CELL--ECM INTERACTIONS IN MYOCARDIAL FORM AND FUNCTION

细胞：细胞外基质(ECM)的相互作用起着关键作用 在早期的心脏形态发生中，但已知的是 来自鸟类模型。对这些相互作用的解释 哺乳动物模型是必不可少的，如果我们要开始研究 先天性心脏病的发病机制。我们建议描绘出， 在哺乳动物胚胎(小鼠和大鼠)中，分布和 已知的主要成分的发育外观 细胞外基质(I型、III型、IV型胶原、透明质酸、 层粘连蛋白、纤维连接蛋白、基底膜硫酸肝素 蛋白多糖)，因为它们与心脏前细胞早期迁移有关 心脏形态发生。A)我们将使用完整的胚胎安装在 与特异性抗体标记的连续切片相结合 ECM的每个组件以定义发育外观 以及这些分子的三维分布之间的关系 到心脏前中胚层。B)然后我们将就地利用 与相关细胞外基质基因的cDNA探针杂交 成分(I型、III型、IV型胶原、纤维连接蛋白、层粘连蛋白)定位 细胞的时间和地理分布能够 心内直视手术对心脏前细胞数量的影响 早期心脏形态发生。此外，我们还将审查 心脏前间充质的免疫组织化学标记物 将这些细胞与周围的侧板中胚层区分开来 处于迁徙和分化的渐进阶段。 利用这些细胞的独特特性进行迁移 成肌细胞，我们将使用针对成肌细胞的单抗 胚胎肌球蛋白、结蛋白中间丝和成肌细胞 特异性细胞表面表位：a)识别心脏前病变 间充质作为侧板中胚层的一个亚群，b) 定义心脏前细胞迁移的精确区域，以及c) 确定这些表位的表达顺序 心肌前细胞分化。额外的单抗 如果针对心脏前间质，将会开发出 需要，以便识别心脏的早期阶段 形态发生。最后，我们将在现场调查细胞- 细胞外基质相互作用参与心脏前细胞迁移， 哺乳动物的分化和早期收缩功能 胚胎。我们将在接下来的一段时间里培养整个胚胎 在抑制剂存在的情况下通过心环的原肠作用 正常细胞：细胞外基质相互作用。胚胎将被评估为 由此产生的结构变化和治疗的效果将 通过心肌序贯运动分析进行量化 收缩功能。这应该提供对 ECM组分对正常结构的相对贡献 和功能开发。

Cell:extracellular matrix (ECM) interactions play a critical role in early cardiac morphogenesis but what is known has been derived from avian models. Elucidation of these interactions in a mammalian model is essential if we are to begin to investigate the mechanisms of congenital heart disease. We propose to delineate, in the mammalian embryo (mouse and rat), the distribution and developmental appearance of the major known components of the extracellular matrix (collagen types I, III, IV, hyaluronic acid, laminin, fibronectin, basement membrane heparan sulfate proteoglycan) as they relate to precardiac cell migration an early cardiac morphogenesis. A) We will employ whole embryo mounts in combination with serial sections labelled with antibodies specific to each component of the ECM to define the developmental appearance and three dimensional distribution of these molecules in relation to the precardiac mesoderm. B) We will then utilize in situ hybridization with cDNA probes for the mRNA's of relevant ECM components (Collagen I,III,IV, fibronectin, laminin) to localize the temporal and geographical distribution of cells capable of influencing the precardiac cell population during the period of early cardiac morphogenesis. In addition, we will examine immunohistochemical markers for precardiac mesenchyme that will distinguish these cells from the surrounding lateral plate mesoderm at progressive stages in their migration and differentiation. Exploiting the unique character of these cells as migrating myoblasts, we will use monoclonal antibodies directed against embryonic myosins, desmin intermediate filaments, and myoblast specific cell surface epitopes to: a) identify the precardiac mesenchyme as a subpopulation of the lateral plate mesoderm, b) define the precise domain of precardiac cell migration, and c) determine the sequence in which these epitopes are expressed during precardiac cell differentiation. Additional monoclonal antibodies directed against precardiac mesenchyme will be developed, if needed, in order to recognize earlier stages of cardiac morphogenesis. Finally, we will investigate, in situ, the cell- ECM interactions involved in precardiac cell migration, differentiation, and early contractile function in the mammalian embryo. We will culture whole embryos during the period from gastrulation through cardiac looping in the presence of inhibitors of normal cell:ECM interaction. Embryos will be evaluated for resulting structural alteration and the effects of treatment will be quantified through sequential motion analysis of myocardial contractile function. This should provide evaluation of the relative contributions of the ECM components to normal structural and functional development.