Regulation of Ciliated Cells that Control Cardiac Laterality

描述(申请人提供)：在胚胎发育过程中，心脏形成功能性左-右(LR)不对称。心脏下缘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了作用。老鼠和斑马鱼模型的研究表明，一组保守的胚胎‘LR纤毛’产生不对称的流体流动，这是脊椎动物胚胎正常的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，不对称的液体流动是由一个叫做库普弗氏囊泡(KV)的器官中的纤毛上皮产生的。与其他脊椎动物不同，在KV中产生LR纤毛的细胞是可以接触到的，并可以在斑马鱼胚胎中进行研究。我们正在使用斑马鱼来描述控制KV形成和心脏不对称的基因和机制。利用正向和反向遗传筛选，我们已经确定了在KV中涉及三个新的调控LR纤毛细胞的基因：1)Rho激酶信号，2)细胞极性和3)离子泵介导的离子通量。本项目的具体目标是确定这三条通路在建立功能性KV和正常心脏偏侧性方面的作用。这项研究的长期目标是促进我们对心脏如何发育不同的左右两侧的理解，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。为了实现这个项目的目标，我们将使用突变体、反义吗啡基因敲除和小分子抑制剂来分析基因功能。我们已经开发了一种方法，专门将吗啡递送到KV细胞系，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特定基因在KV细胞中的作用与它在胚胎中其他细胞类型中的作用。为了分析KV中的LR纤毛细胞，我们将利用斑马鱼提供的一套独特的技术和工具。这些包括活胚胎中KV发育的实时成像，KV细胞的免疫染色和揭示KV细胞结构的一系列标记物，以及不对称液体流动的视频显微镜。该项目的结果将定义调节LR纤毛和心脏偏侧的基因和机制，并潜在地提供对心脏病的洞察。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，但对许多病例来说，潜在的原因尚不清楚。这个项目的重点是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。

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.