Intraciliary calcium directs cardiac left-right asymmetry

纤毛内钙引导心脏左右不对称

• 批准号: 9268566
• 负责人: MARTINA BRUECKNER
• 金额: $ 56.05万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9268566
• 关键词: Address; Affect; Ankyrin Repeat; Automobile Driving; Binding; Biology; Calcium; Calcium Channel; Calcium Oscillations; Calcium Signaling; Calmodulin; Cardiac; Cations; Cells; Cilia; Complex; Congenital Abnormality; Cultured Cells; Cytosol; Data; Development; Embryo; Environment; Epithelial Cells; Failure; Gene Expression; Genes; Goals; Handedness; Human; Image; Infant; Kidney; Left; Link; Liquid substance; Mediating; Methods; Modeling; Molecular; Morphogenesis; Mus; Mutation; PKD2 protein; Pathway interactions; Patients; Pattern; Phenotype; Physiology; Play; Positioning Attribute; Proteins; Reaction Time; Rest; Role; Signal Pathway; Signal Transduction; Situs Inversus; Structure; Syndrome; Techniques; Testing; Zebrafish; cardiogenesis; cell motility; ciliopathy; cilium motility; congenital heart disorder; experimental study; extracellular; in vivo; mRNA Transcript Degradation; novel; public health relevance; response; spatiotemporal; tool

 DESCRIPTION (provided by applicant): Cilia are central to the development of vertebrate left-right (LR) asymmetry, and failure to establish normal LR asymmetry results in the human heterotaxy syndrome (Htx) and severe congenital heart disease (CHD). Htx is amongst the most lethal forms of CHD in humans, and mutations affecting at least 12 genes affecting cilia structure and function have been identified in patients with Htx and other CHD. This proposal investigates the cilium as a distinct calcium signaling compartment, and investigates the role intraciliary calcium plays in heart development. We will define the mechanism by which intraciliary calcium establishes cardiac LR asymmetry. In vertebrate LR development, cilia at the left-right organizer (LRO) beat to generate directional flow of extraembryonic fluid, which is sensed by cilia and directs asymmetrical gene expression. Several lines of evidence link calcium, cilia and LR development: the ciliary calcium channel polycystin-2 (Pkd2) at the LRO is essential for LR development, and in mouse and zebrafish, a left-biased increase of cytoplasmic calcium correlates with LR development and is dependent on both flow and Pkd2. Despite the established importance of cilia, flow, polycystins and cytoplasmic calcium signaling in LR development, the mechanisms linking these components, and further to the downstream signaling cascade driving asymmetric cardiac morphogenesis, are not understood. We have developed a novel method to visualize and manipulate intraciliary calcium in cultured cells and zebrafish embryos. In this proposal, we will use this to address the physiology and function of intraciliary calcium in LR and heart development. We hypothesize that the cilium is a distinct cellular compartment with respect to calcium signaling, and that intraciliary calcium is the link between ciliary motility at the LRO and asymmetric cardiac morphogenesis. In Aim 1, we will characterize intraciliary calcium in-vivo in cultured cells and zebrafish embryos and examine the response of intraciliary calcium to ciliary motility and whether intraciliary calcium requires the polycystin channel in LR development. In Aim 2, we will dissect how intraciliary calcium regulates LR development by analyzing how intraciliary calcium regulates asymmetric gene expression. In Aim 3, we will test whether the ankyrin-repeat protein Inversin transduces intraciliary calcium in cultured renal epithelial cells, zebrafish and mice with mutations affectin inversin. In summary, these experiments provide a new paradigm for ciliary signaling in LR and heart development, and develop techniques that will be broadly applicable to the study of cilia biology.

 描述（由适用提供）：纤毛是脊椎动物左右（LR）不对称性的发展，以及未建立正常LR不对称性导致人类异质综合征（HTX）和严重的先天性心脏病（CHD）导致正常的LR不对称性。 HTX是人类中最致命的冠心病形式之一，在HTX和其他CHD患者中已经确定了影响至少12种影响纤毛结构和功能的基因的突变。该提案将纤毛作为一个独特的钙信号室研究，并研究了内部钙在心脏发育中的作用。我们将定义钙内钙建立心脏LR不对称性的机制。在脊椎动物LR的发育中，左右组织者（LRO）的纤毛摇摆以产生胚外流体的有向流，这是由Cilia感应的，并指导不对称的基因表达。几条证据连接钙，纤毛和LR的发育：LRO的睫状钙通道多囊蛋白-2（PKD2）对于LR的发育至关重要，在小鼠和斑马鱼中，左偏置的细胞质钙与LR的发展相关，并且依赖于流量和PKD2。尽管在LR发育中，纤毛，流量，多囊素和细胞质信号传导具有确定的重要性，但连接这些成分的机制，以及与下游信号级联反向驱动不对称心脏形态发生的机制，尚不清楚。我们开发了一种新颖的方法来可视化和操纵培养细胞和斑马鱼胚胎中的内部钙。在此提案中，我们将使用它来解决LR和心脏发育中内钙的生理和功能。我们假设纤毛是相对于钙信号传导的独特细胞室，并且内部钙是LRO处纤毛运动与非对称心脏形态发生之间的联系。在AIM 1中，我们将表征内部钙的体内培养细胞和斑马鱼胚胎，并检查内部钙对纤毛运动的响应，以及内部钙是否需要LR发育中的多囊菌通道。在AIM 2中，我们将通过分析内部钙如何调节不对称基因表达来剖析内部钙如何调节LR的发育。在AIM 3中，我们将测试Ankyrin-Repeat蛋白非洲蛋白毒素是否会在培养的肾上皮细胞，斑马鱼和具有突变的脑膜上的小鼠中转导钙内钙。总而言之，这些实验为LR和心脏发育中的纤毛信号传导提供了新的范式，并开发了将广泛适用于纤毛生物学研究的技术。