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。在过去的几年中，我们已经完成了详细的分析野生型肝脏发育的斑马鱼，并进行了大规模的正向遗传筛选基因调节肝脏发育的转基因线表达GFP的内胚层和内胚层衍生的器官。在这个遗传筛查中，我们确定了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），它似乎阻止了肝脏的特化。我们已经定位克隆的prt基因，并发现它编码的Wnt 2b同源。此外，基因表达和移植分析表明，在侧板中胚层细胞诱导肝特化表达Wnt 2bb/Prt。其他数据表明，Wnt 2bb/Prt通过β-连环蛋白调节肝脏特化。这些关于典型Wnt信号传导在肝特化中的关键作用的数据是令人惊讶的，鉴于该领域中先前的工作已经在该过程中涉及Bmp和Fgf信号传导。我们建议继续研究斑马鱼肝脏发育，具体目标如下： 1)恢复和分析在我们的屏幕中识别的肝脏突变体，目标是通过库存中心向社区提供它们。 2)更详细地研究Wnt 2b、Bmp和Fgf信号在肝细胞特化中的作用。我们对prt突变体的分析导致了Wnt 2b信号传导对于肝细胞特化是必不可少的这一假设，这一假设将被进一步检验。我们还将进一步测试Bmp和Fgf信号传导在肝细胞特化中的作用。 3)详细研究在我们的筛选中发现的似乎影响肝脏特异性的四种突变：s415，s436，s818和s853。这些突变将进行深入分析，包括详细的表型分析和受影响基因的分离。 总之，这些研究应该提供有价值的信息，以帮助我们指导内胚层细胞向肝细胞谱系分化。

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.