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Zebrafish Genetic Screens
Two of the advantages of using zebrafish to study vertebrate development are that mutations are relatively easy to obtain and to screen. Finding mutations in birds (the chick), frogs (Xenopus), and mammals (mice) is very difficult, and breeding them takes time. In zebrafish, mutants can be easily identified and their parents can still be bred to keep the mutation in the heterozygotes. By mating these heterozygotes, the mutant phenotype will continue to be expressed and therefore available to be studied.
Two types of large-scale genetic screens can be performed in zebrafish. The more traditional screening (modeled after large scale screens in Drosophila) begins when the male parental fish are treated with a chemical mutagen that will cause mutations randomly in their germ cells. Each mutagenized male is then mated to a wild-type female fish to generate F1 lines. The individual fish in the F1 generation carry the mutations inherited from his or her father. If the mutation is dominant, it will become expressed in the F1 generation. If these mutations are recessive, the F1 fish will not show a mutant phenotype, since the wild-type allele will mask the mutation. The sibling F1 fish are mated to each other to produce the F2 generation. In this generation, some males and some females will have the mutated allele.
The F2 fish in each family are then mated to each other. When F2 parents contain the same mutation as a recessive, there will be a 25% chance that their offspring will show the mutant phenotype (Figure 1). Since the development occurs in the open (not in an opaque shell or within the mother), abnormal developmental stages can be readily observed (Driever et al., 1996; Haffter et al., 1996).
A second way of screening zebrafish for developmental mutations involves a particular trick one can do on fish eggs. As in the traditional screening, males are mutagenized and mated to wild-type females. However, instead of crossing siblings to produce the F2 generation, the eggs from the F1 females are gently squeezed from the females and are mixed with sperm that has been treated with ultraviolet irradiation to destroy their DNA. The irradiated sperm fertilize the eggs and activate their development. However, these sperm do not give any genetic material to the eggs. Rather, the eggs develop under the guidance of the female genome only. (Such an embryo is called a gynogenetic haploid.) If the F1 female carried a mutant allele, it would be seen in 50% of these embryos. The haploid embryos only live about three days, so this technique can be used only to screen for mutations of early development (see Cheng and Moore, 1997). Both these screening strategies have provided hundreds of developmental mutations in zebrafish—more than the number of graduate students and post docs available to analyze them!
Cheng, K. C. and Moore, J. L. 1997. Genetic dissection of vertebrate processes in the zebrafish: a comparison of uniparental and two-generation screens. Biochem Cell Biol. 75: 525-533.
Driever, W., and eleven others. A genetic screen for mutations affecting development in zebrafish. Development 123: 37-46.
Haffter, P., and sixteen others. 1996. The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio. Development 123: 1-36.
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