【作者】 王金虎；

【导师】 丁小燕；

【作者基本信息】 中国科学院研究生院（上海生命科学研究院） ， 发育生物学， 2007， 博士

【摘要】 脊椎动物的脊柱来源于体节,体节是胚胎发生过程中的过渡性结构。体节的形成过程被称为体节发生,体节发生受到严格的时间和空间控制。功能上,正确的分节保证了脊椎动物具有正常的体轴。因此,对体节发生的分子机制的研究是发育生物学中的重要课题。研究证明,Wnt,FGF和RA信号途径以及转录因子Mespo等参与了体节发生,但具体的分子机制目前所知甚少。通过对下游转录因子Mespo的研究,本文探讨了在体节发生中, Wnt,FGF和RA信号途径控制体节发生的可能作用方式。我们用Morpholino基团修饰的寡核苷酸(Mo)阻断Mespo在体内的功能,发现爪蟾的体节发生被破坏。同时,对Wnt在爪蟾轴旁中胚层表达区域的确定,以及改变Wnt信号途径的活性检查体节发生,证明Wnt信号途径存在于预决定体节中胚层,而且,Mespo的表达受到Wnt信号途径的调节。通过克隆Mespo 5’上游调控序列,用转基因实验证明了该序列包含指导Mespo在预决定体节中胚层表达所需的调控元件。该序列上存在一个LEF/TCF结合位点,此位点是Mespo在预决定体节中胚层表达所必需,在体内,LEF1蛋白结合在此位点上。进一步的研究发现,FGF信号下游作用因子AKT存在于预决定体节中胚层,在该区域AKT存在活化的磷酸化形式;而且,在预决定体节区域, PI3-K/AKT信号途径通过调节?-catenin的入核,参与了Mespo的表达调控。通过分析Mespo 5’上游不同缺失片段,初步研究了RA对Mespo表达的可能调控方式,实验证明RA信号通路调控Mespo的表达的顺式元件存在于Mespo 5’上游调控区一段326 bp DNA序列里。本文揭示了转录因子Mespo在爪蟾体节发生中的作用,以及Mespo表达的转录调控机制。Wnt信号途径在爪蟾体节发生中的作用,Wnt信号途径通过调控Mespo这个关键转录调控因子的表达控制了体节发生。PI3-K/AKT和RA信号也通过调控Mespo的表达参与体节发生。因此,Wnt,PI3-K/AKT以及RA信号途径通过协同调节Mespo的表达,从而使体节发生过程受到严格的时间和空间调控。更多还原

【Abstract】 The vertebral column is derived from somites, which are transient segments of the paraxial mesoderm that are present in developing vertebrates. The strict spatial and temporal regulation of somitogenesis is of crucial developmental importance. Signals such as Wnt, FGF and RA play roles in somitogenesis, but many details of how these signals function in this process remain unclear. Mespo, a member of bHLH transcription factor family plays important roles in specification of the paraxial mesoderm and regulation of segmentation. However, mechanisms that regulate the expression of Mespo are unclear. Some evidence has shown that Mespo might be regulated by these signals. To address these questions, we isolated a genomic DNA sequence containing 4317 bp of Mespo 5’flanking region in Xenopus. Luciferase assays show that this upstream sequence has a transcription activity. A transgenic assay shows that this genomic contig is sufficient to recapitulate the dynamic stage- and tissue-specific expression pattern of endogenous Mespo from the gastrula to the tailbud stage. Luciferase assay shows that this upstream sequence can respond to Wnt signaling. Transgenic analysis of the Mespo promoter identifies Mespo as a direct downstream target of Wnt/?-catenin signaling pathway. We also show that activity of Wnt/?-catenin signaling in somitogenesis can be enhanced by the PI3-K/AKT pathway. Our data illustrate that Wnt/?-catenin signaling in conjunction with PI3-K/AKT pathway plays a key role in controlling development of the paraxial mesoderm. We further mapped a 326 bp DNA sequence responding to RA signaling. These results shed light on how regulation of Mespo expression is controlled, and this finding provides a powerful tool to study how signaling pathways interact to regulate development of paraxial mesoderm.更多还原