MOLECULAR/CELLULAR BIOLOGY OF CARDIOMYOCYTE DEVELOPMENT

这个项目针对的是两个相互关联的问题， 脊椎动物胚胎的心脏发育：1.细胞鉴定 谱系和发育信号，调节分化， 鸡原肠胚前和原肠胚期的心源性前体细胞 胚胎发生2.最早期横纹肌原纤维的组装 在鸡心脏形成区可识别的肌细胞 胚胎（阶段4-10）。血统分析将使用一个新的复制品- 缺陷型逆转录病毒载体（CXL），其中含有β-gal作为报告基因 基因它来源于脾坏死病毒， 鸡的几乎所有细胞中的感染性和β-gal表达 胚胎不改变正常发育。在第4阶段感染后， 5的发展，胚胎将固定在不同的年龄，染色， 计算机辅助三维图像用的X线半乳糖苷酶和连续切片 分析.在其他实验中，我们将引入基因，如myoDl。 myogenin、ski、myc或ras进入心脏前体，以改变 心肌细胞的分化。肌原纤维发生的研究将 重点放在两组实验：a）肌膜下粘附的作用 鼠疫（SAP）在肌节形成中的作用，和B） 肌球蛋白相关蛋白（C蛋白，86 kD蛋白和肌联蛋白）， A带对齐和与I-Z-I复合物的连接。第一个项目将 包括通过反射率和DIC分析培养心肌细胞中SAP 显微镜以记录和测量锚的SAP之间的距离 当附着的肌原纤维伸长时， 肌节插入此外，将用荧光显微镜显微注射肌细胞。 α-辅肌动蛋白、肌联蛋白、黏着斑蛋白或结蛋白的抗体，或质粒 编码这些蛋白质的截短形式以改变体内功能。 活细胞将在视频增强荧光下可视化 显微镜下观察肌原纤维组装。最后一组实验 包括分析编码C-蛋白和86 kD心脏同种型的cDNA 蛋白质，其骨骼肌形式已被克隆和测序 在我们的实验室里。这些蛋白质含有III型纤连蛋白和C2 免疫球蛋白具有N-CAM家族的特征。我们将隔离 并对心脏cDNA进行测序，表达蛋白质或选择的结构域， E.鉴定与肌球蛋白或肌联蛋白相互作用的结合结构域， 制备用于细胞内肌细胞表达研究的突变cDNA。这些 研究将有助于我们了解人类心脏畸形 在早期发展中。

This project is directed at two interrelated problems central to early cardiac development in vertebrate embryos: 1. The identification of cell lineages and developmental signals which regulate differentiation of cardiogenic precursor cells at pre-gastrula and gastrula stages of chick embryogenesis. 2. The assembly of striated myofibrils in the earliest myocytes that can be identified in the heart-forming region of chick embryos (stages 4-10). Lineage analysis will utilize a new replication- defective, retroviral vector (CXL), which contains beta-gal as a reporter gene. Derived from the spleen necrosis virus, it exhibits high levels of infectivity and beta-gal expression in virtually all cells of the chick embryo without altering normal development. After infection at stages 4 and 5 of development, embryos will be fixed at different ages, stained with X-gal, and serial sections used for computer-assisted, 3-dimensional image analysis. In other experiments, we will introduce genes such as myoDl. myogenin, ski, myc or ras into cardiac precursors to alter the differentiation of cardiac myocytes. The studies of myofibrillogenesis will focus on two sets of experiments: a) the role of sub-sarcolemmal adhesion plagues (SAPs) in sarcomere formation, and b) the roles of myosin-associated proteins (C-protein, 86 kD protein and titin) in both A-band alignment and linkage with I-Z-I complexes. The first project will involve analysis of SAPs in cultured myocytes by reflectance and DIC microscopy to record and measure the distance between SAPs which anchor myofibrils to the plasmalemma as the attached myofibrils elongate by sarcomere insertion. Also, myocytes will be microinjected with fluorescent antibodies to alpha-actinin, titin, vinculin or desmin, or with plasmids encoding truncated forms of these proteins to alter in vivo functions. Living cells will be visualized under video-enhanced fluorescence microscopy to follow myofibril assembly. The last set of experiments involve analysis of cDNAs encoding cardiac isoforms of C-protein and 86 kD protein, the skeletal muscle forms of which have been cloned and sequenced in our laboratory. These proteins contain type III fibronectin and C2 immunoglobulin features characteristic of the N-CAM family. We will isolate and sequence the cardiac cDNAs, express the proteins or selected domains in E. Coli, identify binding domains which interact with myosin or titin, and prepare mutant cDNAs for intracellular myocyte expression studies. These studies will contribute to our understanding of human cardiac malformations during early development.