A colour preference technique to evaluate acrylamide-induced toxicity in zebrafish.

The zebrafish has become a commonly used vertebrate model for toxicity assessment, of particular relevance to the study of toxic effects on the visual system because of the structural similarities shared by zebrafish and human retinae. In this article we present a colour preference-based technique that, by assessing the functionality of photoreceptors, can be used to evaluate the effects of toxicity on behaviour. A digital camera was used to record the locomotor behaviour of individual zebrafish swimming in a water tank consisting of two compartments separated by an opaque perforated wall through which the fish could pass. The colour of the lighting in each compartment could be altered independently (producing distinct but connected environments of white, red or blue) to allow association of the zebrafish's swimming behaviour with its colour preference. The functionality of the photoreceptors was evaluated based on the ability of the zebrafish to sense the different colours and to swim between the compartments. The zebrafish tracking was carried out using our algorithm developed with MATLAB. We found that zebrafish preferred blue illumination to white, and white illumination to red. Acute treatment with acrylamide (2mM for 36h) resulted in a marked reduction in locomotion and a concomitant loss of colour-preferential swimming behaviour. Histopathological examination of acrylamide-treated zebrafish eyes showed that acrylamide exposure had caused retinal damage. The colour preference tracking technique has applications in the assessment of neurodegenerative disorders, as a method for preclinical appraisal of drug efficacy and for behavioural evaluation of toxicity.

Evolutionary Characterization of the Retinitis Pigmentosa GTPase Regulator Gene.

PURPOSE: The evolutionary conservation of the retinitis pigmentosa GTPase regulator (RPGR) gene was examined across vertebrate and invertebrate lineages to elucidate its function. METHODS: Orthologous RPGR sequences from vertebrates and invertebrates were selected. Multiple sequence alignments, phylogenetic analyses, synteny, and gene structure comparisons were carried out. Expression of the alternatively spliced constitutive (RPGR(const) or RPGR(ex1-19)) and RPGR(ORF15) isoforms was examined in developing and adult zebrafish. RESULTS: Phylogenetic analyses and syntenic relationships were consistent with the selected sequences being true orthologues, although whole genome duplications in teleost fish resulted in a more complex picture. The splice form RPGR(const) was present in all vertebrate and invertebrate species but the defining carboxyl (C)-terminal exon of RPGR(ORF15) was absent from all invertebrates. The regulator of chromosome condensation (RCC1)-like domain adopts a seven-bladed ß-propeller structure, which was present in both major splice forms and strongly conserved across evolution. The repetitive acidic region of RPGR(ORF15) showed a high rate of in-frame deletions/insertions across nine primate species, compared with flanking sequences, consistent with an unstable and rapidly evolving region. In zebrafish, RPGR(const) transcripts were most strongly expressed in early development, while the RPGR(ORF15) isoform showed highest expression in adult eye. CONCLUSIONS: The regulator of chromosome condensation 1-like domain of RPGR was conserved in vertebrates and invertebrates, but RPGR(ORF15) was unique to vertebrates, consistent with a proposed role in the ciliary-based transport of cargoes such as rhodopsin, which is ∼10 times more abundant in vertebrate than invertebrate photoreceptors. The repetitive acidic region of RPGR(ORF15) shows a rapid rate of evolution, consistent with a mutation "hot spot."

The Role of RPGR and Its Interacting Proteins in Ciliopathies.

Ciliopathies encompass a group of genetic disorders characterized by defects in the formation, maintenance, or function of cilia. Retinitis pigmentosa (RP) is frequently one of the clinical features presented in diverse ciliopathies. RP is a heterogeneous group of inherited retinal disorders, characterized by the death of photoreceptors and affecting more than one million individuals worldwide. The retinitis pigmentosa GTPase regulator (RPGR) gene is mutated in up to 20% of all RP patients. RPGR protein has different interacting partners to function in ciliary protein trafficking. In this review, we specifically focus on RPGR and its two interacting proteins: RPGRIP1 and RPGRIP1L. We summarize the function of the three proteins and highlight recent studies that provide insight into the cellular function of those proteins.

Knockout of RP2 decreases GRK1 and rod transducin subunits and leads to photoreceptor degeneration in zebrafish.

Retinitis pigmentosa (RP) affects about 1.8 million individuals worldwide. X-linked retinitis pigmentosa (XLRP) is one of the most severe forms of RP. Nearly 85% of XLRP cases are caused by mutations in the X-linked retinitis pigmentosa 2 (RP2) and RPGR. RP2 has been considered to be a GTPase activator protein for ARL3 and to play a role in the traffic of ciliary proteins. The mechanism of how RP2 mutations cause RP is still unclear. In this study, we generated an RP2 knockout zebrafish line using transcription activator-like effector nuclease technology. Progressive retinal degeneration could be observed in the mutant zebrafish. The degeneration of rods' outer segments (OSs) is predominant, followed by the degeneration of cones' OS. These phenotypes are similar to the characteristics of RP2 patients, and also partly consistent with the phenotypes of RP2 knockout mice and morpholino-mediated RP2 knockdown zebrafish. For the first time, we found RP2 deletion leads to decreased protein levels and abnormal retinal localizations of GRK1 and rod transducin subunits (GNAT1 and GNB1) in zebrafish. Furthermore, the distribution of the total farnesylated proteins in zebrafish retina is also affected by RP2 ablation. These molecular alterations observed in the RP2 knockout zebrafish might probably be responsible for the gradual loss of the photoreceptors' OSs. Our work identified the progression of retinal degeneration in RP2 knockout zebrafish, provided a foundation for revealing the pathogenesis of RP caused by RP2 mutations, and would help to develop potential therapeutics against RP in further studies.

Pathogenesis of X-linked RP3: insights from animal models.

Retinitis Pigmentosa (RP) is a genetically heterogeneous disorder characterized by rod and cone photoreceptor cell dysfunction. X-linked RP (XLRP) is one of the most severe forms of human retinal degeneration, as determined by age-of-set and progression, and accounts for six to 20 % of all RP cases. At least six XLRP loci have been identified, but RP3 is the major subtype of XLRP, accounting for 70 to 80 % of affected families. The RPGR gene is responsible for the RP3 form of XLRP and is mutated in 10-20 % of all RP patients. The pathogenesis of retinitis pigmentosa GTPase regulator (RPGR) mutant-causing RP is not clear, different animal models have been used to understand the pathogenesis of these diseases. In this brief review, we will summarize the functional characterization of RPGR and highlight recent studies in animal models, which will not only shed light on the disease mechanisms in XLRP but will also provide therapeutic strategies for RP treatment.

Zebrafish model for the genetic basis of X-linked retinitis pigmentosa.

Retinitis pigmentosa (RP) affects 1/4000 individuals in most populations, and X-linked RP (XLRP) is one of the most severe forms of human retinal degeneration. Mutations in both the retinitis pigmentosa GTPase regulator (RPGR) gene and retinitis pigmentosa 2 (RP2) gene account for almost all cases of XLRP. The functional roles of both RPGR and RP2 in the pathogenesis of XLRP are unclear. Due to the surprisingly high degree of functional conservation between human genes and their zebrafish orthologues, the zebrafish has become an important model for human retinal disorders. In this brief review, we summarize the functional characterization of XLRP-causing genes, RPGR and RP2, in zebrafish, and highlight recent studies that provide insight into the cellular functions of both genes. This will not only shed light on disease mechanisms in XLRP but will also provide a solid platform to test RP-causing mutants before proposing XLRP gene therapy trials.