CELLULAR AND MOLECULAR MECHANISMS OF HEART DEVELOPMENT

Axolotls(火蜥蜴)中的隐性心脏突变基因c提供了一种 这是研究心脏诱导分子生物学的极佳模型。 当纯合子时，该基因会导致原肌球蛋白减少，即缺失 肌原纤维，以及心肌不能启动 宫缩。该基因似乎通过异常诱导发挥作用。 前内胚层的突起，因为变异的心脏可以被“拯救” 通过在正常内胚层存在的情况下进行器官培养，已知的有效心脏 脊椎动物的肌肉感受器组织。此外，已经确定了 从正常前内胚层获得的RNA组分的添加 或从培养液中“调理”出正常内胚层才能纠正突变。 体外心脏；这些被拯救的突变心脏具有正常量的 原肌球蛋白结合到正常收缩的肌原纤维中。这个 目前的研究旨在阐明细胞和细胞因子的序列 指导正常肌纤维形成的分子事件和机制(S) 识别、表征和确定以下诱导因素的作用 调节心肌细胞分化。具体目标如下：(1) 我们将提纯和鉴定正常胚胎产生的RNA 前内胚层将静止的变异心脏变成充满活力的- 收缩“正常”器官。我们的假设是正常的前房 Axolotl胚胎的内胚层产生一种可扩散的RNA，它促进 (诱导)心脏分化；(2)编码活跃分子的基因 心脏诱导RNA将被克隆和测序。这将有助于我们测试 我们的假设是这个单一的基因突变改变了诱导性 突变型轴突对前内胚层功能的影响 产生可扩散的RNA；(3)原肌球蛋白，其表达为 显然是受心脏致命突变的影响，将在 用Northern印迹原位研究正常、突变和被挽救的突变心脏 杂交和体外翻译实验。这项研究将 提供了关于归纳的机制的重要新信息(S) 负责正常肌细胞分化的相互作用。基因 突变的axolotl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，即正常情况下收缩突变人的心脏。因此， 拟议的研究应提供对 心肌诱导和正常肌原纤维形成的调节 基因水平。理解能够将一种 “非肌肉”细胞收缩成肌肉可能是巨大的；如果这可能 适用于人类，即心肌组织受损的人 由于心肌梗死可能会使组织重新分化 再次转化为功能性肌肉。在更广泛的生物学意义上，这 脊椎动物的“先天缺陷”有可能为 与之相关的现代生物学和医学中尚未解决的重大问题 胚胎发育过程中基因表达的调控。

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.