Seeing through the eyes of zebrafish mutants and morphants: functional approaches to vision

Abstract

In the presented thesis the zebrafish visual system has been approached from two different angles using forward and reverse genetics. In a classic forward genetic screen functional tools were employed to isolate novel mutants with inner retina defects. We designed a screening strategy combining morphological assessment, behavioral testing and electrophysiological recordings to identify larval mutants with inner retina malfunction and adult mutants with eye defects. Our efforts resulted in the identification of 16 larval as well as 8 adult mutant families of which three selected mutants have been characterized in detail. The NH028 mutant is unable to perceive motion, but has an intact and light-responsive retina. leto larvae are completely blind despite a normally layered retina. In asteria mutants, a degeneration of inner organs accompanies a progressive loss of vision during larval stages. The zebrafish leberknodel (lbk) mutant had been identified in a previous screen. Severe impairments in visual function, abnormal RPE (retinal pigment epithelium) morphology and additional phenotypes in the liver, intestine and innate immune system have been shown to result from a general vesicle defect. We found that lbk larvae are mutated in the vam6/vps39 gene, a component of a protein complex essential for vesicle tethering and fusion. We propose that vam6 is a novel candidate gene for vesicle-associated pathologies in human patients. CRALBP (cellular retinaldehyde binding protein) is a chaperone for retinoids in the visual cycle, the biochemical pathway for chromophore regeneration. We identified two CRALBP orthologs in the zebrafish with differential expression in the retina. Cralbp a is expressed in the RPE (retinal pigment epithelium) and Cralbp b in Muller glia cells. The canonical visual cycle is located in the RPE, while Muller glia cells have been suggested to be the compartment for the cone-specific pathway. The observed subfunctionalization event allowed us to separately assess the impact of loss of either one or both proteins on 11-cis-retinal recycling and visual system performance. We have shown that cone photoreceptors use not only the canonical pathway, but cone vision is also affected by blocking Cralbp in Muller cells. Hence, cones use both the canonical as well as an additional cone-specific visual cycle located to Muller glia cells to regenerate their pigment. Both pathways involve the Cralbp 5 protein. Therefore we have identified the first molecular component of the proposed cone visual cycle. Finally, we conducted an expression study of the zebrafish orthologs of RGS9 (regulator of G protein signalling 9) and its binding protein R9AP. We detected RGS9a and R9APa in photoreceptors where they likely function in the termination of phototransduction, analogous to what has been found in mammals. In contrast to that, RGS9b and R9APb are not expressed in the eye.