CELLULAR AND MOLECULAR MECHANISMS OF HEART DEVELOPMENT

心脏发育的细胞和分子机制

• 批准号: 3353618
• 负责人: LARRY F LEMANSKI
• 金额: $ 15.93万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-09-30 至 1995-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3353618
• 关键词: RNA; Urodela; cell differentiation; complementary DNA; cytoskeleton; endoderm; freeze etching; gel electrophoresis; gene expression; gene induction /repression; genetic manipulation; hamsters; heart cell; histochemistry /cytochemistry; histogenesis; immunochemistry; immunoelectron microscopy; immunofluorescence technique; laboratory mouse; laboratory rabbit; mammalian embryology; microinjections; monoclonal antibody; muscle proteins; mutant; myocardium disorder; myofibrils; myogenesis; nonmammalian vertebrate embryology; tissue /cell culture

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.

Axolotls（salamanders）中隐性心脏突变基因C提供 研究心脏诱导分子生物学的优秀模型。 当纯合子时，该基因会导致肌动蛋白的减少 肌原纤维，心肌失败 收缩。 该基因似乎通过异常诱导作用 由于突变心可以“救出”，因此内胚层的过程 通过在正常内胚层的存在下进行器官培养，这是一种已知有效的心脏 脊椎动物中的肌肉电感器组织。 此外，它已被确定 从正常前内胚层获得的RNA分数的添加 或通过正常内胚层从培养基“调节”可以纠正突变体 体外心；这些被救出的突变心有正常的数量 肌球蛋白掺入正常收缩的肌原纤维中。 这 目前的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，表征和确定归纳因素的作用 调节心肌分化。 具体目的如下：（1） 我们将纯化并表征正常胚胎产生的RNA 前内胚层将静态突变心变成剧烈的内胚层 - 收缩“正常”器官。 我们的假设是正常的前方 Axolotl胚胎中的内胚层产生可扩散的RNA，可促进 （诱导）心脏的区别； （2）主动编码的基因 诱导RNA的心脏将被克隆并测序。 这将帮助我们测试 我们的假说，即这种单基因突变改变了归纳 突变轴突中前内胚层的能力，通过影响 产生扩散的RNA； （3）tropomyosin，其表达是 显然将通过心脏致死突变调节，在 正常，突变和被救出的北印迹研究的救助心脏。 杂交和体外翻译实验。 这项研究会 提供有关归纳机制的重要新信息 造成正常心肌分化的相互作用。 遗传 突变体axolotl系统的异常可以用作重要工具 在这些研究中，由于有一个明确定义的生物测定终点 各种实验，即通常会收缩突变心。 因此， 拟议的研究应提供有关该研究的重大见解 调节心肌诱导和正常的肌原纤维化。 基因水平。 理解能够转变的健康相关性 进入收缩肌肉的“非肌肉”细胞可能是巨大的。如果可以的话 被应用于人类，心脏肌肉中损坏组织的人 由于心肌梗塞可能能够使组织重新分化 再次进入功能性肌肉。 从更广泛的生物学意义上讲，这 脊椎动物“先天缺陷”可能能够为 现代生物学和医学中的主要未解决问题与 控制胚胎发育过程中基因表达。