Intraciliary calcium directs cardiac left-right asymmetry

 描述(由申请人提供)：纤毛是脊椎动物左-右不对称发展的核心，未能建立正常的左-右不对称会导致人类异位综合征(HTX)和严重先天性心脏病(CHD)。HTX是人类最致命的CHD形式之一，已在HTX和其他CHD患者中发现了至少12个影响纤毛结构和功能的基因突变。这项建议调查纤毛作为一个不同的钙信号间隔，并调查了纤毛内钙在心脏发育中的作用。我们将定义肌内钙建立心脏LR不对称性的机制。在脊椎动物LR的发育中，位于左右组织器(LRO)的纤毛产生定向的胚外液体流动，纤毛被纤毛感知并指导不对称的基因表达。有几条证据表明钙、纤毛和LR的发育有关：LRO上的纤毛钙通道多囊蛋白-2(PKD2)对于LR的发育是必不可少的，而在小鼠和斑马鱼中，细胞质钙的左偏增加与LR的发育相关，并依赖于Flow和PKD2。尽管纤毛、Flow、多囊蛋白和胞质钙信号在LR发育中的重要性已被证实，但连接这些成分以及进一步到驱动心脏形态不对称发生的下游信号级联的机制尚不清楚。我们开发了一种新的方法来可视化和操作培养细胞和斑马鱼胚胎中的内钙。在这项建议中，我们将利用这一点来阐述睫状肌内钙在LR和心脏发育中的生理和功能。我们假设纤毛是一个关于钙信号的不同细胞隔室，而纤毛内钙是LRO纤毛运动和心脏形态发生不对称之间的联系。在目标1中，我们将研究培养细胞和斑马鱼胚胎体内的纤毛内钙的特性，并研究纤毛运动对纤毛内钙的响应，以及在LR发育过程中，纤毛内钙是否需要多囊蛋白通道。在目标2中，我们将通过分析胞内钙如何调节不对称基因表达来剖析胞内钙如何调控LR的发育。在目标3中，我们将测试在培养的肾上皮细胞、斑马鱼和突变影响反转素的小鼠中，Ankyrin-Repeat Protein Inversin是否转导纤毛内钙。综上所述，这些实验为LR和心脏发育中的纤毛信号提供了一个新的范例，并开发了将广泛应用于纤毛生物学研究的技术。

 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.