wnt Signaling in Liver Development and Disease

描述（由申请人提供）： 肝病是美国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我们了解正常肝功能和疾病至关重要。斑马鱼是研究早期脊椎动物发育的极好模型，因为它在发育过程中具有不透明性，并且能够产生大量后代。我们的实验室通过利用正向遗传学的优势识别斑马鱼发育中重要的基因做出了重大贡献，最近在遗传筛选中鉴定了几种易患癌症的突变体。最近的报告已经证明了斑马鱼系统用于肝脏发育研究的可行性。肝脏形态发生已经通过功能基因组学方法和荧光转基因鱼系的使用进行了描述。一些研究表明，wnt通路，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形成非常重要。为了检验这一假设，在特异性目标1中，将产生在肝脂肪酸结合蛋白的肝特异性启动子的控制下过表达与绿色荧光蛋白融合的稳定化β-连环蛋白的转基因斑马鱼。这些鱼将使用组织学，免疫组织化学和原位杂交进行检查，以分析肝脏特异性基因的表达模式。与野生型鱼相比，荧光标记允许通过FACS和随后的微阵列分析进行肝细胞分离。将研究在热休克启动子下表达wnt途径抑制剂的其他转基因菌株和wnt途径的小分子抑制剂，以了解wnt信号传导减少对肝脏发育的影响。具体目标2将通过使用转基因β-连环蛋白鱼和其他表达wnt通路效应物细胞周期蛋白D1和myc的转基因鱼来确定wnt信号传导在肝肿瘤形成中的重要性。将使用先进的多光谱荧光成像技术和超声检测所产生的肿瘤，并通过组织学，细胞遗传学和微阵列分析进行分析。将携带肿瘤抑制因子p53和APC的突变基因的鱼与转基因鱼交配，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.