Origin of vertebrate left-right asymmetry

描述（由申请人提供）：沿左右轴的非随机不对称发展是脊椎动物发育的独特特征。在小鼠和人类中，这一过程中的缺陷通常会影响心脏的发育并导致严重的先天性心脏异常。本研究的目的是了解胚胎纤毛产生和发出左右位置信息的机制。胚胎纤毛的运动是由轴突动力蛋白左右驱动的。Lrd对于左右不对称的发展是必要的和特定的。当它有缺陷时，原肠胚形成时在胚胎淋巴结（组织者）上发现的正常运动的单纤毛会瘫痪。活动的淋巴结纤毛产生围绕淋巴结的胚胎外液定向流动（结流），而lrd缺陷小鼠没有结流。这些观察结果表明，活动的9+0节点纤毛在LR不对称的发生中起直接作用。多囊蛋白基因Pkd2在肾单纤毛中作为一种机械换能器，通过介导细胞内钙信号响应肾小管的流体流动，LR不对称的发展也在多囊蛋白基因Pkd2缺陷的小鼠中出现异常。我们已经表明，多囊蛋白-2蛋白定位于非运动的单纤毛的一个子集。最后，我们在e7.75小鼠胚胎中观察到不对称的乳周钙信号。这些数据表明，可能需要胚胎纤毛来产生和感觉节流。在本提案中，我们将首先测试节点处是否存在两类单纤毛。首先，我们将利用免疫荧光和透射电镜研究单纤毛节点的结构。使用Cre-lox技术，我们将生成删除所有节点单纤毛的小鼠，并将其LR发育与仅缺乏活动节点单纤毛的小鼠进行比较。我们将通过观察影响结流的突变和人工结流对不对称结周钙信号的影响，来确定不对称钙信号在LR不对称发展中的作用。通过研究人工引入钙信号对培养胚胎LR表型的影响，将检验不对称乳周钙信号在LR发育途径中的地位。最后，我们将评估钙透性阳离子通道多囊蛋白-2在LR发育中的作用。将检查Pkd2-/-小鼠的结纤毛形态和不对称的结周钙信号，以确定结流是否通过多囊蛋白通道产生不对称的结周钙信号。

DESCRIPTION (provided by applicant): The development of non-random asymmetry along the left-right axis is a unique feature of vertebrate development. Defects in this process in mouse and man commonly affect the development of the heart and result in severe congenital cardiac anomalies. The goal of this proposal is to understand the mechanism by which embryonic cilia create and signal left-right positional information. Motility of embryonic cilia is driven by the axonemal dynein, left right dynein (lrd). Lrd is essential and specific to the development of left-right asymmetry. When it is defective, the normally motile monocilia found on the node (organizer) of the embryo at the time of gastrulation are paralyzed. Motile node cilia generate directional flow of the extraembryonic fluid surrounding the node (nodal flow), and there is no nodal flow in mice with defective lrd. These observations suggest a direct role for motile 9+0 node cilia in the initiation of LR asymmetry. The development of LR asymmetry is also abnormal in mice with defects in the polycystin gene Pkd2, which functions in kidney monocilia as a mechanotransducer by mediating an intracellular calcium signal in response to fluid flow in the renal tubule. We have shown that Polycystin-2 protein localizes to a subset of node monocilia that are non-motile. Finally, we observe an asymmetric perinodal calcium signal in e7.75 mouse embryos. These data suggest that embryonic cilia may be required to both create and sense nodal flow. In this proposal, we will first test whether there are two classes of monocilia at the node. First, we will study the structure of node monocilia by immunofluorescence and transmission electron microscopy. Using Cre-lox technology, we will generate mice that have deleted all node monocilia, and compare their LR development to that observed in mice lacking only motile node monocilia. We will determine the role of asymmetric calcium signaling in the development of LR asymmetry by observing the effect of mutations affecting nodal flow, and of artificial nodal flow, on the asymmetric perinodal calcium signal. The position of asymmetric perinodal calcium signaling in the pathway of LR development will be tested by examining the effect of artificially introduced calcium signals on LR phenotype in cultured embryos. Finally, we will evaluate the role of the calcium-permeable cation channel polycystin-2 in LR development. Pkd2-/- mice will be examined for morphology of node cilia, and for asymmetric perinodal calcium signals in order to determine whether nodal flow produces asymmetric perinodal calcium signals via the polycystin channel.