes. METHODS: Analyses by light and transmission electron microscopy, coupled with molecular tracers of fluid flow, were used to identify and study the sites of aqueous humor secretion and absorption in adult zebrafish eyes. RESULTS: Zebrafish eyes show aqueous humor secretion primarily from the dorsal ciliary region and outflow through a ventral canalicular network that connects with an aqueous plexus and veins of the choroidal rete. CONCLUSIONS: Vectorial flow of zebrafish aqueous humor is in contrast to that in mammals in which secretion and absorption of aqueous humor are circumferential around and through the iridocorneal angle. However, local anatomy and ultrastructure of the tissues and cells specialized for aqueous humor dynamics in zebrafish show conservation with that of mammals. These observations suggest that zebrafish can serve as a useful genetic model to help understand the regulation and cellular basis of normal and abnormal aqueous humor dynamics in humans.

Mutations in laminin alpha 1 result in complex, lens-independent ocular phenotypes in zebrafish.

We report phenotypic and genetic analyses of a recessive, larval lethal zebrafish mutant, bal(a69), characterized by severe eye defects and shortened body axis. The bal(a69) mutation was mapped to chromosome 24 near the laminin alpha 1 (lama1) gene. We analyzed the lama1 gene sequence within bal(a69) embryos and two allelic mutants, bal(arl) and bal(uw1). Missense (bal(a69)), nonsense (bal(arl)), and frameshift (bal(uw1)) alterations in lama1 were found to underlie the phenotypes. Extended analysis of bal(a69) ocular features revealed disrupted lens development with subsequent lens degeneration, focal cornea dysplasia, and hyaloid vasculature defects. Within the neural retina, the ganglion cells showed axonal projection defects and ectopic photoreceptor cells were noted at inner retinal locations. To address whether ocular anomalies were secondary to defects in lens differentiation, bal(a69) mutants were compared to embryos in which the lens vesicle was surgically removed. Our analysis suggests that many of the anterior and posterior ocular defects in bal(a69) are independent of the lens degeneration. Analysis of components of focal adhesion signaling complexes suggests that reduced focal adhesion kinase activation underlies the anterior segment dysgenesis in lama1 mutants. To assess adult ocular phenotypes associated with lama1 mutations, genetic mosaics were generated by transplanting labeled bal cells into ocular-fated regions of wild-type blastulas. Adult chimeric eyes displayed a range of defects including anterior segment dysgenesis and cataracts. Our analysis provides mechanistic insights into the developmental defects and ocular pathogenesis caused by mutations in laminin subunits.

Morphogenesis of the anterior segment in the zebrafish eye.

BACKGROUND: The ocular anterior segment is critical for focusing incoming light onto the neural retina and for regulating intraocular pressure. It is comprised of the cornea, lens, iris, ciliary body, and highly specialized tissue at the iridocorneal angle. During development, cells from diverse embryonic lineages interact to form the anterior segment. Abnormal migration, proliferation, differentiation, or survival of these cells contribute to diseases of the anterior segment such as corneal dystrophy, lens cataract, and glaucoma. Zebrafish represent a powerful model organism for investigating the genetics and cell biology of development and disease. To lay the foundation for genetic studies of anterior segment development, we have described the morphogenesis of this structure in zebrafish. RESULTS: As in other vertebrates, the zebrafish anterior segment derives from diverse origins including surface ectoderm, periocular mesenchyme, and neuroepithelium. Similarly, the relative timing of tissue differentiation in the anterior segment is also conserved with other vertebrates. However, several morphogenic features of the zebrafish anterior segment differ with those of higher vertebrates. These include lens delamination as opposed to invagination, lack of iris muscles and ciliary folds, and altered organization in the iridocorneal angle. In addition, substantial dorsal-ventral differences exist within the zebrafish anterior segment. CONCLUSION: Cumulatively, our anatomical findings provide a reference point to utilize zebrafish for genetic studies into the mechanisms of development and maintenance of the anterior segment.

Human immunodeficiency virus type 2 (HIV-2) vector-mediated in vivo gene transfer into adult rabbit retina.

PURPOSE: To evaluate the potential usefulness of HIV-2 viral vector in in vivo retinal gene therapy. METHODS: An HIV-2 virus based viral vector was constructed and administered subretinally and intravitreally into rabbit eyes. After viral vector administration, the eyes were closely monitored for any adverse effects by slit lamp, indirect ophthalmoscopy, and fundus photography. Eyes were enucleated at specified times after injection, and reporter gene expression was identified within cell types and graded by the pattern and distribution of staining cells using fluorescent microscopy. RESULTS: The HIV-2 viral vector demonstrated efficient gene transfer into many types of retinal cells without apparent cytotoxicity. Notably with subretinal injection, the HIV-2 vector resulted in higher efficiency of transduction of photoreceptor cells than of the other cell types (p < 0.05). With the intravitreal administration of HIV-2 viral vectors, cellular transduction and transgene expression in the ganglion cell layer was the dominant finding. CONCLUSIONS: HIV-2 viral vector may be a useful gene delivery vehicle for retinal photoreceptor cells and ganglion cells. It deserves further exploration to investigate its potential merit in long term gene therapy protocols and in other animal species.