CARDIAC SPECIFIC HOMEOBOX GENES IN ZEBRAFISH DEVELOPMENT

斑马鱼发育中的心脏特异性同源框基因

• 批准号: 2445027
• 负责人: Kyu-Ho Lee
• 金额: $ 7.39万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-07-12 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2445027
• 关键词: cell differentiation; complementary DNA; developmental genetics; embryo /fetus culture; embryogenesis; gene expression; genetic library; genetic regulation; heart cell; histogenesis; homeobox genes; immunocytochemistry; in situ hybridization; laboratory rabbit; messenger RNA; molecular cloning; nucleic acid sequence; phenotype; ribozymes; transcription factor; zebrafish

This project is directed at understanding the role of the homeobox transcription factor Nkx2.5 in the regulation of vertebrate cardiac development. There is compelling evidence that Nkx2.5-like genes are mediators of commitment and differentiation in the cardiac cell lineage, and of cardiac morphogenesis. This investigation is broadly based on the hypothesis that normal cardiac ontogeny depends on the timely expression of appropriate Nkx2.5 activity, and that perturbation of that activity will result in abnormal cardiac phenotypes. The proposal is organized around three experimental Aims, each designed to answer a specific set of questions about the biology of Nkx2.5 as it pertains to cardiac development. Aim l is to define the gene and cDNA sequences of the zebrafish Nkx2.5 ortholog, zNkx2.5. How many NK and homeodomain genes are present in zebrafish, and which of these is the true Nkx2.5 ortholog? Which sequences can be used for the construction of specific probes for the true ortholog? Aim 2 is to determine the pattern of expression of zNkx2.5 in zebrafish. How early and in what cardiac precursors is zNkx2.5 expressed? How does the pattern of expression of zNkx2.5 vary with time? Are there temporal or spatial differences between mRNA and protein expression for zNkx2.5? How does timing of expression compare to that of other cardiac determining gene products? Aim 3 is to determine the dependence of cardiac development and overall body plan on appropriate zNkx2.5 expression in zebrafish by perturbing mRNA levels via direct injection of zNkx2.5 mRNA or anti-zNkx2.5 mRNA ribozyme. What is the aim of function phenotype? What is the loss of function phenotype? To what extent can we alter the timing and location of cardiac precursor determination? This research applies molecular strategies to the analysis of vertebrate cardiac development. It is being pursued in zebrafish because a variety of critical features makes this experimental animal exceptionally well- suited to this purpose. The applicant expects to gain some expertise in basic vertebrate embryology, and to learn specific techniques of embryo manipulation including collection, manipulation, microinjection of RNA or protein agents, and in situ assay for gene product expression.

该项目旨在了解同型人的作用 转录因子NKX2.5在脊椎动物心脏调节中 发展。有令人信服的证据表明NKX2.5样基因是 心脏细胞谱系中承诺和分化的介体， 和心脏形态发生。这项调查广泛基于 正常心脏个体发育的假设取决于及时的表达 适当的NKX2.5活动，该活动的扰动 将导致心脏表型异常。 该提案围绕三个实验目的组织，每个目标旨在 回答有关NKX2.5生物学的一组特定问题 与心脏发展有关。目的L是定义基因和cDNA 斑马鱼NKX2.5直系同源物的序列，Znkx2.5。多少个NK和 斑马鱼中存在同源域基因，其中哪一个是真实的 NKX2.5直系同源物？哪些序列可用于构建 真正的直系同源物的特定探针？目标2是确定模式 斑马鱼中Znkx2.5的表达。多早期和哪个心脏 前体是否表达Znkx2.5？表达方式如何 Znkx2.5随时间而变化吗？之间存在时间或空间差异 Znkx2.5的mRNA和蛋白表达？表达时间如何 与其他确定基因产物的心脏确定的相比？目标3是 确定心脏发展和整体身体计划对 通过通过扰动mRNA水平通过 直接注射Znkx2.5 mRNA或抗Znkx2.5 mRNA核酶。是什么 功能表型的目的？功能表型的损失是什么？到 我们可以在多大程度上改变心脏前体的时间和位置 决心？ 这项研究将分子策略应用于脊椎动物的分析 心脏发展。 它是在斑马鱼中追求的，因为 关键特征使这种实验动物异常良好 适合此目的。申请人希望获得一些专业知识 基本的脊椎动物胚胎学，并学习胚胎的特定技术 操纵包括收集，操纵，RNA的显微注射或 蛋白质药物，以及用于基因产物表达的原位测定。