Zfp36l2在胚胎体轴形成中的作用机理研究

During embryonic development, a precise and tightly regulated gene expression is necessary to ensure the proper formation of all cell types and organs. RNA binding proteins play key roles in post-transcriptional regulation of gene expression, and recent studies have shown that these proteins are involved in many developmental processes. Axis formation is an important process in embryonic development. The main signal pathway and many important transcription factors involved in axis formation have been identified, but little information is available to date regarding the functions of RNA binding proteins in vertebrate axis formation. .We have identified Zfp36l2, a RNA-binding protein, in frog (Xenopus laevis) and zebrafish (Danio rerio), and whole mount in situ hybridization showed that Zfp36l2 is specifically expressed in mesoderm. We also found that Zfp36l2 mRNA overexpression resulted in head defects. In addition, luciferase experiment revealed that Zfp36l2 was able to regulate Wnt signal pathway, and knockdown of Zfp36l2 led to dorsalized embryos with enlarged head and truncated tail. Taken together, these preliminary results suggest that Zfp36l2 may play a role in axis formation, possibly by targeting RNAs involved in canonical Wnt singal pathway. However, its precise function and mode of action remain enigmatic, and are worth studying. .The aim of this proposal is thus to explore the mechanism of Zfp36l2 function in axis formation. We plan to screen putative target RNAs of Zfp36l2 by RNA immunoprecipitation (RIP), to examine the precise role of Zfp36l2 and its target RNA in developmental processes by classical gain-of-function and loss-of-function experiments, to determine its binding sites in target RNAs by RIP, to explore how Zfp36l2 regulates its target genes, then how this regulates canonical Wnt signal pathway in the end affecting axis formation. We also plan to identify proteins which may interact with Zfp36l2. Eventually, we will elucidate the mechamism of Zfp36l2 in developmental processes, especially in axis formation. These analyses should provide novel insight into the mechanisms underlying axis formation.

RNA结合蛋白在早期发育中起重要调控作用，但这些转录后调控因子在脊椎动物体轴形成中的作用还不清楚。我们已克隆了斑马鱼和非洲爪蛙的Zfp36l2，发现其在中胚层特异表达，过量表达时胚胎头部发育缺陷，基因敲降时胚胎出现背部化表型，报告基因实验结果表明其可能影响经典Wnt信号通路，提示Zfp36l2可能是通过经典Wnt信号通路调节体轴形成的新基因。但其具体作用机制尚不明确。因此，我们拟首先采用RIP-差异筛选Zfp36l2的候选靶RNA；采用过量表达与基因敲降方法系统研究Zfp36l2与靶RNA的功能、与Wnt信号通路的关系以及它们在发育过程中的相互作用；采用报告基因、Western、RIP等方法确定Zfp36L2与靶RNA的结合位点及作用机制；同时筛选可能与Zfp36l2结合的蛋白。由此，阐明Zfp36l2在体轴形成中的作用机制，确定其在经典Wnt信号通路所处位置，完善体轴形成基因调控网络。

RNA 结合蛋白在早期发育中起重要调控作用，但这些转录后调控因子在脊椎动物体轴形成中的作用还不清楚。我们发现斑马鱼和非洲爪蛙的Zfp36l2在中胚层特异表达，过量表达时胚胎头部发育缺陷，基因敲降时胚胎出现背部化表型，报告基因实验结果表明其可能影响经典Wnt 信号通路。但其具体作用机制尚不明确。我们首先采用RIP-差异筛选到多个Zfp36l2 的候选靶RNA；并通过原位杂交研究了筛选的候选靶基因中的Ly6家族等多个基因的表达模式，并通过过量表达与基因敲降方法研究了靶RNA 的功能、报告基因实验结果表明Zfp36l2没有转录活性，能够抑制Tcf1以及Wnt3a激活的Wnt信号通路，并且Zfp36l2也能够抑制β-catenin激活的Wnt信号通路。Zfp36l2的羧基端对于其功能是必需的。进一步的研究表明Zfp36l2能够调节Cox2 和Xnr3 mRNA的稳定性，而与Cox2相互作用的位点是在Cox2 mRNA的3’-UTR的富含AU区。酵母双杂交筛选到可能与Zfp36l2结合的蛋白，并通过Talens技术建立了Zfp36l2敲除的F2代纯合突变体，之后将用于确定母源性Zfp36l2的功能。由此，我们确定 Zfp36l2 通过直接抑制β-catenin激活的信号通路以及通过Xnr3和Cox2 mRNA3’-UTR中富含AU序列的区域调控其稳定性影响Wnt信号通路从而在体轴形成中起作用。这是Zfp36l2除免疫、血细胞发生之外的一个新功能。也是体轴形成过程中鉴定到的通过转录后调控起作用的新因子。