The Role of NDRG4 in Myocardial Development

描述（由申请人提供）：最近，我们已经从人、小鼠和斑马鱼中克隆并表征了NDRG 4，NDRG家族（N-myc下游调节基因）的新成员，并证明了NDRG家族的这种遗传学上不同的成员仅在鱼和小鼠的心脏和脑中表达。NDRG 4主要在胚胎和成人心肌中表达，尽管在发育中的脑中检测到NDRG 4蛋白的多种亚型，但在发育中的心脏中仅检测到一种亚型（NDRG 4-S）。在斑马鱼中进行的Morpholino敲除实验导致胚胎心肌显著变薄，肌细胞数量减少，异常循环和心力衰竭。此外，小鼠杂合子的一个亚型Ndrg 4等位基因显示显着的躯体和心肌生长迟缓后不久出生。我们假设NDRG 4在心脏形态发生过程中调节心肌细胞增殖，并在维持成人成熟分化的心肌细胞表型中发挥重要作用。因此，我们建议：1）明确ndrg 4在斑马鱼体内心脏发育早期的作用。将反义吗啉代寡核苷酸设计到ndrg 4的5 '-非翻译区，并注射到斑马鱼胚胎中。此外，将使用反向遗传TILLING筛选来获得Ndrg 4中的错义和无义突变。这些方法将用于评估心脏循环和腔室形成的改变，并确定心肌细胞增殖、凋亡和肌细胞基因表达的正常程序是否存在扰动。2)描述Ndrg 4在体外对小鼠心脏发育的作用。将使用Ndrg 4无效突变纯合的小鼠ES细胞（Ndrg 4/），利用肌细胞群的免疫荧光分选、qRT-PCR和周期调节的FACs分析，确定Ndrg 4在肌细胞分化的胚状体模型中增殖、细胞周期控制和顺序心肌细胞基因表达中的作用。采用LC-MS-MS的蛋白质组学策略将用于鉴定NDRG 4相关蛋白，以便将NDRG 4置于适当的蛋白质相互作用网络中进行系统水平分析。3)确定Ndrg 4在产前和产后体内心脏发育中的作用。条件性loxP Ndrg 4等位基因纯合的动物将与在大鼠肌钙蛋白T启动子控制下的Cre重组酶的心肌特异性表达和多西环素诱导的cTnT-nrtTA小鼠系结合使用，以确定Ndrg 4的组织和时间特异性缺失在发育中的心肌和成年小鼠心脏中的作用。将通过评价在Ndrg 4和Ndrg 2等位基因中具有复合杂合和纯合无效突变的胚胎来分析Ndrg 4和Ndrg 2之间的上位相互作用。公共卫生相关性：我们的初步数据表明，NDRG 4，NDRG家族的一个在遗传学上不同的成员，几乎只在斑马鱼和小鼠的心脏和大脑中表达。在斑马鱼中进行的Morpholino敲除实验导致胚胎中心肌显著变薄、肌细胞数量减少、异常成环和心力衰竭。因此，本项目的目标是确定这种新的细胞质蛋白在心脏发育和出生后心肌生长中的作用。

DESCRIPTION (provided by applicant): Recently, we have cloned and characterized NDRG4, a novel member of the NDRG family (N-myc downstream-regulated gene), from human, mouse and zebrafish and documented that this phylogenetically distinct member of the NDRG family is expressed exclusively in the heart and brain of both fish and mouse. NDRG4 is expressed predominantly in the embryonic and adult myocardium and although multiple isoforms of NDRG4 protein were detected in developing brains, only one isoform (NDRG4-S) was detected in the developing heart. Morpholino knockdown experiments in zebra fish resulted in dramatic thinning of the myocardium, decreased myocyte number, abnormal looping and cardiac failure in the embryo. In addition, mice heterozygous for a hypomorphic Ndrg4 allele show dramatic somatic and myocardial growth retardation shortly after birth. We hypothesize that NDRG4 plays a role in regulating cardiomyocyte proliferation during cardiac morphogenesis and plays an essential role in maintenance of the mature, differentiated myocyte phenotype in the adult. We therefore propose to 1) Define the role of ndrg4 in early heart development of zebrafish in vivo. Antisense morpholino oligonucleotides will be designed to the 5'-untranslated region of ndrg4 and injected into zebrafish embryos. In addition a reverse genetic TILLING screen will be used to obtain missense and nonsense mutations in Ndrg4. These approaches will be used to evaluate alterations in cardiac looping and chamber formation and determine if there are perturbations in the normal program of cardiomyocyte proliferation, apoptosis, and myocyte gene expression. 2) Delineate the role of Ndrg4 on murine cardiac development, in vitro. Mouse ES cells homozygous for a null mutation in Ndrg4 (Ndrg4 / ) will be used to determine the role of Ndrg4 in proliferation, cell cycle control, and sequential cardiomyocyte gene expression in the embryoid body model of myocyte differentiation utilizing immunofluorescent sorting of the myocyte population, qRT-PCR and FACs analysis of cycle regulation. A proteomic strategy employing LC-MS-MS will be used to identify NDRG4 associated proteins in order to place NDRG4 within the appropriate protein interaction networks for systems level analysis. 3) Determine the role of Ndrg4 in prenatal and postnatal cardiac development, in vivo. Animals homozygous for a conditional loxP Ndrg4 allele will be used in conjunction with myocardial specific expression of Cre recombinase under control of the rat troponin T promoter and the doxycyline inducible cTnT-nrtTA mouse line to determine the effects of tissue and temporal specific deletion of Ndrg4 in developing myocardium and adult mouse heart. Epistatic interactions between Ndrg4 and Ndrg2 will be analyzed by evaluation of embryos with compound heterozygous and homozygous null mutations in the Ndrg4 and Ndrg2 alleles. PUBLIC HEALTH RELEVANCE: Our preliminary data shows that NDRG4, a phylogenically distinct member of the NDRG family is expressed almost exclusively in the heart and brain of both zebra fish and mouse. Morpholino knockdown experiments in zebra fish result in dramatic thinning of the myocardium, decreased myocyte number, abnormal looping and cardiac failure in the embryo. Therefore, the goal of this project is to determine the role of this novel cytoplasmic protein in cardiac development and postnatal myocardial growth.