Tol2 Transposon-Mediated Mutagenesis And Characterization Of A Mutant Stac In Zebrafish

Embryogenesis is a complicated process involving in precise control of the expression of many genes. Generation and molecular characterization of mutants defective in specific developmental processes is an effective approach to reveal developmental mechanisms. As an excellent model organism, zebrafish has been extensively used for generation of developmental mutants. In this study, Tol2 transposon-mediated gene-trap approach was adopted to generate developmental mutants in zebrafish, and functions of interrupted genes in the mutants were further investigated.This study generated 66 transgenic fish lines that expressed the green fluorescent protein (GFP). In less than one third of the lines, GFP was expressed in specific tissues or at specific stages during embryonic development. Intercrosses between F1 siblings identified 6 lines that produced mutant phenotypes in the offsprings. One of these mutant lines, stagnant and curly (stac), which was derived from BGS055 line, was further characterized.The GFP expression in embryos of BGS055 line occurred ubiquitously at early stages, and since then it maily happened in the brain and the spinal cord. Mutant embryos exhibited discernible cell death as early as 20 hpf, and extensive cell death in the head and spinal cord and upward curly tail at later stages. The stac mutants were deformed at 4-5 days postfertilization. In stac mutants, the Tol2 transposon element was inserted into the second exon of the cenph locus and resulted in loss of its product. The cenph-encoded protein (Cenph) is a centromere protein and plays essential roles in chromosome segregation during mitosis. The stac mutant phenotype was rescued by cenph mRNA overexpression, and mimicked by cenph knockdown with antisense morpholinos, suggesting the responsibility of cenph for stac mutants. Analysis of stac embryos by immunostaining showed that many embryonic cells contain hypercondensed chromatins and apparent DNA fragmentation, indicative of extensive apoptosis. Further study demonstrated that the intrinsic apoptosis pathway was hyperactivated in stac mutants and that p53 knockdown temporarily blocked excess apoptosis in stac mutants. Immunofluorescence and flow cytometry revealed that mitotic cells in stac mutants showed chromosome missegregation, spindles misalignment and cell cycle arrested in G2/M phase. Additionally, the carcinogenesis analysis with carcinogen exposure suggested that heterozygous stac fish develop invasive tumors at a dramatically reduced level compared to wildtype siblings.Taken together, this project demonstrates that Tol2 transposon-mediated gene trapping is an effective way to investigate functions of developmental genes. And this study also reveals an essential role of cenph in mitosis and embryonic development and its association with tumor development.