MOLECULAR GENETICS OF LIVER DEVELOPMENT

描述（由申请人提供）：我们的目标是识别和了解调节肝脏发育的基因的功能。我们正在斑马鱼中进行这些研究，斑马鱼是一种非常适合胚胎学和遗传学研究的脊椎动物模型系统。在过去的几年里，我们完成了斑马鱼野生型肝脏发育的详细分析，并利用在内胚层和内胚层衍生器官中表达GFP的转基因系，对调节肝脏发育的基因进行了大规模的前向遗传筛选。在这个基因筛选中，我们发现了37个影响肝脏发育的隐性突变。筛选出来的最引人注目的突变之一是prometheus (prf)，它似乎阻断了肝脏的分化。我们对prt基因进行了定位克隆，发现其编码Wnt2b同源物。此外，基因表达和移植分析表明，侧板中胚层细胞通过表达Wnt2bb/Prt诱导肝脏分化。其他数据表明，Wnt2bb/Prt通过β -连环蛋白调节肝脏规格。这些关于典型Wnt信号在肝脏特化中的关键作用的数据令人惊讶，因为之前的研究表明Bmp和Fgf信号在这一过程中有牵连。我们建议继续我们对斑马鱼肝脏发育的研究，具体目标如下：

DESCRIPTION (provided by applicant): Our goals are to identify and understand the function of genes regulating liver development. We are conducting these investigations in zebrafish, a vertebrate model system well suited for embryological and genetic studies. Over the past few years, we have completed a detailed analysis of wild-type liver development in zebrafish, and conducted a large-scale forward genetic screen for genes regulating liver development using a transgenic line expressing GFP in the endoderm and endoderm-derived organs. In this genetic screen, we identified 37 recessive mutations affecting liver development. One of the most striking mutations to come out of the screen is prometheus (prf), which appears to block hepatic specification. We have positionally cloned the prt gene and found that it encodes a Wnt2b homologue. Furthermore, gene expression and transplantation analyses indicate that cells in the lateral plate mesoderm induce hepatic specification by expressing Wnt2bb/Prt. Additional data indicate that Wnt2bb/Prt regulates hepatic specification through beta-catenin. These data about the critical role of canonical Wnt signaling in hepatic specification are surprising in light of previous work in the field that has implicated Bmp and Fgf signaling in this process. We propose to continue our studies of zebrafish liver development with the following specific aims: 1) Recover and analyze the liver mutants that were identified in our screen with the goals of making them available to the community through the stock center. 2) Investigate in more detail the role of Wnt2b, Bmp and Fgf signaling in hepatocyte specification. Our analysis of the prt mutant has led to the hypothesis that Wnt2b signaling is essential for hepatocyte specification and this hypothesis will be tested further. We will also further test the role of Bmp and Fgf signaling in hepatocyte specification. 3) Investigate in detail four mutations identified in our screen that appear to affect hepatic specification: s415, s436, s818 and s853. These mutations will be analyzed in depth including detailed phenotypic analysis and isolation of the affected gene. Altogether, these studies should provide valuable information to help us direct endodermal cells to differentiate towards the hepatocyte lineage.