Gucy2f Zebrafish Knockdown - A Model for GUCY2D-related Leber Congenital Amaurosis

Abstract

Purpose: : In this study we will demonstrate the feasibility of using gene-targeting in the zebrafish as a model for the photoreceptor-specific guanylate cyclase (GUCY2D)-related LCA1, by reporting the visual phenotype and retinal histology resulting from Gucy2f-knockdown.

Methods: : Gucy2f zebrafish LCA-orthologous cDNA was identified and isolated by PCR amplification. The expression pattern of Gucy2f was determined by whole-mount in-situ hybridization and its function was studied by gene knockdown utilizing two different morpholino-modified oligo (MO), one that blocks translation of Gucy2f and one that blocks splicing of Gucy2f. Visual function was assessed with an optomotor assay (moving grating of 0.16 cycles/degree) on 6 days post-fertilization larvae, and by analyzing changes in retinal histology.

Results: : Gucy2f knockdown resulted in significantly lower vision as measured by the optomotor response compared to uninjected zebrafish larvae and morpholino (MO)-controls. Histological changes in the Gucy2f-knockdown larvae included loss and shortening of cone and rod outer segments, retinal pigment epithelium thickening and loss of rod nuclei.

Conclusions: : A zebrafish model of Gucy2f-related LCA1 displays early visual dysfunction and photoreceptor layer dystrophy. This study serves as proof of concept for the use of zebrafish as a simple, inexpensive model with excellent vision on which further study of LCA-related genes is possible.

Keywords: gene/expression • genetics • mutations