Abstract
Abstract: :
Purpose: The zebrafish represents an excellent model organism for developmental biology and is rapidly expanding to adult phenotypes with resemblance to human disease. We seek to use zebrafish to study gene interactions important for normal development and maintenance of ocular function and, in particular, as it applies to glaucoma phenotypes. Along this path, we identified and studied zebrafish homologs of several genes known to play an important role in mammalian eye development/ function. Here we present studies of a zebrafish homolog of the human optineurin (OPTN) gene that was shown to be involved in glaucoma. Methods: Analysis of public nucleotide databases was performed using BLASTN and BLASTX engines. Multiple sequence alignments were carried out using BCM Search Launcher suite. RNA extractions, RT–PCR, in situ hybridization, cloning and sequencing were performed using standard techniques. Results:Examination of public databases identified several zebrafish genomic sequences with homology to human optineurin. Oligonucleotides were designed based on sequences corresponding to the most 5' and 3' ends of the gene and used in RT–PCR reactions to amplify mRNA. The resultant transcript was ∼1,800 nt in length, containing the complete coding region sequence. The predicted open reading frame encodes a 522– amino–acid protein with overall 46% identity to the human and 44% to the mouse OPTN proteins; identity between the mouse and human proteins is 77%. The genomic structure of zebrafish optn was found to be similar to mammalian genes with the coding region divided into thirteen exons. Comparative analysis of OPTN proteins showed conservation of E50 but not M98 or R545 amino acids across species. Based on literature analysis, E50K mutation is strongly associated with familial normal–tension glaucoma. The other mutations appear to likely represent population–specific variants. Expression analysis of the zebrafish optn gene showed a dynamic pattern during embryonic development with transcripts first observed in 3–8 hpf embryos and the most strong expression in 72 hpf embryos. Zebrafish optineurin mRNA was also detected in adult tissues, with most abundant expression in the eye. Conclusions: We identified a new zebrafish gene with strong homology to human optineurin, which is associated with glaucoma. Expression of zebrafish optn in the adult eye suggests this gene is important for normal ocular function, which can now be investigated using a multitude of techniques offered by this powerful vertebrate.
Keywords: neuroprotection • gene/expression • intraocular pressure