MOLECULAR GENETICS OF CARDIAC CELL DIFFERENTIATION

描述(申请人提供)：本项目的长期目标是 了解心肌细胞分化的机制。的具体目标 这项建议是为了确定心脏瓣膜形成的重要基因 斑马鱼，分析它们的功能。心脏瓣膜在几个阶段形成 脊椎动物心管的位置，包括房室(AV) 边界问题一直是我们研究的重点。在反病毒的过程中 瓣膜形成，房室交界处的心肌细胞诱导 潜在的心内膜细胞将经历上皮向间充质的转变 (EMT)，形成心内膜垫。这个垫子后来被改装成一个 阀门。对鸡和小鼠胚胎的研究已经导致了鉴定和 瓣膜形成过程中几个重要步骤的分子分析 但显然，对这一过程至关重要的额外分子仍有待于 已确认身份。 此前在斑马鱼中进行的基因筛选已经导致了两个 影响瓣膜形成的突变，Jekyll和心脏恐惧症。我们最近做了 通过定位克隆的方法分离了Jekyll基因，发现 它编码UDP-葡萄糖脱氢酶，这是一种启动要添加的糖的酶 进入糖胺多聚糖链。恶作剧链条是 透明质酸和蛋白多糖也是共价连接的。为了 了解Jekyll和心脏ofk在调节瓣膜形成中的作用，我们 提出以下具体目标：1)进一步分析其瓣膜表型 通过组织学分析、基因和蛋白质表达研究以及细胞 移植实验；2)利用在我们的 Lab，在包括心内膜在内的所有内皮细胞中表达GFP，以 实时检测野生型和突变型胚胎的瓣膜形成； 3)对心脏恐惧进行遗传作图，并通过定位来启动其分离 克隆方法。我们还将进行大规模的二倍体遗传筛查 胚胎以确定影响心脏瓣膜形成的其他突变。我们 将开始分析定义新基因座的突变，以便剖析 这些基因在心脏瓣膜形成中发挥作用。 这些心脏瓣膜形成的分子遗传学研究应该会导致 确定这一过程的关键调节器以及详细的 了解它们的功能。这一信息应该会增强我们的 理解并有能力诊断并可能纠正阀门和 人类的隔膜缺陷。

DESCRIPTION (provided by applicant): The long-term goal of this project is to understand mechanisms of cardiac cell differentiation. The specific goals of this proposal are to identify genes important for cardiac valve formation in zebrafish and analyze their function. Cardiac valves are formed at several places along the vertebrate heart tube including the atrioventricular (AV) boundary which has been the focus of our studies. During the process of AV valve formation, myocardial cells at the AV boundary induce a subset of the underlying endocardial cells to undergo an epithelial to mesenchymal transition (EMT) and form an endocardial cushion. This cushion is later remodeled into a valve. Studies in chick and mouse embryos have led to the identification and analysis of several molecules important for distinct steps in valve formation but clearly additional molecules critical for this process remain to be identified. Previous genetic screens in zebrafish have led to the identification of two mutations that affect valve formation, jekyll and cardiofunk. We have recently isolated the Jekyll gene through a positional cloning approach and found that it encodes UDP-glucose dehydrogenase, an enzyme that primes sugars to be added into glycosaminoglycan (GAG) chains. GAG chains are the building blocks of Hyaluronic acid and are also covalently linked to proteoglycans. In order to understand the role of jekyll and cardiofunk in regulating valve formation, we propose the following specific aims: 1) further analyze their valve phenotype through histological analyses, gene and protein expression studies, and cell transplantation experiments; 2) utilize a transgenic line, generated in our lab, that expresses GFP in all endothelial, including endocardial, cells to examine valve formation in real time both in wild-type and in mutant embryos; and 3) map cardiofunk genetically and initiate its isolation by a positional cloning approach. We will also conduct large-scale genetic screens in diploid embryos to identify additional mutations affecting cardiac valve formation. We will begin to analyze the mutations defining new loci in order to dissect the role these genes play in cardiac valve formation. These molecular genetic studies of cardiac valve formation should lead to the identification of critical regulators of this process as well as to a detailed understanding of their function. This information should enhance our understanding and ability to diagnose, and possibly correct, valve and septation defects in humans.