GENETIC ANALYSIS OF ZEBRAFISH EMBRYO DEVELOPMENT

Human Genome Research

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Abstract

Zebrafish (Danio rerio) has emerged as ?Drosophila of the vertebrates? to study genetic controls of development and disease. It has several advantages over mice due to its small size, large progeny, and short reproduction cycle. Transparent embryos and ex vivo development allows noninvasive observations of embryonic development. Furthermore, the ability to do saturation mutagenesis using ENU (N-ethyl-N-nitrosourea) followed by forward and reverse genetic technologies to isolate mutants has a great potential in functional genomics. [unreadable] In order to identify novel genes required for embryonic hematopoiesis, particularly those affecting early stages as well as myelopoiesis, we conducted a whole mount RNA in situ hybridization screen of ENU-mutagenized F2 haploid embryos with antisense probes for cbfb and l-plastin, genes expressed in the hematopoietic stem cells and myeloid lineage cells, respectively. One mutant line, mummy, was identified from the screen that showed defects at or above the level of hematopoietic stem cells. Genetic mapping and positional cloning identified the mutated gene as dhx8, which encodes an RNA helicase. The yeast homolog of dhx8, prp22, functions during mRNA splicing. Our results demonstrate that mutations in an RNA helicase involved in splicing may show tissue specific developmental defects. In addition, we have identified two other mutant lines from the screen that have similar hematopoietic defects. Genetic mapping indicated that the mutated genes in these two mutants are not dhx8 and are not one of the genes known to be involved in hematopoiesis. Strong efforts are ongoing in the lab to clone these two mutated genes. [unreadable] Gene knockout technology has yet to be developed in the zebrafish, mainly due to the fact that embryonic stem cells have not been isolated. As an alternative, we have developed the approach of sequencing a large number of ENU-mutated fish genomes to identify mutations in the genes of interest. This approach has proven to be feasible and is amendable to high-throughput development of a large number of fish lines with a series of mutations in any gene of interest. We have generated a mutant library archived as a sperm bank and a DNA bank from ~3400 ENU-mutagenized F1 males. DNA samples are used to screen for mutations by sequencing or other heteroduplex screening methods. The corresponding sperm samples are used to revive the mutant fish by in vitro fertilization. We have screened this library for mutations in runx1 and gata1, two genes playing important roles in mammalian hematopoiesis and in human leukemia. We identified one point mutation in runx1 that results in an early truncation of the encoded protein, and two point mutations in the gata1 gene that substitutes conserved amino acid residues in the DNA binding domain of the encoded protein. We have recovered fish lines carrying these three mutations and have been analyzing their phenotypes. Preliminary data suggest that the mutations affect hematopoiesis as would be predicted from the functions of the genes, and at least one of the gata1 mutations is a hypomorphic one, i.e., it encodes a protein with reduced function. We plan to continue our analysis of these mutant lines and use them in additional mutagenesis screens to identify genetic modifiers.