CELLULAR AND MOLECULAR MECHANISMS OF HEART DEVELOPMENT

蝾螈（蝾螈）中的隐性心脏突变基因c提供了一种 这是研究心脏诱导的分子生物学的极好模型。 纯合子时，该基因导致原肌球蛋白减少， 以及心肌无法启动 宫缩 该基因似乎通过异常诱导 因为突变的心脏可以被“拯救”， 通过在正常内胚层存在下进行器官培养， 脊椎动物的肌肉感应组织。 此外，已确定 添加从正常前内胚层获得的RNA组分 或来自由正常内胚层“条件化”的培养基可以纠正突变体 心脏在体外;这些获救的突变心脏有正常数量的 并入正常收缩的肌原纤维中的原肌球蛋白。 的 本研究旨在阐明细胞和 指导正常肌原纤维发生的分子事件和机制， 识别、表征和确定诱导因素的作用， 调节肌细胞分化。 具体目标如下：（1） 我们将纯化和鉴定正常胚胎细胞产生的RNA， 前内胚层，将静止的突变心脏变成活跃的- 收缩“正常”器官。 我们假设正常的前额叶 蝾螈胚胎的内胚层产生一种可扩散的RNA， （2）心脏的分化;（3）心脏的分化。 心脏诱导RNA将被克隆和测序。 这将帮助我们测试 我们的假设是，这个单一的基因突变改变了 能力的前内胚层在突变蝾螈通过影响 产生可扩散的RNA;（3）原肌球蛋白，其表达是 明显受到心脏致死突变的调节，将在 原位北方印迹法研究正常、突变和挽救突变心脏 杂交和体外翻译实验。 这项研究将 提供有关诱导机制的重要新信息 负责正常肌细胞分化的相互作用。 遗传 突变蝾螈系统的异常可以作为一个重要的工具， 在这些研究中，由于存在明确定义的生物测定终点， 各种实验，即正常收缩的变异心脏。 因此，在本发明中， 拟议的研究应提供重要的见解， 调节心肌诱导和正常肌原纤维发生 基因水平。 了解能够将一个人转变为一个人的健康相关性， “非肌肉”细胞进入收缩的肌肉可能是巨大的;如果这可以 应用于人体，那些心肌组织受损的人， 因为心肌梗塞的病人也许能让组织重新分化 变成功能性肌肉 从更广泛的生物学意义上讲， 脊椎动物的“出生缺陷”有可能提供答案， 现代生物学和医学中未解决的主要问题 控制胚胎发育过程中的基因表达。

Recessive cardiac mutant gene c in axolotls (salamanders) provides an excellent model for studying the molecular biology of heart induction. When homozygous, the gene results in a reduction of tropomyosin, an absence of myofibrils, and a failure of the cardiac muscle to initiate contractions. The gene appears to exert its effect via abnormal inductive processes from the anterior endoderm since mutant hearts can be "rescued" by organ-culturing in the presence of normal endoderm, a known potent heart muscle inductor tissue in vertebrates. Furthermore, it has been determined that the addition of an RNA fraction obtained from normal anterior endoderm or from medium "conditioned" by the normal endoderm can correct mutant hearts in vitro; these rescued mutant hearts have normal amounts of tropomyosin incorporated into myofibrils that contract normally. The present investigation is designed to elucidate the sequence of cellular and molecular events and mechanism(s) directing normal myofibrillogenesis and to identify, characterize and determine the role of inductive factors which regulate myocyte differentiation. The specific aims are as follows: (1) we will purify and characterize the RNA produced by normal embryonic anterior endoderm that turns the quiescent mutant hearts into vigorously- contracting "normal" organs. It is our hypothesis that the normal anterior endoderm in axolotl embryos produces a diffusible RNA which promotes (induces) differentiation of the heart; (2) The gene coding for the active heart inducing RNA will be cloned and sequenced. This will help us test our hypothesis that this single gene mutation alters the inductive capability of the anterior endoderm in mutant axolotls by affecting the production of a diffusible RNA; (3) Tropomyosin, whose expression is apparently modulated by the cardiac lethal mutation, will be analyzed in normal, mutant and rescued-mutant hearts by Northern blot studies, in situ hybridization, and in vitro translation experiments. This research will provide significant new information on the mechanism(s) of inductive interactions responsible for normal myocyte differentiation. The genetic abnormalities of the mutant axolotl system can be used as an important tool in these studies since there is a clearly-defined bioassay end point for the various experiments, namely, normally contracting mutant hearts. Thus, the proposed studies should provide significant insights into the regulation of heart muscle induction and normal myofibrillogenesis at the gene level. The health relevance of understanding the being able to turn a "nonmuscle" cell into contracting muscle could be tremendous; if this could be applied in humans, people who have damaged tissue in their heart muscle due to myocardial infarcts might be able to have the tissue redifferentiate into functional muscle again. In a broader biological sense, this vertebrate "birth defect" is potentially capable of providing answers to major unsolved problems in modern biology and medicine related to the control of gene expression during embryonic development.