INDUCTION OF EARLY EMBRYONIC HEART DEVELOPMENT

这项研究旨在识别和描述已知和未知的增长 调节胚胎心脏发育的因素。 结果 这项工作将有助于了解的机制， 先天性心脏病，并将建议策略，诱导 成年人受损的成熟心肌细胞的再生。 我们有 已经确定了两个已知的生长因子家族--激活素 和成纤维细胞生长因子（FGF）--可能在 心源性过程 我们已经证明，激活素大约包括 40%的胚胎内胚层分泌产物，细胞中， 诱导心脏发生，且激活素单独能够实现 在限定培养基中的心脏发生。 并且，使用同种型特异性抗体， 我们已经表明FGF家族成员aFGF、FGFK、FGF 5和bFGF是 集中在最早的心肌细胞中。 我们还表明， 寡脱氧核苷酸（ODN）介导的bFGF缺失抑制心脏 生长发育，bFGF，像激活素，足以支持心脏 文化的发展。 在更新期间，这些调查结果将 通过确定这些的单独和组合功能来扩展 心脏发育过程中的因素。 物种同源探针 激活素和FGF的同种型将从cDNA表达中分离 文库并测序。 激活素和FGF mRNA的量和位置 和蛋白质同种型将使用mRNA杂交进行评估， 免疫化学分析。 每个因素的作用将直接 通过在心脏细胞中添加/删除这些蛋白质进行测试， 在培养物和胚胎中的发育。 将执行删除操作 通过（i）反义ODN介导的mRNA敲除和（ii）抗体 中和蛋白质。 FGF诱导心脏发生的假说 通过上调血清反应因子（SRF）蛋白， 肌节肌动蛋白基因的表达。 心肌细胞 激活素和FGF的膜受体将通过PCR表征 （聚合酶链反应）扩增和DNA测序; 也将确定受体缺失对心脏发生的影响。 最后， 这项研究的另一个主要目标是识别和表征 新的，未知的心源性诱导分子， 内胚层 一种未知的、主要的25 kD内胚层分泌的抗体 我们已经鉴定的蛋白质，以及次要的内胚层蛋白质， 将制备用于文库筛选、免疫组织化学作图和 心脏发生过程中的功能分析。

This research seeks to identify and characterize known and unknown growth factors that regulate development of the embryonic heart. Results from this work will contribute to an understanding of the mechanisms of congenital heart disease and will suggest strategies for inducing the regeneration of injured mature cardiac myocytes in the adult. We have already determined that two known growth factor families -- the activins and the fibroblast growth factors (FGFs) -- may have a major role in the cardiogenic process. We have shown that activin comprises approximately 40% of the secretory product of embryonic endoderm, cells in which may induce cardiogenesis, and that activin alone is able to effect cardiogenesis in defined medium. And, using isoform-specific antibodies, we have shown that FGF family members aFGF, FGFK, FGF5 and bFGF are concentrated in the earliest myocardial cells. We have also shown that oligodeoxynucleotide (ODN)-mediated deletion of the bFGF inhibits heart development, and that bFGF, like activin, is sufficient to support heart development in culture. During the renewal years, these findings will be extended by determining the individual and combined function of these factors during heart development. species' homologous probes for isoforms of activin and FGF will be isolated from a cDNA expression library and sequenced. The amount and location of activin and FGF mRNA and protein isoforms will be assessed using mRNA hybridization and immunochemical analyses. The function of each factor will be directly tested by adding/deleting these proteins to/from heart cells during development in culture, and in the embryo. Deletions will be performed by (i) antisense ODN-mediated knockout of mRNA and (ii) antibody neutralization of protein. The hypothesis that FGF induces cardiogenesis by up-regulating serum response factor (SRF) protein, which causes expression of sarcomeric actin genes, will be tested. Myocardial cell membrane receptors for activin and FGF will be characterized by PCR (polymerase chain reaction) amplification and DNA sequencing; the effects of receptor deletion on cardiogenesis will also be determined. Finally, another major goal of this research is to identify and characterize novel, unknown cardiogenic inducer molecules that are secreted by endoderm. Antibodies to an unknown, major 25 kD endoderm-secreted protein that we have identified, as well as minor endodermal proteins, will be prepared for library screening, immunohistochemical mapping and functional analysis during cardiogenesis.