wnt Signaling in Liver Development and Disease

描述(由申请人提供)： 在美国，肝病是发病率和死亡率的常见原因，了解肝脏发育过程中的关键途径对于我们了解正常的肝功能和疾病是至关重要的。斑马鱼是研究脊椎动物早期发育的优秀模型，因为它在发育过程中是半透明的，并且能够产生大量的后代。我们的实验室利用正向遗传学的优势识别了在斑马鱼发育中重要的基因，并在最近的遗传筛查中鉴定了几个易患癌症的突变。最近的报道证明了斑马鱼系统用于肝脏发育研究的可行性。功能基因组学方法和荧光转基因鱼类品系的使用已经描述了肝脏的形态发生。一些研究表明，WNT途径，特别是β-连环蛋白，对肝脏生长、肝细胞和胆管分化以及肿瘤的形成具有重要作用。为了验证这一假设，在肝脏特异性启动子肝脏脂肪酸结合蛋白的控制下，将产生高表达稳定的β-连环素与绿色荧光蛋白的转基因斑马鱼。这些FISH将通过组织学、免疫组织化学和原位杂交进行检测，以分析肝脏特异基因的表达模式。与野生型鱼类相比，荧光标记允许通过FACS和随后的微阵列分析来分离肝细胞。其他转基因菌株，在热休克启动子下表达WNT途径的抑制剂，以及WNT途径的小分子抑制剂，将被研究减少WNT信号对肝脏发育的影响。具体目的2将通过使用转基因β-连环蛋白FISH和其他表达WNT途径的效应物Cyclin D1和myc的转基因FISH来确定WNT信号在肝肿瘤中的重要性。由此产生的肿瘤将使用先进的多光谱荧光成像技术和超声波进行检测，并通过组织学、细胞遗传学和微阵列分析进行分析。携带肿瘤抑制基因P53和APC突变基因的FISH将与转基因FISH交配，以研究与WNT途径的协同效应。这些研究将为WNT信号通路在肝脏发育和肿瘤形成中的作用提供新的见解，并可能为肝脏疾病的治疗确定新的治疗靶点。

DESCRIPTION (provided by applicant): Liver disease is a common cause for morbidity and mortality in the U.S., and understanding the critical pathways during liver development is essential for our knowledge of normal liver function and disease. The zebrafish is an excellent model to study early vertebrate development because of its translucency during development and the ability to produce large numbers of offspring. Our laboratory has made significant contributions by identifying genes important in zebrafish development using the advantage of forward genetics, and recently characterized several cancer-prone mutants in a genetic screen. Recent reports have demonstrated the feasibility of the zebrafish system for the study of liver development. Liver morphogenesis has been described by functional genomics approaches and use of fluorescent transgenic fish lines. Several studies suggest that the wnt pathway, especially beta-catenin, is of great importance for liver growth, hepatocyte and biliary differentiation and neoplasia. To test this hypothesis, in Specific Aim 1 transgenic zebrafish will be created overexpressing stabilized beta-catenin fused with green fluorescent protein under the control of the liver specific promoter of liver fatty acid binding protein. These fish will be examined using histology, immunohistochemistry and in situ hybridization to analyze the expression patterns of liver-specific genes. The fluorescent labeling allows for hepatocyte isolation by FACS and subsequent microarray analysis in comparison to wild-type fish. Other transgenic strains, expressing inhibitors of the wnt pathway under a heatshock promoter, and small molecule inhibitors of the wnt pathway will be studied for the effects of decreased wnt signaling for liver development. Specific Aim 2 will determine the importance of wnt signaling in liver neoplasia by using the transgenic beta-catenin fish and other transgenic fish expressing the effectors of the wnt pathway, cyclin D1 and myc. Resulting tumors will be detected using advanced multispectral fluorescence imaging technology and ultrasound, and analyzed by histology, cytogenetics, and microarray analysis. Fish carrying mutant genes of the tumor suppressors p53 and APC will be mated to transgenic fish to study synergistic effects with the wnt pathway. These studies will provide new insight into the role of the wnt signaling pathway during liver development and neoplasia, and may identify new therapeutic targets for the treatment of liver disease.