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J. M. Fadool, H. P. Robinson, C. Saade, J. R. Willer, K. Alvarez-Delfin; Mutation of the Doughnut Locus Leads to Rod and Cone Dystrophy and Vestibular Defects in Zebrafish. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5205.
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© ARVO (1962-2015); The Authors (2016-present)
The zebrafish has become an increasingly important model for investigating mechanisms of human disease including those affecting the visual system. In a forward genetic screen of free swimming zebrafish larvae, we have identified several loci regulating photoreceptor cell fate specification and survival. In the present study we describe a novel mutation, doughnut (dou), leading to photoreceptor cell dystrophy and vestibular defects.
To identify genes essential for photoreceptor development, we screened 5- to 6-day postfertilization (dpf) zebrafish larvae for ethyl nitrosourea (ENU) -induced mutations leading to alterations in rod photoreceptors. Defects were detected by in situ immunolabeling using a rod specific antibody. The dou mutant phenotype was identified by disrupted immunolabeling for rod photoreceptors. The phenotype was further characterized by histology, immunolabeling with cell specific markers, and behavioral methods. Cell death was detected by TUNEL. To identify the molecular lesion, linkage analysis was performed using SSLPs.
In the developing zebrafish retina, photoreceptors first appear in the ventral retina. Differentiation then spreads to the central and dorsal retina. The spatial and temporal pattern of photoreceptor development was similar in dou mutant and wildtype embryos. This was followed by progressive photoreceptor degeneration in mutant larvae beginning in the central retina and moving towards the periphery, leaving a ring of immunolabeled cells adjacent to the retinal margin. Retinal lamination was unaffected, and labeling for ganglion cells, amacrine cells, bipolar cells and Mullar glia did not reveal any additional alterations. The dou mutant larvae failed to demonstrate an optokinetic reflex. Mutant larvae could be reared to juvenile or adult stages when housed separately from wildtype siblings. However, the mutant juveniles and adults demonstrated swimming deficits characterized by a circling behavior consistent with vestibular defects. The dou mutation was mapped to a 0.6Mb interval on Chromosome 5.
Mutation of the dou locus leads to progressive photoreceptor dystrophy and vestibular defects similar to those observed in human Usher Syndrome. These data demonstrate the utility of a genetic screen for late onset mutations affecting the retina in otherwise normal appearing larvae as models of diseases affecting the human visual system.
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