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C. Nicholas, B.D. Perkins, B.A. Link, J.E. Dowling; Mapping and Characterization of Dazed, a Mutation Affecting Rod Differentiation and Survival in Zebrafish . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3069.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To better understand the mechanisms controlling vertebrate retinal development, we have used a forward genetic screen in zebrafish to identify recessive mutants with abnormal eye morphology. One mutant, dazed, was isolated which exhibited small eyes and retinal degeneration during development. We have genetically mapped the mutation and characterized the phenotype with respect to retinal development. Methods: The genomic position of the dazed locus was mapped using recombinant linkage analysis. The onset and progression of retinal cell death was measured by acridine orange staining. Histological analysis was performed at various timepoints to study retinal lamination and retinal anatomy. The existence and localization of retinal cell types was verified by immunohistochemistry with cell type specific markers. In situ hybridizations were done to study the expression patterns of retinogenesis genes such as rx2, pax6, six6, and sonic hedgehog. A behavioral assay for the escape response in adult fish was used to test for night blindness in heterozygous fish. Results: Linkage analysis has shown that dazed maps to linkage group 3 and is close to marker z30645. The retinas of dazed embryos had approximately 30% fewer cells than wild type. Histological analysis indicated that the retina was laminated but disorganized by 3 dpf, and cell death was noted by 2 dpf. This was confirmed by acridine orange studies, which showed cell death beginning at 36 hours post fertilization (hpf) in the retina and the forebrain. Antibody studies showed that rods failed to differentiate by 56 hpf and few rods were present by 72 hpf, indicating a failure of rod differentiation rather than rod degeneration. A 2-fold increase in the percentage of cone photoreceptors was seen in mutant retinas, but approximately 2-fold decreases in the percentages of amacrine and ganglion cells relative to wild type. There was no detectable difference between wild type and dazed embryos in the expression of various retinogenesis genes. Almost 40% of adult dazed heterozygous fish were night blind when screened using a behavioral assay, indicating rod dysfunction. Conclusions: We identified a mutation that disrupts the normal development of the zebrafish retina. The dazed mutation resulted in a significant decrease in the number of rods, and may represent a mutation that affects rod differentiation and survival. This mutation could provide important clues as to the cell fate choices of multi-potent retinal progenitor cells and the signals required to maintain the differentiated retina.
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