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

Tomoya Kotani, and Koichi Kawakami

 Maternal factors, which are transmitted from mother to offspring through eggs, play important roles during early development. We aim to identify novel vertebrate maternal factors by performing a forward genetics approach in zebrafish. First, we created random insertions of the Tol2-based gene trap construct containing the gfp gene as a reporter in the zebrafish genome and isolated transgenic fish expressing GFP in one-cell stage embryos. In these lines, the insertions are thought to be integrated in genes expressed maternally and capture their transcripts. 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 hr post fertilization (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. 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 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.