MECHANISMS OF HEART INDUCTION AND MYOFIBRILLOGENESIS

心脏发育将在正常和心脏突变的Axolotls和 在正常和心肌病仓鼠中。我们的目标是澄清 肌原纤维形成的事件和机制(S) 确定胚胎诱导相互作用调节和 在细胞和分子水平上指导心脏分化。 发育中的心脏细胞将通过免疫组织化学方法进行检查 确定各种不同的 收缩/细胞骨架蛋白。深度冷冻蚀刻技术在中国的应用 与免疫电子显微镜的结合将允许三维 肌原纤维形成过程中蛋白质分布的分析。 双向凝胶电泳研究和Western blotts将有助于 确定病毒的早期外观和相对浓度 分化细胞中的蛋白质。“新”收缩/细胞骨架 将通过制备特定的多克隆和单克隆来探索蛋白质 用二维凝胶检测未知蛋白抗体 电泳法。微量注射荧光标记的特异性蛋白 将被用来检测它们的掺入 进入生物的肌原纤维和/或细胞骨架元素 分化细胞。此外，针对各种病毒的抗体 蛋白质将被注入培养的细胞中，以帮助确定 特定的蛋白质在肌原纤维组织中发挥作用。心脏感应 调节正常肌原纤维形成的过程将在 细胞和分子水平。为了理解这一点，Aaxolotls中的c基因将 被用作研究中的“工具”。由于来自正常组织的可扩散的RNA 前内胚层通过以下方式纠正心脏变异心肌细胞的缺陷 促进肌原纤维形成，将进行各种生化实验 执行以确定这种归纳关系的机制(S) 在细胞和分子水平上。这两种独特基因的使用 突变应该提供必要的生物学“工具”来决定 正常肌原纤维形成和肌原纤维形成的事件顺序和机制(S) 心脏感应。通过了解基本机制(S)的心路历程 细胞形成正常的功能单位，各种疾病会改变 这些过程是可以理解的，或许可以颠倒过来。在更广泛的范围内 从某种意义上说，提出的研究可能有助于回答一大悬而未决的问题 现代生物学中的问题：基因表达的控制 发展。

Heart development will be studied in normal and cardiac mutant axolotls and in normal and cardiomyopathic hamsters. Our objectives are to elucidate the sequence of events and mechanism(s) of myofibrillogenesis and to determine the roles of embryonic inductive interactions regulating and directing heart differentiation at the cellular and molecular levels. Developing heart cells will be examined by immunohistochemical methods to establish the appearance and precise locations of the various contractile/cytoskeletal proteins. Deep freeze etching methods in combination with immunoelectron microscopy will permit three-dimensional analyses of the protein distributions during myofibril formation. Two-dimensional gel electrophoresis studies and Western blots will aid in determining the early appearance and relative concentrations of the proteins in the differentiating cells. "New" contractile/cytoskeletal proteins will be probed by preparing specific polyclonal and monoclonal antibodies against unknown proteins detected by two-dimensional gel electrophoresis. Micro-injection of fluorescently-tagged specific proteins into cultured heart cells will be employed to examine their incorporation into the myofibrils and/or cytoskeletal elements of the living differentiating cells. In addition, antibodies against the various proteins will be injected into cultured cells to help determine roles that specific proteins play in myofibril organization. Heart inductive processes which regulate normal myofibrillogenesis will be studied at the cellular and molecular levels. To accopmlish this, gene c in axolotls will be employed as a "tool" in the studies. Since a diffusible RNA from normal anterior endoderm corrects the defect in cardiac mutant heart cells by promoting myofibrillogenesis, a variety of biochemical experiments will be performed to determine the mechanism(s) of this inductive relationship at the cellular and molecular levels. The use o these two unique genetic mutations should provide the biological "tools" necessary for determining the sequence of events and mechanism(s) of normal myofibrillogenesis and heart induction. By understanding the basic mechanism(s) of how heart cells form into normal functional units, the various diseases which alter these processes can be understood, and, perhaps, reversed. In a broader sense, the proposed research may help to answer one of the major unsolved problems in modern biology: the control of gene expression during development.