RA下游因子Aplnra/b在斑马鱼器官左右不对称模式建成中的时空调控作用及机制研究

The organ left-right(LR) asymmetry defect always results in or is acommpanied with many congenital diseases. Although Retinoic acid (RA) has been reported to be involved in regulating organ LR asymmetry patterning, the downstream factors as well as the detailed mechanism underlying are largely unknown. Our preliminary data in zebrafish showed that down-regulation of RA signal resulted in increased expression of aplnra/b. In addition, knock-down of agtrl1a caused the heart and liver asymmetry defects, which is dependent on Nodal signal.These results suggested that aplnra/b might act as relay of RA to mediate the special role of RA in regulating organ LR asymmetry patterning. In this project, firstly we will valuate the special role of aplnra/b in organ LR asymmetry pattering by Talen mediated gene knock-out and some other technologies; then we will continue to construct the transgenic zebrafish line expressing dominant negative protein, and use specific gene knock-down in dorsal forerunner cells (DFCs) and other critical methods to study the spatial-temporal role of aplnra/b in regulating organ LR asymmetry patterning. Afterwards, we will focus on studying the mechanism how aplnra/b is involved in regulating organ LR asymmetry by using specific location knockdown, Fluro-beads movement tracing, CoIP and other experiments. Finally, we will study the relationship between aplnra/b and RA in this process. The result from this project will help us understand the mechanism how organ LR asymmetry is patterned in embryogenesis, as well as provide an avenue for preventing and curing the LR asymmetry related diseases.

器官左右不对称模式建成紊乱与许多先天性疾病相关。RA信号参与了器官左右不对称模式建成的调控，但其下游因子及具体调控机制不明。本项目前期数据表明在斑马鱼中Aplnra/b受RA信号负调控,且Aplnra以Nodal依赖性形式参与了心脏和肝脏左右不对称模式的建立，提示Aplnra/b可能介导了RA对器官左右不对称建成的调控。故本项目首先运用MO注射等研究Aplnra/b在器官左右不对称建成中的作用，并运用转基因及DFCs细胞特异性敲降等技术研究Aplnra/b对器官左右不对称建成的时空调节模式，然后运用基因定位敲降、荧光beads KV示踪等实验探究其时空特异性作用的具体调控机制，最后探讨Aplnra/b与RA在左右不对称模式建成中的关系。项目研究结果既加深了我们对器官左右不对称模式建成分子调控机制的认识，也有望为器官左右不对称模式紊乱相关疾病的防治提供理论依据。

器官左右不对称模式建成紊乱与许多先天性疾病相关。RA信号参与了器官左右不对称模式建成的调控，但其下游因子及具体调控机制不明。本项目前期数据表明在斑马鱼中Aplnra/b受RA信号负调控,且Aplnra以Nodal依赖性形式参与了心脏和肝脏左右不对称模式的建立，提示Aplnra/b可能介导了RA对器官左右不对称建成的调控。本项目后续研究发现aplnra和aplnrb在胚胎早期相互协调、互为补充的调控器官左右不对称模式建成的发生。其中aplnra主要通过背部前迁移细胞细胞自主性的调控KV及KV中纤毛的发生，从而调控Nodal信号而调控左右模式建成；aplnrb在早期左右模式建成中不起主导作用，他通过细胞非自主性模式调控了nodal信号从而调控左右模式建成。另外我们发现apela而非apln与aplnr受体结合调控了早期KV及KV中纤毛的发生，而在晚期apln及apela共同调控了middle line的结构功能。在于RA作用的关系上我们发现aplnr信号通路并非完全介导RA在左右不对称模式建成中的作用。在以上研究的基础上，我们发现aplnr下游PRK参与了早期原肠运动细胞迁移的调控；也发现另一G蛋白偶联受体Cxcr4a可能以相似的作用机制调控了器官左右不对称模式的建成。总之项目研究结果既加深了我们对器官左右不对称模式建成分子调控机制的认识，也有望为器官左右不对称模式紊乱相关疾病的防治提供理论依据。