Tol2Transposon-Mediated Zebrafish Mutagenesis And Nup107Mutant Study

Birth defects threaten human health.3to4out of100babies are born witha birth defect in the world. Most birth defects are a combinational result ofgenetic and environmental factors, such as chromosome abnormalities, singlegene defects, or a teratogenic exposure. To search for genes with vital functionsin early development, we use zebrafish as a model to uncover them. In this study,we performed a Tol2transposon-mediated gene trap and screen to achievezebrafish mutants, and studied the functions and molecular mechanisms of thetrapped genes in embryonic development.This study generates195transgenic zebrafish lines, most of which expressEGFP maternally and zygotically. Among them, only about40lines possessunique EGFP patterns.4mutants are isolated, all of which are zygoticallyhomozygous recessive mutants.In this study, we focused on the nup107tsu068Gtmutant line. In this lineEGFP is maternally expressed and its zygotic expression becomes prominent inthe head region and the intestine from24hours postfertilization (hpf) onward.The nup107locus is disrupted by an insertion of Tol2transposon element in thefirst intron and as a result it fails to produce normal transcripts. Homozygousnup107tsu068Gtmutant embryos exhibit tissue-specific defects after3dayspostfertilization (dpf), including loss of the pharyngeal skeletons, degenerationof the intestine, absence of the swim bladder, and smaller eyes. These mutantsdie at5-6dpf. Nup107knockdown with antisense morpholinos mimicks thephenotype of nup107tsu068Gtmutant, suggesting that Nup107deficiency isresponsible for the mutant defects. Extensive apoptosis occurs in the affectedtissues, which is partially dependent on p53apoptotic pathways. In cells of thedefective tissues, FG-repeat nucleoporins are disturbed and nuclear pore numberis reduced, leading to impaired translocation of mRNAs from the nucleus to thecytoplasm, while protein import is left unaffected. In mitotic event, spindleassembly and sister chromosomes segregation keep normal. So do cell cycle andproliferation. The nup107tsu068Gtmutant shows a much higher level of γ-H2A.X and more sensitivity to UV radiation and hydroxyurea (a DNA replicationinhibitor), indicating a role of nup107in resolving DNA replication errors andmaintaining genome stability and integrity. ATM-Chk2-p53DNA damageresponse is activated, which together with the defects of NPC assembly andfunction account for the apoptosis in these disrupted tissues. Our findings shednew light on developmental function of nup107in vertebrates.