The Role of NDRG4 in Myocardial Development

描述（由申请人提供）：最近，我们从人类、小鼠和斑马鱼中克隆并鉴定了NDRG家族（N-myc下游调节基因）的新成员NDRG4，并记录了这种系统发育上独特的NDRG家族成员仅在鱼类和小鼠的心脏和大脑中表达。NDRG4主要在胚胎和成人心肌中表达，尽管在发育中的大脑中检测到NDRG4蛋白的多种异构体，但在发育中的心脏中仅检测到一种异构体（NDRG4- s）。斑马鱼的Morpholino基因敲除实验导致胚胎心肌急剧变薄，心肌细胞数量减少，心肌环异常和心力衰竭。此外，低形Ndrg4等位基因杂合的小鼠在出生后不久就表现出明显的体细胞和心肌生长迟缓。我们假设NDRG4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ndrg4在斑马鱼体内早期心脏发育中的作用。将在ndrg4的5'-非翻译区设计反义morpholino寡核苷酸并注射到斑马鱼胚胎中。此外，反向遗传TILLING筛选将用于获得Ndrg4的错义和无义突变。这些方法将用于评估心环和心室形成的改变，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)阐明Ndrg4在体外小鼠心脏发育中的作用。Ndrg4零突变纯合子小鼠胚胎干细胞（Ndrg4 /）将用于确定Ndrg4在增殖、细胞周期控制和心肌细胞序列基因表达中的作用，在胚胎体模型中，利用肌细胞群体的免疫荧光分选、qRT-PCR和FACs分析周期调节。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置于适当的蛋白质相互作用网络中进行系统级分析。3)在体内确定Ndrg4在产前和产后心脏发育中的作用。条件loxP Ndrg4等位基因纯合的动物将与大鼠肌钙蛋白T启动子和强力霉素诱导的cTnT-nrtTA小鼠系控制下Cre重组酶的心肌特异性表达结合使用，以确定Ndrg4在发育心肌和成年小鼠心脏中的组织和时间特异性缺失的影响。通过对Ndrg4和Ndrg2等位基因复合杂合和纯合零突变的胚胎进行评估，分析Ndrg4和Ndrg2之间的互作作用。公共卫生相关性：我们的初步数据显示，NDRG4是NDRG家族在系统发育上不同的成员，几乎只在斑马鱼和小鼠的心脏和大脑中表达。斑马鱼的Morpholino敲除实验导致胚胎心肌急剧变薄，心肌细胞数量减少，环异常和心力衰竭。因此，这个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.