CARDIOVASCULAR DEVELOPMENT IN XENOPUS LAEVIS

（自申请人摘要）先天性缺陷遗传基础研究的快速进展，增加了对脊椎动物发育过程中操纵特定基因表达的快速和经济有效方法的紧迫性。非洲爪蟾（Xenopus laevis）是研究得最好的脊椎动物之一，为早期心脏发育的检查提供了许多优势。利用绿色荧光蛋白（GFP）的表达来控制转录的转基因载体制作转基因青蛙胚胎的方法为研究心血管基因表达的控制提供了一个机会。这些载体将被构建为使用哺乳动物和两栖动物启动子来检查组织和时间特异性基因表达控制的保护。荧光显微镜可以在不牺牲转基因胚胎的情况下监测转基因胚胎的GFP表达。三个特定的基因，已经涉及到心血管缺陷或发展也将使用mRNA和反义寡核苷酸注射到非洲爪蟾胚胎成熟的方法进行检查。Msx-1是一个含有抑制蛋白的同源盒，与唐氏综合征相关的房室间隔缺陷（AVSD）有关。在一些唐氏症个体中，Msx-1水平与其调节的基因之间的重要平衡被破坏的假设将通过故意修改爪蟾胚胎中Msx-1同源物的水平来验证。Betaglycan， tgf - β -2结合蛋白在1号染色体的一个小区域中被发现，与一些非唐氏综合征相关的AVSD病例有关。研究人员将验证一种假设，即心脏中特定的突变或β -多糖水平的改变会导致瓣膜缺陷。最后，研究人员将研究血管内皮生长因子（VEGF）的控制。VEGF是血管组织建立和维持的重要信号分子。VEGF mRNA在青蛙卵母细胞中的存在，加上卵母细胞操作的便便性，为单细胞检测VEGF控制提供了机会。非洲爪蟾胚胎的自由生活和非常明显的发育将为研究不同VEGF亚型的作用和VEGF在心血管发育过程中的激活提供一种手段。研究人员还将研究缺氧对VEGF表达的影响，以开始开发一种方法来测定环境条件如何在没有特定突变的情况下导致缺陷。

(Adapted from the Applicant's Abstract) The rapid progress in studies that identify the genetic basis for congenital defects has increased the urgency for rapid and cost effective ways to manipulate specific gene expression during vertebrate development. One of the best studied vertebrates, Xenopus laevis offers many advantages for the examination of early cardiac development. Methods to make transgenic frog embryos provide an opportunity to look specifically at the control of cardiovascular gene expression using transgenic vectors that report the control of transcription by expression of green fluorescent protein (GFP). These vectors will be constructed to use either mammalian and amphibian promoters to examine conservation of control of tissue and temporal specific gene expression. GFP expression transgenic embryos can be monitored by fluorescence microscopy without sacrificing the transgenic embryo. Three specific genes, already implicated in cardiovascular defects or development will also be examined using the well established method of mRNA and antisense oligonucleotide injection into Xenopus embryos. Msx-1, a homeobox containing repressor protein, is implicated in Down's Syndrome related atrial-ventricular septal defects (AVSD). The hypothesis that the important balance of Msx-1 levels with the genes it regulates is disrupted in some Down's individuals will be tested by deliberate modification of the levels of the Msx-1 homologue in Xenopus embryos. Betaglycan, the TGFbeta-2 binding protein is found in a small region of chromosome 1 associated with some cases of non-Down's syndrome related AVSD. The investigators will test the hypothesis that specific mutations or altered levels of betaglycan in the heart lead to valvular defects. Finally, the investigators will investigate the control of vascular endothelial growth factor (VEGF). VEGF is an essential signaling molecule for the establishment and maintenance of vascular tissue. The presence of VEGF mRNA in frog oocytes, coupled with the ease of oocyte manipulation provides an opportunity to examine VEGF control in single cell assays. The free living, very visible development of the Xenopus embryo will provide a means to examine the roles of different VEGF isoforms and the activation of VEGF during cardiovascular development. The investigators will also examine the effect of hypoxia on VEGF expression to begin to develop a way to assay how environmental conditions may lead to defects ev en when no specific mutation is present.