【作者】 田甜；

【导师】 孟安明；

【作者基本信息】 清华大学 ， 生物学， 2009， 博士

【摘要】 斑马鱼胚胎的早期发育过程是一个复杂的生物学过程,有多条信号通路及通路中的众多关键基因参与并调控着胚胎发育的全过程。通过大规模化学诱变(ENU诱变)和反转录病毒插入诱变,已经筛选到很多斑马鱼胚胎发育的突变体,揭示了一批相关基因的功能。本论文的研究是利用Tol2转座子介导的基因捕获的方法产生GFP表达特异的转基因鱼家系,并筛选斑马鱼胚胎早期发育的突变体,继而分析相关突变基因在胚胎发育过程中可能的功能。本研究中共筛选到45个转基因鱼家系,其中一些转基因鱼家系胚胎的GFP的表达在发育过程中呈现出一定的时空特异性。但其胚胎并没有明显的突变表型。TZBGS010转基因鱼家系的荧光分布具有一定的特异性,胚胎受精后24小时时GFP荧光主要集中在嗅窝,耳泡,神经底板和肾管中。TAIL-PCR分析表明,在该转基因鱼家系中转座子所捕获的基因为foxj1b基因。Foxj1是一个转录因子,已有研究报道表明,其在脊椎动物中Foxj1与系统性自身免疫和纤毛的生成有关。斑马鱼的基因组表达两个foxj1同源基因,foxj1a和foxj1b。在我们的研究工作中,通过在单细胞时期注射morpholino抑制foxj1a或foxj1b表达会导致早期的左右不对称的标志性基因lefty2, southpaw, pitx2c和后期的内部器官的标志性基因tpm4-tv1, cmlc2, cp等表达出现随机分布。通过注射体外合成的mRNA过量表达foxj1a或foxj1b同样会扰乱胚胎正常的左右不对称发育。这些结果表明,在斑马鱼的胚胎发育中两个foxj1基因对于正常的左右不对称发育是必须的。不同于foxj1b,在背部前驱细胞中特异将foxj1a敲除,也会使得左右不对称的标志性基因表达出现随机分布的情况。因此,foxj1a通过背部前驱细胞以细胞自主性调控的方式调节左右不对称发育而foxj1b基因的作用则是非自主性的。更多还原

【Abstract】 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.更多还原