Hooked! Modeling human disease in zebrafish

Abstract

Zebrafish have been widely used as a model system for studying developmental processes, but in the last decade, they have also emerged as a valuable system for modeling human disease. The development and function of zebrafish organs are strikingly similar to those of humans, and the ease of creating mutant or transgenic fish has facilitated the generation of disease models. Here, we highlight the use of zebrafish for defining disease pathways and for discovering new therapies.

Figure 1. Drawing depicting zebrafish.

Shown are larval (A) and adult (B) zebrafish organs. Larval age is 3 to 5 days post fertilization (dpf).

Figure 2. Forward genetics approaches to generate zebrafish disease models.

Chemical mutagenesis (left). Adult males that have been mutagenized by treatment with ENU are crossed to a wild-type female to create an F1 generation that contains a random set of point mutations in their genome. In a diploid-based screen, members of the F1 are outcrossed to wild type to increase the number of fish carrying specific recessive mutations. The F2 generation is subsequently intercrossed to generate F3 progeny, which can be analyzed phenotypically for recessive defects. One-fourth of the F2 family intercrosses will produce mutant progeny in one-fourth of the F3 embryos. In a haploid screen, eggs obtained from F1 females are fertilized with UV-treated sperm to generate haploid embryos. The haploid clutch derived from a heterozygous female (+/m) will contain 50% mutant and 50% wild-type embryos. Insertional mutagenesis (right). Virus is injected into 1,000- to 2,000-cell–stage embryos. F1 fish carrying more then 3 insertions are subsequently bred. The F2 generation is screened employing the same breeding scheme used for the chemical-based mutagenesis.

Figure 3. Reverse genetic approaches to generating zebrafish disease models.

(A) Morpholinos or mRNA are injected into embryos to transiently knock down or overexpress a potential disease gene. Phenotypic analysis is performed on these embryos. Alternatively, mRNA encoding ZFN or TALEN is injected into the embryos. Putative founders are raised to adulthood and outcrossed to identify carriers. Founders carrying the allele of interest are then outcrossed to generate an F1 population. Heterozygous F1 carriers are identified by genotyping. (B) TILLING: adult males that have been mutagenized by treatment with ENU are crossed to wild-type females. A DNA library of tailfin clips or frozen sperm is prepared from F1 males. PCR amplification of exons of a specific gene of interest followed by sequencing is performed on the genomic library. Once a mutation is identified, the fish carrying the mutation will be recovered by crossing the corresponding fish from the live library to a wild-type fish or by thawing the cryopreserved sperm and using it for in vitro fertilization (IVF). The resulting progeny are then intercrossed to generate heterozygous and homozygous mutants for the disease gene.