The Role of NDRG4 in Myocardial Development

描述(申请人提供)：最近，我们从人、小鼠和斑马鱼中克隆并鉴定了NDRG家族的一个新成员NDRG4(N-myc下游调节基因)，并证明这个在系统发育上不同的NDRG家族成员仅在鱼和小鼠的心脏和脑中表达。NDRG4主要在胚胎和成年心肌中表达，虽然在发育中的脑中检测到多种亚型的NDRG4蛋白，但在发育中的心脏中只检测到一种亚型(NDRG4-S)。在斑马鱼上进行的吗啉基因敲除实验导致心肌显著变薄，心肌细胞数量减少，胚胎出现异常环状和心力衰竭。此外，Ndrg4等位基因杂合子的小鼠在出生后不久就表现出显著的躯体和心肌生长迟缓。我们推测NDRG4在心脏形态发生过程中起着调节心肌细胞增殖的作用，并且在维持成人成熟的、分化的心肌细胞表型方面起着重要作用。因此，我们建议1)确定ndrg4在斑马鱼体内早期心脏发育中的作用。反义吗啉寡核苷酸将被设计在ndrg4的5‘非翻译区，并注射到斑马鱼胚胎中。此外，反向遗传筛选将被用于获得Ndrg4的错义和无义突变。这些方法将被用来评估心脏循环和心腔形成的变化，并确定心肌细胞增殖、凋亡和心肌细胞基因表达的正常程序是否存在扰动。2)体外研究Ndrg4在小鼠心脏发育中的作用。Ndrg4(Ndrg4/)纯合子小鼠ES细胞将被用来确定Ndrg4在心肌细胞分化的类胚体模型中的增殖、细胞周期控制和顺序心肌细胞基因表达中的作用，利用心肌细胞群体的免疫荧光分类、qRT-PCR和周期调节的FACS分析。采用LC-MS-MS的蛋白质组学策略将用于鉴定NDRG4相关蛋白质，以便将NDRG4放置在适当的蛋白质相互作用网络中进行系统水平分析。3)体内测定Ndrg4在出生前和出生后心脏发育中的作用。条件loxP Ndrg4等位基因纯合的动物将在大鼠肌钙蛋白T启动子和多西环素诱导的cTnT-nrtTA小鼠系的控制下，结合Cre重组酶的心肌特异性表达，以确定Ndrg4在发育中的心肌和成年小鼠心脏中的组织和时间特异性缺失的影响。Ndrg4和Ndrg2等位基因存在复合杂合和纯合子零突变的胚胎将通过评估来分析Ndrg4和Ndrg2之间的上位性相互作用。与公共卫生相关：我们的初步数据显示，NDRG4是NDRG家族中一个在系统发育上截然不同的成员，几乎只在斑马鱼和小鼠的心脏和大脑中表达。在斑马鱼上进行的吗啉基因敲除实验会导致斑马鱼的心肌显著变薄，心肌细胞数量减少，胚胎出现异常环状和心力衰竭。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.