Tol2 Transposon-Mediated Gene Trap And The Functional Analysis Of Foxj1 Gene In Zebrafish

The early embryonic development of zebrafish is a complicated biological process, which involves many key genes and regulating signal pathways.And the mutant screening in large scale in zebrafish by traditional chemical mutagenesis (ENU) and retrovirus insertional mutagenesis have already generated many mutants in zebrafish and implied many functions of the related genes.In our study, we utilized Tol2 transposon mediated-gene trap method to generate some transgenic fish families in which the GFP expression was specific in some organs or tissues.We also screened for mutant phenotypes in those offspring embryos, and analyzed possible functions of the mutated genes.45 transgenic fish lines have been generated, and the embryos in some families expressed GFP specifically.However, we did not find any mutants in those offspring embryos.The GFP expression in TZBGS010 transgenic fish family is specific in olfactory pits, otic vesicle, floor plate and the pronephic ducts at 24 hpf. TAIL-PCR analysis showed that the gene trapped by Tol2 transposon in this transgenic family was foxj1b gene.The HNF-3/HFH-4/Foxj1, a transcription factor, has been reported to be involved in systemic autoimmunity and cilia genesis in vertebrates. The zebrafish genome expressed two paralogous foxj1 genes, foxj1a and foxj1b. In this study, we demonstrate that down-regulation of either foxj1a or foxj1b by injecting antisense morpholino at the one-cell stage results in randomized expression of the early left-right (LR) asymmetric markers lefty2, southpaw,pitx2c and the later internal organ markers tpm4-tv1, cmlc2, cp in zebrafish embryos. Overexpression of foxj1a and foxj1b by injecting synthetic mRNAs in vitro also disrupts normal LR asymmetries in zebrafish embryos.These data indicate that the two foxj1 genes are required for normal laterality development in zebrafish embryos. In contrast to foxj1b knockdown exclusively in dorsal forerunner cells (DFCs) that has little effect on laterality, foxj1a knockdown in DFCs randomizes the LR patterns of the markers. Thus, foxj1a regulates asymmetric development through DFCs in a cell-autonomous fashion but foxj1b functions in a non cell-autonomous way.