Intraciliary calcium directs cardiac left-right asymmetry

纤毛内钙引导心脏左右不对称

• 批准号: 8964912
• 负责人: MARTINA BRUECKNER
• 金额: $ 58.55万
• 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/8964912
• 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; Life; 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; extracellular; in vivo; mRNA Transcript Degradation; novel; public health relevance; research study; 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（由申请人提供）：纤毛是脊椎动物左右（LR）不对称发展的核心，未能建立正常的LR不对称导致人类内脏异位综合征（Htx）和严重的先天性心脏病（CHD）。Htx是人类CHD中最致命的形式之一，并且已经在Htx和其他CHD患者中鉴定了影响纤毛结构和功能的至少12个基因的突变。这项建议调查纤毛作为一个独特的钙信号室，并调查纤毛内钙在心脏发育中的作用。我们将明确睫状体内钙离子建立心脏左室舒张不对称的机制。在脊椎动物LR发育中，左右组织者（LRO）的纤毛跳动以产生胚外液的定向流动，这种流动被纤毛感知并指导不对称的基因表达。几条证据将钙、纤毛和LR发育联系起来：LRO处的纤毛钙通道多囊蛋白-2（Pkd 2）对LR发育至关重要，在小鼠和斑马鱼中，细胞质钙的左偏增加与LR发育相关，并依赖于流量和Pkd 2。尽管纤毛，流量，多囊蛋白和细胞质钙信号在LR发展的重要性，连接这些组件的机制，并进一步驱动不对称心脏形态发生的下游信号级联，不清楚。我们开发了一种新的方法来可视化和操纵培养细胞和斑马鱼胚胎中的纤毛内钙。在本提案中，我们将利用这一点来解决LR和心脏发育中纤毛内钙的生理学和功能。我们假设，纤毛是一个独特的细胞隔室相对于钙信号，和纤毛内钙是纤毛运动在LRO和不对称的心脏形态发生之间的联系。在目的1中，我们将在培养细胞和斑马鱼胚胎中表征体内纤毛内钙，并检查纤毛内钙对纤毛运动的反应，以及纤毛内钙是否需要LR发育中的多囊蛋白通道。在目标2中，我们将通过分析纤毛内钙如何调节不对称基因表达来剖析纤毛内钙如何调节LR的发育。在目标3中，我们将测试锚定重复蛋白Inversin是否在培养的肾上皮细胞、斑马鱼和具有影响inversin突变的小鼠中转导纤毛内钙。总之，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例，并开发了将广泛适用于纤毛生物学研究的技术。