The transposon-mediated gene trap approach identified novel genes that expressed maternally and involved in embryogenesis

Tomoya Kotani, Saori Nagayoshi, and Koichi Kawakami

National Institute of Genetics, Mishima, Japan

　Genes expressed maternally are thought to play important roles in early development. Some maternal mutants have been identified by ENU-based chemical mutagenesis. However, it is difficult to identify the genes responsible for the phenotypes of maternal mutants by positional cloning, compared with those of zygotic mutants. We have been developing a gene trap method in zebrafish using the Tol2 transposon system. The insertions of a transposon-based gene trap vector containing a splice acceptor and the promoter-less GFP gene caused the GFP-expression in temporally and spatially restricted patterns during development. We found that a third of the GFP-expressions was also caused in the fertilized eggs. In order to identify novel maternal genes and to reveal their functions during zebrafish development, we analyzed the fish lines expressing GFP in the fertilized eggs.
　The gene trap fish line, SAG20, expressed GFP in the fertilized eggs. Zygotic expression of GFP was first observed in notochord at 12 hpf. The trapped transcript was identified by 5’RACE, and the full-length cDNA was cloned by 3’RACE. The gene was tentatively named sag20. In situ hybridization revealed that the sag20 gene was indeed expressed maternally and in notochord. The sag20 gene product is highly conserved among vertebrates and has a coiled coil domain in the C-terminal region. Transcription of the sag20 gene was undetectable by RT-PCR in homozygous fish. The maternal homozygous embryos showed defective phenotypes in mesenchymal-to-epithelial transformation and in myoD expression during gastrulation and somitogenesis. Also, we have identified six genes, five of which are novel, trapped in the fish lines expressing GFP maternally. These results show that our gene trap method is useful for identifying novel maternal genes and analyzing their functions during zebrafish development.