Lazy eyes zebrafish mutation affects Müller glial cells, compromising photoreceptor function and causing partial blindness.

A behavioral assay based on the optokinetic reflex was used to screen chemically mutagenized zebrafish larvae for deficits in visual function. A homozygous recessive mutation, lazy eyes (lze), was isolated based on the observation that 5-day postfertilization (dpf) mutants displayed weaker and less frequent eye movements than wild-type fish in response to moving stripes. Electroretinographic (ERG) recordings revealed that mutants had severely reduced a- and b-wave amplitudes relative to wild-type fish, indicating outer retinal dysfunction. Retinal lamination and cellular differentiation were normal in the lze retina; however, mutant photoreceptor cells had small outer segments and pyknotic nuclei were occasionally observed in the outer retina and the marginal zone of lze. Cone, rod, amacrine, bipolar, and Müller cell marker analyses indicated that the typical lze retina contained fewer rod photoreceptors and fewer Müller cells than wild-type fish at 5 dpf. At 3 dpf, however, mutant retinas had normal numbers of rod photoreceptors and Müller cells, suggesting that the initial differentiation of these cell types occurred normally. Rod photoreceptor histology was normal at this early stage, but Müller cells were often hypertrophied, suggesting that they were unhealthy. Constant light rearing of mutant animals accelerated the Müller cell degeneration, severely worsened the visual deficit, but had no obvious affect on the photoreceptors. When ERG responses and Müller cell degeneration from the same mutant animals were analyzed, the extent of the Müller cell loss matched closely the degree to which ERG responses were reduced. In summary, the lze gene appears to be required for Müller cell viability and normal visual function. The lze mutant may be a model for the study of the involvement of Müller cells in photoreceptor development and function.

Transgenic expression of a GFP-rhodopsin COOH-terminal fusion protein in zebrafish rod photoreceptors.

To facilitate the identification and characterization of mutations affecting the retina and photoreceptors in the zebrafish, a transgene expressing green fluorescent protein (GFP) fused to the C-terminal 44 amino acids of Xenopus rhodopsin (Tam et al., 2000) under the control of the 1.3-kb proximal Xenopus opsin promoter was inserted into the zebrafish genome. GFP expression was easily observed in a ventral patch of retinal cells at 4 days postfertilization (dpf). Between 45-50% of the progeny from the F1, F2, and F3 generations expressed the transgene, consistent with a single integration event following microinjection. Immunohistochemical analysis demonstrated that GFP is expressed exclusively in rod photoreceptors and not in the UV, blue, or red/green double cones. Furthermore, GFP is localized to the rod outer segments with little to no fluorescence in the rod inner segments, rod cell bodies, or rod synapse regions, indicating proper targeting and transport of the GFP fusion protein. Application of exogenous retinoic acid (RA) increased the number of GFP-expressing cells throughout the retina, and possibly the level of expressed rhodopsin. When bred to a zebrafish rod degeneration mutant, fewer GFP-expressing rods were seen in living mutants as compared to wild-type siblings. This transgenic line will facilitate the search for recessive and dominant mutations affecting rod photoreceptor development and survival as well as proper rhodopsin expression, targeting, and transport.

Transgenic expression of a GFP-rhodopsin COOH-terminal fusion protein in zebrafish rod photoreceptors.

To facilitate the identification and characterization of mutations affecting the retina and photoreceptors in the zebrafish, a transgene expressing green fluorescent protein (GFP) fused to the C-terminal 44 amino acids of Xenopus rhodopsin (Tam et al., 2000) under the control of the 1.3-kb proximal Xenopus opsin promoter was inserted into the zebrafish genome. GFP expression was easily observed in a ventral patch of retinal cells at 4 days postfertilization (dpf). Between 45-50% of the progeny from the F1, F2, and F3 generations expressed the transgene, consistent with a single integration event following microinjection. Immunohistochemical analysis demonstrated that GFP is expressed exclusively in rod photoreceptors and not in the UV, blue, or red/green double cones. Furthermore, GFP is localized to the rod outer segments with little to no fluorescence in the rod inner segments, rod cell bodies, or rod synapse regions, indicating proper targeting and transport of the GFP fusion protein. Application of exogenous retinoic acid (RA) increased the number of GFP-expressing cells throughout the retina, and possibly the level of expressed rhodopsin. When bred to a zebrafish rod degeneration mutant, fewer GFP-expressing rods were seen in living mutants as compared to wild-type siblings. This transgenic line will facilitate the search for recessive and dominant mutations affecting rod photoreceptor development and survival as well as proper rhodopsin expression, targeting, and transport.