母源因子Hwa及互作蛋白在斑马鱼体轴诱导中的分子机理

Axis induction and dorsal-ventral specification is critical for the establishment of the fate map of zebrafish embryo. This process is regulated not only by the products of zygotic genome, but also by important maternal factors, such as the maternal β-catenin signaling pathway. So far, how maternal β-catenin signaling is activated specifically at the dorsal side is unclear...In our primary results, we get a maternal mutant huluwa(hwa), characterized by no axis formation and severe defect of dorsal-ventral patterning. The mutant gene is a novel protein-coding gene, so we name it hwa. Through experiments in mammalian cell lines and zebrafish embryos, we demonstrate that Hwa protein induce axis formation by activating the organizer specific genes in a maternal β-catenin dependent way. However, the detailed mechanism of how it works needs further study to disclose...In the present project, we set out to indentify a batch of interacting proteins of Hwa through Yeast Two-hybrid screening and Co-Immunoprecipitation, disclose their relationship and regulation mechanism, demonstrate how they work together to regulate the maternal β-catenin signaling and other signaling pathway, and uncover the molecular network of axis induction and dorsal-ventral specification.

体轴诱导和背腹分化对于斑马鱼胚胎发育命运图谱的建立至关重要。此过程不仅受到合子基因组转录产物的调控，还受到重要母源因子的调控，如母源β-catenin信号通路。但目前母源β-catenin信号背部区域特异激活机制尚不清楚。..在前期工作中，我们筛选到一个母源突变体huluwa (hwa)，表现为无体轴生成，并伴随严重的背腹分化缺陷，经鉴定突变基因为一个未注释的全新蛋白编码基因，命名为hwa。结合哺乳动物细胞系和斑马鱼胚胎实验，我们证明了Hwa蛋白通过母源β-catenin信号通路激活组织中心特异基因的表达，参与体轴诱导。详细分子机理尚待研究。..本项目拟通过酵母双杂交、免疫共沉淀等方式捕获和鉴定Hwa蛋白的互作因子，明确它们的互作关系及调控机制，论证它们如何协同调控母源β-catenin信号通路，以及与其他早期发育调控信号通路的交互关系，揭示早期发育过程中体轴诱导和背腹分化的调控分子网络。

脊椎动物胚胎体轴诱导及形成不仅受到合子基因组转录产物的调控，还受到重要母源因子的调控，如母源β-catenin信号通路。在前期工作中，我们发现Huluwa（Hwa）蛋白通过母源β-catenin信号通路激活组织中心特异基因的表达参与体轴诱导，但信号转导和信息交互的详细分子机理尚不清楚。本项目结合哺乳动物细胞系和斑马鱼胚胎实验，我们进一步证明了Hwa蛋白通过结合并促进Tankyrase介导的Axin降解，可作为一个背部决定因子在斑马鱼胚胎背侧卵裂球中激活母源β-catenin信号；通过系统性蛋白突变分析，揭示了Hwa蛋白本身重要调控序列位点（PPNSP、RRSST）和关键翻译后修饰（磷酸化）对Hwa信号转导活性的重要性；通过pull-down/MS鉴定并验证了多个Hwa蛋白互作因子；明确了Cdk激酶对Hwa磷酸化及Hwa/β-catenin信号激活的调控作用。这些研究结果不仅揭示了Hwa蛋白本身及下游信号转导更为细致的调节机理，还证明了细胞周期相关调控激酶在Hwa/β-catenin信号通路调控及体轴诱导中的重要贡献。