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

脊椎动物胚胎用来传递左右信息的复杂信号通路在过去十年里已经开始被揭开，在老鼠和斑马鱼中使用正向和反向遗传方法，或者在青蛙和小鸡中通过实验胚胎学。在这个项目中，我们通过遗传学和实验胚胎学的方法，使用青竹鱼这一新兴的模式生物来研究LR规范的机制。在分离到的五个LR缺陷青竹突变株中，我们分离了三个突变株KTU、ABC和aA90的相关基因。前两个编码新基因，其余两个编码细胞质动力蛋白重链2(Dhc2)。在这个项目中，我们重点研究了KTU基因，因为它从单细胞生物体到具有活动纤毛/鞭毛的人类都是高度保守的。青竹KTU突变体是一种典型的随机突变体，定位克隆已证实KTU是一种新的细胞质蛋白。在没有KTU的情况下，大多数动力蛋白的外臂和内臂在轴丝中缺失，导致运动性丧失。生化数据显示，KTU是长期寻找的蛋白质之一，在IFT将动力蛋白臂复合体装载到睫状室之前，它参与了细胞质中动力蛋白臂复合体的预组装(提交的论文)。在研究过程中，我们分离了硬骨鱼库普弗氏囊泡(KV，相当于小鼠结节的器官)中最早的不对称基因。这个基因被称为Charon，编码Cerberus/Dan家族蛋白的一个成员。青竹纤毛呈不对称表达，右侧表达较强，受KV上游动纤毛产生的液流直接调节。该基因可能是鱼类LR不对称的一个可靠的基因标记，因此将有助于对流体流动下游的早期步骤进行分析。

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.