CARDIOVASCULAR DEVELOPMENT IN XENOPUS LAEVIS

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(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.