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）：在胚胎发生期间，心脏发展左右（LR）不对称性。心脏LR不对称或侧向的扰动通常会导致先天性心脏缺陷。有强有力的证据表明纤毛细胞在建立心脏横向性方面起作用。小鼠和斑马鱼模型的工作表明，一组保守的胚胎“ LR纤毛”会产生一种不对称的流体流，这是脊椎动物胚胎的正常LR模式所必需的。但是，LR纤毛生成LR信息的机制尚不清楚。在斑马鱼中，不对称流体流是由一个称为库普弗囊泡（KV）的器官中的纤毛上皮产生的。与其他脊椎动物不同，可以访问KV中LR纤毛的细胞，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。我们使用前向遗传筛选和反向遗传筛选，已经确定了在KV中调节LR纤毛细胞的三个新途径的基因：1）Rho激酶信号传导，2）细胞极性和3）离子泵介导的离子通量。该项目的具体目的是表征这三个途径在建立功能性KV和正常心脏侧向的作用。这项研究的长期目标是促进我们对心脏如何发展左右两侧的理解，并提供候选基因，以帮助诊断和治疗人类先天性心脏缺陷。为了实现该项目的目标，我们将使用突变体，反义形态基因敲低和小分子抑制剂分析基因功能。我们已经开发了一种将形态学特异性传递到KV细胞谱系的方法，以分析这些蛋白质在LR纤毛细胞中的功能丧失。这种方法使我们能够将特定基因在KV细胞中的作用与其在胚胎中其他细胞类型中的作用区分开。为了分析KV中的LR纤毛细胞，我们将利用斑马鱼中提供的一套独特的技术和工具。其中包括对活胚胎中KV发育的实时成像，具有揭示KV细胞结构和不对称流体流动视频显微镜检查的标记的KV细胞免疫染色。该项目的结果将定义调节LR纤毛和心脏侧向的基因和机制，并有可能对心脏病的见识。公共卫生相关性：先天性心脏病是最常见的先天缺陷。在理解心脏缺陷方面取得了进展，但是在许多情况下，根本原因是未知的。该项目着重于控制心脏发展的机制。我们的目标是确定可能有助于诊断，治疗或预防先天性心脏病的基因。