Age-Related Cone Abnormalities in Zebrafish with Genetic Lesions in Sonic Hedgehog

PURPOSE. Sonic hedgehog (Shh) signaling is essential for photoreceptor differentiation and retinal cell survival in embryonic zebrafish. The study was conducted to determine whether adult heterozygous carriers of mutant alleles for the shh gene display retinal abnormalities. METHODS. Retinal cryosections from young, middle-aged, and senescent wild-type and sonic-you ϩ/Ϫ (syu ϩ/Ϫ ) zebrafish were probed with retinal cell type-specific markers. Contralateral retinal flatmounts from these fish, and from adult albino zebrafish subjected to light-induced photoreceptor damage followed by regeneration, were hybridized with blue cone opsin cRNA for quantitative analysis of the blue cone pattern. Retinal expression of shh mRNA was measured by quantitative RT-PCR. RESULTS. Regions of cone loss and abnormal cone morphology were observed in the oldest syu ϩ/Ϫ zebrafish, although no other retinal cell type was affected. This phenotype was agerelated and genotype-specific. Cone distribution in the oldest syu ϩ/Ϫ zebrafish was predominantly random, as assessed by measuring the short-range pattern, whereas that of wild-type fish and the younger syu ϩ/Ϫ zebrafish was statistically regular. A measure of long-range pattern revealed atypical cone aggregation in the oldest syu ϩ/Ϫ zebrafish. The light-treated albino zebrafish displayed random cone patterns immediately after light toxicity, but showed cone aggregation on regeneration. Retinas from the syu ϩ/Ϫ fish showed reduced expression of shh mRNA compared with those of wild-type siblings. 9 In addition, Shh stimulates proliferation of rat Müller glia and the generation of rhodopsin-expressing cells in vitro, and in vivo subsequent to retinal damage. CONCLUSIONS. The The use of anamniote vertebrate models has uncovered important roles for Hedgehog signaling in the development of rod and cone photoreceptors and the RPE. In zebrafish embryos, reduction in Shh signaling through molecular, pharmacologic, or genetic techniques results in multiple retinal abnormalities including reduced rod and cone photoreceptor differentiation. Many inherited retinal degenerative diseases, including those related to age, are characterized by rod and/or cone photoreceptor death. 18 As a complement to the mouse models, zebrafish offer several advantages for studying retinal disorders. For example, zebrafish have a duplex retina with a substantial number of cone photoreceptors arranged in a precise mosaic that can be quantitatively assessed. 23 Zebrafish are also a model for aging and age-related disease