Zebrafish With the brass Mutation Show Glaucoma-Associated Phenotypes

Abstract

Purpose:: Zebrafish brass mutants are hypopigmented, have raised intraocular pressure (IOP) and exhibit iris hypoplasia. These fish are thus candidates to model glaucoma-related phenotypes. In the current study we have sought the causative mutation for brass and investigated the retinal phenotypes in aged animals.

Methods:: Servo-null electrophysiology was used to measure the IOP of wild-type and brass fish in Oregon AB (AB) and Long Fin (LF) strains. To characterize the brass phenotype eye size to body length measures, histology and retinal cell counts, transmission electron microscopy, quantitative RT-PCR, recombinant linkage mapping, DNA sequencing and morpholino knockdown techniques were used.

Results:: In both AB and LF strains brass fish exhibit elevated IOP. The ratio of eye size to body length in aged (2 year) brass fish in the LF strain was 1.4x greater than wild-type siblings. Cell counts of central retinal sections (n=13 animals) revealed a significant increase in cell density in aged brass fish as compared to wild-type siblings. Interestingly, the proportion of retinal ganglion cells was unaltered. Linkage analysis and fine mapping localized the brass mutation to chromosome 13 and identified lyst (lysosomal trafficking regulator) as a strong candidate for underlying brass phenotypes. When mutated in humans and mice, one of the characteristic phenotypes observed is enlarged melanosomes. Ultrastructural analyses of brass eyes revealed similar phenotypes in which melanosomes were irregularly shaped, unusually large, and vacuolated. Quantitative RT-PCR showed changes in lyst transcript levels between brass and wild-type fish. DNA sequencing of the lyst coding sequence in brass fish did not identify an obvious causative mutation. However, analysis of a splice disrupting lyst morpholino showed that partial knockdown of the normal lyst transcript was sufficient to produce a fully penetrant embryonic phenotype, suggesting that normal regulation of lyst expression may be disrupted in brass fish.

Conclusions:: Zebrafish with the brass mutation show glaucoma-associated phenotypes including iris hypoplasia, raised IOP, and age-related buphthalmia. However, brass mutants do not show retinal ganglion cell loss. Genetically, the brass mutation maps to the lyst gene and brass fish display characteristics similar to lyst mutants in other species. Cumulatively, these data suggest lyst mutations may render individuals susceptible to glaucoma-related phenotypes.

Keywords: aging • retina • linkage analysis