Abstract
Purpose::
Retinal guanylate cyclase-1 (RetGC-1) is expressed primarily in cones, but also in rods, and facilitates the opening of cGMP-gated channels to allow the influx of extracellular calcium during phototransduction. Mutations in RetGC-1 cause heritable forms of retinal degeneration, including autosomal dominant cone-rod dystrophy 6 (CORD6). This disease onsets during adolescence and early adulthood, when few tissue samples are available for examination, making the histopathology of disease progression difficult to study. Our goal is to establish an in vivo model for CORD6 by generating transgenic zebrafish expressing mutant human RetGC-1 specifically in cones.
Methods::
Tol2 transposon-based transgenic constructs were generated placing the human wild type retGC-1 gene, or E837D R838S mutant variant, under the control of the native zebrafish cone transducin promoter and constructs were injected into zebrafish eggs to generate transgenic lines. Transgenic zebrafish were identified by PCR of genomic DNA, and human retGC-1 mRNA expression was confirmed using real time RT-PCR. Cone morphology was examined by immunohistochemistry on multiple sections using zpr-1 (red-green cone-specific) antibody, and cone width and length were measured at peripheral and central retinal regions. DAPI staining was used to count cell nuclei in each retinal layer. Larval zebrafish were tested for visual function using the optokinetic response (OKR) assay.
Results::
Transgenic lines of zebrafish express wild type and mutant forms of human retGC-1 mRNA respectively. Preliminary data show normal OKR responses of wild type and mutant retGC-1 transgenics at 5 days post-fertilisation. However, adult mutant retGC-1 transgenics have abnormal cone morphologies, with a reduction in the width and length of cone inner segments, appearance of holes in the photoreceptor cell layer, and a decrease in the number of ONL (photoreceptor) nuclei. These abnormalities worsen as the photoreceptors age. Wild type retGC-1 transgenics have normal cone morphologies.
Conclusions::
Transgenic expression of human mutant retGC-1 in zebrafish cone photoreceptors leads to progressive cone degeneration. This phenotype will be further examined by immunohistochemistry and electron microscopy.
Keywords: transgenics/knock-outs • retinal degenerations: hereditary • photoreceptors