Analysis of vertebrate left-right axis formation using medaka fish as a model

在过去的十年里，脊椎动物胚胎用来传递左右信息的复杂信号通路已经开始被揭示出来，通过小鼠和斑马鱼的正向和反向遗传方法，或者通过青蛙和小鸡的实验胚胎学。在本项目中，我们通过遗传学和实验胚胎学的方法，研究了medaka鱼（一种新兴的模式生物）LR规范的机制。在分离的5个因LR缺陷而产生的medaka突变体中，我们分离出了ktu、abc和aA90 3个突变体的致病基因。前两个编码新基因，其余编码细胞质动力蛋白重链2 （dhc2）。在这个项目中，我们专注于ktu基因，因为它从单细胞生物到具有运动纤毛/鞭毛的人类高度保守。medaka ktu突变体是一种典型的随机突变体，定位克隆证实ktu是一种新的细胞质蛋白。在缺乏Ktu的情况下，轴素中大多数外部和内部动力蛋白臂缺失，导致运动性丧失。生化数据表明，在IFT将动力蛋白臂复合物装载到纤毛室之前，Ktu是细胞质中参与预组装的长期寻找的蛋白质之一（论文提交）。在研究过程中，我们从硬骨鱼库普弗氏囊泡（KV，相当于小鼠淋巴结的器官）中分离出最早的不对称基因。这个基因被称为charon，编码Cerberus/DAN家族蛋白的一个成员。medaka charon呈不对称表达，右侧表达更强烈，受KV上皮上运动纤毛产生的液体流动的直接调节。该基因可能是鱼类LR不对称的可靠基因标记，因此将有助于分析流体流动的早期下游步骤。

The complex signaling pathways that vertebrate embryo uses to convey left-right information have been beginning to be unveiled for the last decade, using forward-and reverse-genetic approaches in both mouse and zebrafish, or via experimental embryology in frog and chick. In this projected we have examined the mechanisms of LR specification using medaka fish, an emerging model organism, through approaches of genetics and experimental embryology.1. Among the five medaka mutants isolated for LR defects, we have isolated the responsible genes for three mutants, ktu, abc and aA90. The first two encodes novel genes and the rest encodes cytoplasmic dynein heavy chain2 (dhc2). In this project, we focused on the ktu gene because this is higly conserved from a unicellular organism to human with motile cilia/flagella. The medaka ktu mutant is a typical randomized mutant, and positional cloning has identified Ktu as a novel cytoplasmic protein. In the absence of Ktu, most outer and inner dynein arms are missing in the axoneme, leading to a loss of motility. Biochemical data demonstrate that Ktu is one of the long-sought proteins involved in pre-assembly of dynein arm complexes in the cytoplasm before IFT loads them for the ciliary compartment(paper submitted).2. During the course of the study, we isolated the earliest asymmetric gene in teleost Kupffer's vesicle(KV, an equivalent organ to the mouse node). This gene is called charon, encoding a member of the Cerberus/DAN family proteins. The medaka charon displays asymmetric expression with more intense expression on the right side and found to be directly regulated by liquid flow generated by motile cilia on the KV epithelium. This gene could be a reliable gene marker for LR asymmetry in fish and thus will be useful for the analysis of the early steps downstream of fluid flow.