Origin of vertebrate left-right asymmetry

脊椎动物左右不对称的起源

• 批准号: 6821825
• 负责人: MARTINA BRUECKNER
• 金额: $ 41.6万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6821825
• 关键词: biological signal transduction; calcium ion; dynein ATPase; electron microscopy; embryogenesis; genetically modified animals; green fluorescent proteins; immunofluorescence technique; intracellular; kidney; laboratory mouse; morphology; sensory receptors; vertebrate embryology; video microscopy

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.

描述（由申请人提供）： 非随机的不对称性沿着左右轴的发展是脊椎动物发展的独特特征。在小鼠和人类中，这一过程的缺陷通常会影响心脏的发育，并导致严重的先天性心脏畸形。这个提议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丝动力蛋白，左右动力蛋白（lrd）驱动。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的节点（组织者）上发现的正常运动的单纤毛被麻痹。能动的淋巴结纤毛产生围绕淋巴结的胚外液的定向流动（淋巴结流），而lrd缺陷的小鼠则没有淋巴结流。这些观察结果表明，在启动LR不对称的运动9+0结纤毛的直接作用。LR不对称性的发展在多囊蛋白基因Pkd 2缺陷的小鼠中也是异常的，多囊蛋白基因Pkd 2通过介导细胞内钙信号响应于肾小管中的流体流动而在肾单眼中作为机械换能器起作用。我们已经表明，多囊蛋白-2蛋白定位于一个子集的节点monocilia是不动的。最后，我们在e7.75小鼠胚胎中观察到不对称的结周钙信号。这些数据表明，胚胎纤毛可能需要创建和感觉结流。在这个建议中，我们将首先测试是否有两个类的单眼在节点。首先，我们将利用免疫萤光及穿透式电子显微镜来研究单纤毛节的结构。使用Cre-lox技术，我们将产生删除所有节点的小鼠，并将其LR发育与仅缺乏活动节点的小鼠进行比较。我们将通过观察影响淋巴结流量的突变和人工淋巴结流量对不对称结周钙信号的影响，确定不对称钙信号在LR不对称发展中的作用。将通过检查人工引入的钙信号对培养胚胎中LR表型的影响来测试不对称结周钙信号在LR发育途径中的位置。最后，我们将评估钙渗透性阳离子通道多囊蛋白-2在LR发展中的作用。将检查PKd 2-/-小鼠的结纤毛形态和不对称结周钙信号，以确定结流是否通过多囊蛋白通道产生不对称结周钙信号。