Gucy2f zebrafish knockdown – a model for Gucy2d-related leber congenital amaurosis

Abstract

Mutations in retinal-specific guanylate cyclase (Gucy2d) are associated with Leber congenital amaurosis-1 (LCA1). Zebrafish offer unique advantages relative to rodents, including their excellent color vision, precocious retinal development, robust visual testing strategies, low cost, relatively easy transgenesis and shortened experimental times. In this study we will demonstrate the feasibility of using gene-targeting in the zebrafish as a model for the photoreceptor-specific GUCY2D-related LCA1, by reporting the visual phenotype and retinal histology resulting from Gucy2f knockdown. Gucy2f zebrafish LCA-orthologous cDNA was identified and isolated by PCR amplification. Its expression pattern was determined by whole-mount in-situ hybridization and its function was studied by gene knockdown using two different morpholino-modified oligos (MO), one that blocks translation of Gucy2f and one that blocks splicing of Gucy2f. Visual function was assessed with an optomotor assay on 6-days-postfertilization larvae, and by analyzing changes in retinal histology. Gucy2f knockdown resulted in significantly lower vision as measured by the optomotor response compared with uninjected and control MO-injected zebrafish larvae. Histological changes in the Gucy2f-knockdown larvae included loss and shortening of cone and rod outer segments. 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: leber congenital amaurosis; animal model; GUCY2D; optomotor assay INTRODUCTION Leber congenital amaurosis (LCA) is a family of severe, early-onset, autosomal-recessive forms of pediatric blindness. LCA has been linked to more than 16 genes, often exhibiting clinical heterogeneity. 1,2 LCA1 is caused by mutations in the gene GUCY2D, which encodes the retinal Gucy2d-1 (GC1) and accounts for B20% of all cases of LCA. 3 LCA2 is caused by mutations in the RPE65 gene, which is expressed in the retinal pigment epithelium. Breakthrough research has demonstrated visual improvement following gene therapy trials in humans with LCA2. 4,5 Human trials relied on a decade of proof-of-concept studies in canine and rodent models. 6-8 Unlike LCA2, in which gene therapy is aimed at correcting an RPE defect, searching for gene therapy for LCA1 will involve an attempt at treating the photoreceptors. European Journal of Human GeneticsResearch in inherited retinal disease relies heavily on animal models, commonly mammals such as mice and dogs. These animal models have significant advantages, including the availability of knockout technology. Two specific drawbacks slow the applicability of rodent or canine models for study of relatively rare genetic diseases such as LCA and other retinal dystrophies. The first difficulty is the high cost and length of time required to create a new knockout line; thus, it is applicable only for comm...

The zebrafish eye—a paradigm for investigating human ocular genetics

“…Knockdown of the zebrafish orthologue gucy2f results in early visual dysfunction with visible outer segment and photoreceptor layer loss. 72 Morpholino-induced knockdown of centrosomal protein 290 kDa (cep290) resulted in delayed intracellular transport and reduced visual acuity despite a fully laminated retina, consistent with human LCA patients. 73 Importantly, injection of cep290 mutants with an N-terminal CEP290 construct rescued visual function, supporting a potential treatment The wild-type retina shows characteristic stratification in three nuclear and two plexiform layers.…”

Functional architecture of the retina: Development and disease

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Human Induced Pluripotent Stem Cells As a Tool to Model a Form of Leber Congenital Amaurosis

“…Animal models are available for modeling some mutations, including chicken (Perrault et al, 2000), mouse (Batten et al, 2004;Dyer et al, 2004;Ramamurthy, 2004;Redmond et al, 1998;Rolling, 2004;Yang et al, 1999), dog (Aguirre et al, 1998), and zebrafish (Stiebel-Kalish et al, 2012), but there is clearly a need for in vitro models that would allow exploration of pathological mechanisms in vitro and could be used in a search for treatment. Induced pluripotent stem cells (iPSCs) produced by genetic reprogramming of somatic cells from human donors could be of interest, because of a clinical phenotype or an identified disease-related mutation (Takahashi et al, 2007), and may be a unique tool to meet those challenges.…”