Regulation of Ciliated Cells that Control Cardiac Laterality

控制心脏偏侧性的纤毛细胞的调节

• 批准号: 7851355
• 负责人: JEFFREY D AMACK
• 金额: $ 34.98万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2011-12-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7851355
• 关键词: Appearance; Architecture; Candidate Disease Gene; Cardiac; Cell Lineage; Cell Polarity; Cells; Cilia; Collection; Complex; Congenital Abnormality; Congenital Heart Defects; Data; Development; Diagnosis; Embryo; Embryonic Development; Epithelial Cells; Epithelium; Genes; Genetic; Genetic Screening; Goals; Handedness; Heart; Heart Diseases; Human; Image; Ion Pumps; Ions; Label; Larva; Left; Life; Link; Mediating; Methods; Microscopy; Modeling; Morphogenesis; Mus; Organ; Pathway interactions; Pattern; Play; Prevention; Proteins; Regulation; Rho-associated kinase; Role; Side; Signal Transduction; System; Techniques; Testing; Time; Tube; Vertebrates; Vesicle; Video Microscopy; Work; Zebrafish; base; cardiogenesis; cell type; congenital heart disorder; fluid flow; gene function; heart function; inhibitor/antagonist; insight; loss of function; mutant; positional cloning; protein complex; public health relevance; small molecule; tool

DESCRIPTION (provided by applicant): During embryogenesis, the heart develops functional left-right (LR) asymmetries. Perturbation of cardiac LR asymmetry, or laterality, often leads to complex congenital heart defects. There is strong evidence that ciliated cells play a role in establishing cardiac laterality. Work from mouse and zebrafish models indicate a conserved group of embryonic 'LR cilia' generate an asymmetric fluid flow that is required for normal LR patterning of the vertebrate embryo. However, the mechanisms by which LR cilia generate LR information remain unclear. In zebrafish, asymmetric fluid flow is produced by a ciliated epithelium in an organ called Kupffer's vesicle (KV). Unlike other vertebrates, the cells that give rise to LR cilia in KV are accessible and can be studied in the zebrafish embryo. We are using zebrafish to characterize genes and mechanisms that control KV formation and heart asymmetry. Using both forward and reverse genetic screens, we have identified genes that implicate three new pathways in the regulation of LR ciliated cells in KV: 1) Rho kinase signaling, 2) cell polarity and 3) ion pump-mediated ion flux. The specific aims of this project are to characterize the role of these three pathways in establishing a functional KV and normal cardiac laterality. The long- term objective of this study is to advance our understanding of how the heart develops distinct left and right sides, and to provide candidate genes that may aid in diagnosis and treatment of human congenital heart defects. To achieve the goals of this project, we will analyze gene function using mutants, antisense morpholino gene knockdowns and small molecule inhibitors. We have developed a method to deliver morpholinos specifically to the KV cell lineage to analyze loss-of-function of these proteins specifically in LR ciliated cells. This approach allows us to distinguish the role of a particular gene in KV cells from its roles in other cell types in the embryo. To analyze LR ciliated cells in KV, we will take advantage of a unique set of techniques and tools available in zebrafish. These include real-time imaging of KV development in live embryos, immunostaining of KV cells with a collection of markers that reveal KV cellular architecture and videomicroscopy of asymmetric fluid flow. Results from this project will define genes and mechanisms that regulate LR cilia and heart laterality, and potentially provide insight into heart disease. PUBLIC HEALTH RELEVANCE: Congenital heart disease is the most common birth defect. Progress has been made in understanding heart defects, but for many cases the underlying cause is unknown. This project focuses on mechanisms that control heart development. Our goal is to identify genes that may aid in diagnosis, treatment or prevention of congenital heart disease.

描述（由申请人提供）：在胚胎发生过程中，心脏出现功能性左右（LR）不对称。心脏 LR 不对称性或偏侧性的扰动通常会导致复杂的先天性心脏缺陷。有强有力的证据表明纤毛细胞在建立心脏偏侧性方面发挥着作用。小鼠和斑马鱼模型的研究表明，一组保守的胚胎“LR 纤毛”会产生不对称的流体流动，这是脊椎动物胚胎正常 LR 模式所必需的。然而，LR 纤毛产生 LR 信息的机制仍不清楚。在斑马鱼中，不对称的流体流动是由称为库普弗囊泡 (KV) 的器官中的纤毛上皮产生的。与其他脊椎动物不同，KV 中产生 LR 纤毛的细胞是容易获得的，并且可以在斑马鱼胚胎中进行研究。我们正在使用斑马鱼来表征控制 KV 形成和心脏不对称的基因和机制。通过正向和反向遗传筛选，我们确定了涉及 KV 中 LR 纤毛细胞调节的三种新途径的基因：1) Rho 激酶信号传导，2) 细胞极性和 3) 离子泵介导的离子流。该项目的具体目标是描述这三种通路在建立功能性 KV 和正常心脏偏侧性方面的作用。这项研究的长期目标是增进我们对心脏如何发育出独特的左侧和右侧的理解，并提供可能有助于诊断和治疗人类先天性心脏缺陷的候选基因。为了实现该项目的目标，我们将使用突变体、反义吗啉代基因敲低和小分子抑制剂来分析基因功能。我们开发了一种将吗啉代特异性递送至 KV 细胞谱系的方法，以分析这些蛋白在 LR 纤毛细胞中的功能丧失。这种方法使我们能够区分 KV 细胞中特定基因的作用与胚胎中其他细胞类型的作用。为了分析 KV 中的 LR 纤毛细胞，我们将利用斑马鱼中可用的一套独特的技术和工具。这些包括活体胚胎中 KV 发育的实时成像、使用一系列标记物对 KV 细胞进行免疫染色以揭示 KV 细胞结构以及不对称流体流动的视频显微镜。该项目的结果将定义调节 LR 纤毛和心脏偏侧性的基因和机制，并有可能提供对心脏病的深入了解。公共卫生相关性：先天性心脏病是最常见的出生缺陷。在了解心脏缺陷方面已经取得了进展，但许多病例的根本原因尚不清楚。该项目重点研究控制心脏发育的机制。我们的目标是识别可能有助于诊断、治疗或预防先天性心脏病的基因。