ROLE OF INTEGRINS IN MAMMALIAN HEART DEVELOPMENT

心脏畸形的发生率为每1，000人中有7至10例 出生使先天性心脏病成为最常见的出生形式 缺损 尽管在诊断和手术方面取得了令人印象深刻的进展， 治疗，目前还没有了解的分子基础， 这些畸形。 许多无疑是细胞错误的结果 迁移或细胞间相互作用发生在早期心脏 发展 一个被称为整合素的细胞表面受体家族 已经显示出介导细胞相互作用， 与细胞外基质结合 干扰这些功能 受体导致细胞在早期 形态发生 我们假设整合素是重要的决定因素 早期心脏形态发生的证据 我们进一步假设它们是 在心脏发育过程中以编程方式表达， 整联蛋白组在不同的时间和位置表达 在心脏形态发生过程中，形态发生至少 部分地不仅依赖于整合素的表达， 在正确的时间和地点表达正确的整合素。 我们将 用小鼠胚胎来检验这些假设。 为了实现这一目标，我们 将首先开发cDNA探针、多克隆抗体和单克隆抗体。 对主要整联蛋白的α亚单位特异的抗体 在小鼠胚胎中表达。 这将通过组装 来自小鼠文库的完整的小鼠cDNA构建体， 杆状病毒系统和使用纯化的蛋白质作为免疫原， 抗体生产。 这种试剂在大多数情况下并不存在， 小鼠整联蛋白。 其次，我们将监测整合素的程序 在小鼠发育早期表达1）通过逆转录酶- 内源性整合素mRNA的PCR扩增。 原位杂交 将用于检测放大消息的蜂窝来源;以及 2)通过使用单克隆多克隆抗体的免疫组织化学 针对小鼠整联蛋白α亚基制备。 三是 确定整合素表达的功能意义， 通过1）破坏早期的 用生物活性抗整联蛋白抗体进行形态发生; 2） 在早期心肌或内皮细胞中阻断整合素合成 使用携带特异于 小鼠整联蛋白α亚基;和3）干扰正常细胞 通过诱导不适当的整合素表达的相互作用 携带完整整联蛋白α亚基cDNA的缺陷型逆转录病毒。 这项工作完成后，我们将准确地确定 整合素对心脏发育各个阶段的重要性。 作为 这些实验将进行组织学评估，我们将知道 整合素在心肌，内皮细胞， 原始心管和早期房室瓣。 我们将测试 这一假说认为，在细胞中存在一个精确的整合素表达程序， 发育中的心脏，并确定在什么时候， 建立整联蛋白表达。 最后，我们将确定 整合素表达和整合素程序的功能重要性 哺乳动物心脏发育过程中整合素的表达。

Malformations of the heart occur at the rate of 7 to 10 cases/1,000 live births making congenital heart disease the most frequent form of birth defect. Despite impressive advances in diagnosis and surgical treatment, there is as yet no understanding of the molecular basis of these malformations. Many are undoubtedly the result of errors in cell migration or cell-cell interactions that occur during early heart development. A family of cell surface receptors known as the integrins has been shown to mediate cellular interactions with one another and with the extracellular matrix. Interference with the function of these receptors results in aberrant behavior of cells during early morphogenesis. We hypothesize that integrins are important determinants of early heart morphogenesis. We further postulate that they are programmatically expressed during heart development such that different groups of integrins are expressed at different times and locations during cardiac morphogenesis and that morphogenesis is at least partially dependent not only on integrin expression, but upon the expression of the right integrin in the right place and time. We will test these hypotheses using the mouse embryo. To accomplish this we will first develop cDNA probes, polyclonal antibodies and monoclonal antibodies specific for the alpha subunits of the major integrins expressed in the mouse embryo. This will be accomplished by assembling complete mouse cDNA constructs from mouse libraries, expressing them in the baculovirus system and using the purified proteins as immunogens for antibody production. Such reagents do not, for the most part, exist for mouse integrins. Second, we will monitor the program of integrin expression during early mouse development 1) by reverse transcriptase- PCR amplification of endogenous integrin mRNA. In situ hybridization will be used to detect the cellular source of the amplified message; and 2) by immunohistochemistry using the monoclonal polyclonal antibodies prepared against mouse integrin alpha subunits. Third, we will determine the functional significance of integrin expression in the developing heart of cultured mouse embryos by 1) disrupting early morphogenesis with biologically active anti-integrin antibodies; 2) blocking integrin synthesis in early myocardial or endocardial cells using retrovirus vectors carrying antisense constructs specific for mouse integrin alpha subunits; and 3) perturbing normal cellular interactions by inducing inappropriate integrin expression using defective retroviruses carrying complete integrin alpha subunit cDNA's. Upon completion of this work, we will have determined precisely the importance of integrins to various stages of heart development. As these experiments will be evaluated histologically, we will know the role of integrins in the formation of the myocardium, the endocardium, primitive heart tube and early A-V valves. We will have tested the hypothesis that there is a precise program of integrin expression in the developing heart and determined at what point the adult pattern of integrin expression is established. Finally, we will have determined the functional importance of both integrin expression and the program of integrin expression during mammalian heart development.