misty somites, a maternally expressed gene identified by the transposon-mediated gene trap method in zebrafish, is required for somite boundary formation

Tomoya Kotani, and Koichi Kawakami

National Institute of Genetics, Mishima, Japan

 Maternal factors, which are transmitted from mother to offspring through eggs, play important roles during early development. Chemical mutagenesis screens to isolate maternal effect mutants have been performed. However, it is difficult to identify genes responsible for mutant phenotypes by positional cloning, especially for maternal effect mutants. We have been developing the gene trap method in zebrafish using the Tol2 transposon. The insertions of the Tol2-based gene trap vector cause GFP-expression in unique patterns during development. We hypothesized that when the insertions were integrated in genes expressed maternally and captured their transcripts, GFP should be expressed in eggs. In fact, we isolated transgenic fish expressing GFP in one-cell stage embryos. Then, we generated females homozygous for the insertions and analyzed embryos from these females for morphological defects during embryonic development. By screening 16 homozygous females, we found that embryos from one homozygous female exhibited defects in somite boundary formation at 13 hpf (the 6-somite stage). The maternal insertional mutation was named misty somites (mis) and further analyzed. In the mis mutant, the gene trap construct was integrated within an intron of a novel gene, and captured its transcript. We performed in situ hybridization using a probe for the mis gene and elucidated that the mis mRNA was indeed accumulated in one-cell stage embryos. To prohibit expression of the Mis protein completely, we injected an antisense morpholino oligonucleotide (MO) that blocks the translation initiation site of the mis gene to the mis homozygous embryos. The MO injection resulted in embryonic lethality. In the MO-injected embryos, the genes expressed during somitogenesis, such as fgf8, deltaC, her1, tbx24 and mespb, were expressed and somite boundaries were observable at 16 hpf (the 14-somite stage). At 20 hpf (the 20-somite stage), however, the boundaries were disappeared in the MO-injected embryos. Thus, we identified a novel gene by the transposon-mediated gene trap method, which encodes a maternal factor required for the maintenance of the somite boundaries during vertebrate development.