MOLECULAR/CELLULAR BIOLOGY OF CARDIOMYOCYTE DEVELOPMENT

这个项目针对的是两个相互关联的问题，这些问题是早期 脊椎动物胚胎心脏发育：1.细胞的鉴定 调控细胞分化的谱系和发育信号 雏鸡原肠前期和原肠胚期的心源性前体细胞 胚胎发生。2.最早的横纹肌原纤维的组装 雏鸡心脏形成区可识别的肌细胞 胚胎(4-10期)。谱系分析将利用一种新的复制- 缺陷型逆转录病毒载体(CXL)，其含有β-半乳糖作为报告基因 吉恩。来源于脾坏死病毒，它表现出高水平的 鸡的感染性和几乎所有细胞中β-半乳糖的表达 胚胎在不改变正常发育的情况下。在感染4和4级后 5发育后，将胚胎固定在不同的年龄，染色 用于计算机辅助三维图像的X-GAL和连续切片 分析。在其他实验中，我们将引入myoDl等基因。 肌生成素、SKI、myc或ras进入心脏前体改变 心肌细胞的分化。肌原纤维形成的研究将 重点研究两组实验：a)肌膜下粘连的作用 瘟疫(唾液)在肌节形成中的作用，以及b) 肌球蛋白相关蛋白(C蛋白、86kD蛋白和肌动蛋白)在两者中的表达 与I-Z-I复合体的A带对齐和连锁。第一个项目将 用反射法和DIC法分析培养心肌细胞中的SAP 用显微镜记录和测量锚定的凹槽之间的距离 当附着的肌原纤维伸长到质膜上时 肌节插入。此外，心肌细胞将被显微注射荧光 抗α-肌动蛋白、肌动蛋白、纽蛋白或结蛋白的抗体，或与质粒结合的抗体 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的荧光下可视化 显微镜下观察肌原纤维组装情况。最后一组实验 涉及编码心脏C蛋白亚型和86kD的cDNA的分析 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型纤维连接蛋白和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，表达蛋白质或选定的结构域 E.Coli，鉴定与肌球蛋白或肌动蛋白相互作用的结合域，以及 为细胞内心肌细胞表达研究准备突变的cDNA。这些 研究将有助于我们理解人类心脏畸形 在早期发育过程中。

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.