CARDIOVASCULAR DEVELOPMENT IN XENOPUS LAEVIS

(改编自申请人的摘要)在确定先天性缺陷的遗传基础的研究中取得的快速进展，增加了在脊椎动物发育过程中快速且具有成本效益的方法来操纵特定基因表达的紧迫性。非洲爪哇是研究最深入的脊椎动物之一，它为早期心脏发育的检查提供了许多优势。方法利用报告绿色荧光蛋白(GFP)表达调控转录的转基因载体，使转基因青蛙胚胎有机会特异性地观察心血管基因表达的调控。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的控制的保守性。在不牺牲转基因胚胎的情况下，可以用荧光显微镜监测转基因胚胎的GFP表达。已经与心血管缺陷或发育有关的三个特定基因也将使用成熟的方法--将mRNA和反义寡核苷酸注射到非洲爪哇胚胎中--进行检测。MSX-1是一种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打破的假设，将通过故意修改非洲爪哇胚胎中MSX-1同源物的水平来检验。Betaglycan是一种转化生长因子β-2结合蛋白，位于1号染色体的一个小区域，与一些与非唐氏综合征相关的AVSD病例有关。研究人员将检验这一假设，即心脏中特定的突变或改变水平的Betaglycan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。血管内皮生长因子是血管组织建立和维持所必需的信号分子。在青蛙卵母细胞中存在血管内皮生长因子mRNA，加上卵母细胞操作的简便性，为在单细胞检测中检验血管内皮生长因子的控制提供了机会。非洲爪哇胚胎的自由生活和非常明显的发育将为研究不同的血管内皮生长因子亚型的作用和血管内皮生长因子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血管内皮生长因子表达的影响，以开始开发一种方法，在没有特定突变的情况下，分析环境条件如何导致缺陷。

(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.