MOLECULAR GENETICS OF LIVER DEVELOPMENT

肝脏具有几种基本功能，包括血液的储存和过滤、胆汁的分泌、氨基酸的合成和分泌以及将糖转化为糖原。尽管有这些基本功能，但人们对导致肝脏形成的细胞和分子机制知之甚少。我们建议在斑马鱼中使用一种正向遗传学方法来研究肝脏发育。在发育中的斑马鱼胚胎中，很难看到肠管及其相关器官，因此在形态屏幕上也不太容易看到。利用在发育中的肠管及其相关器官、肝脏和胰腺中表达GFP的转基因株，我们计划进行大规模的二倍体筛查，以寻找影响这些器官形成的突变。我们未来的研究重点将放在影响肝脏发育的突变上。然而，影响胰腺和/或肠道形成的突变也将在我们的筛查中被识别出来。我们将建立一个系统，在网络上公布这些突变的表型，并在确定后尽快提供。为了利用这个GFP转基因系筛选影响肝脏、胰腺和肠道发育的突变，我们提出了以下具体目标：1)详细描述斑马鱼肝脏的发育。这些研究将包括对GFP转基因株系的进一步分析，以及对野生型和特定突变背景下肝脏基因表达的详细检查。2)筛选影响肝脏、胰腺和肠道形成的突变。我们将利用GFP转基因株系进行大规模的二倍体筛选。3)对肝脏突变进行分类，并对其中的几个突变进行详细分析。互补分析、基因作图和分子特征将使我们能够对这些突变进行分类，并选择几个进行进一步的工作。这些肝脏发育的分子遗传学研究将有助于为进一步研究人类肝脏畸形和功能障碍的病理生理学提供必要的背景，也可能促进以分化为基础的治疗各种疾病状态的策略。

The liver performs several essential functions including the storage and filtration of blood, the secretion of bile, the synthesis and secretion of amino acids, and the conversions of sugars into glycogen. Despite these essential functions, little is known about the cellular and molecular mechanisms leading to the formation of the liver. We propose to use a forward genetic approach in zebrafish to investigate liver development. The gut tube ad its associated organs are not readily visible in the developing zebrafish embryo and thus have not been very accessible to morphological screens. Using a transgenic line which expresses GFP in the developing gut tube and its associated organs, the liver and pancreas, we plan to conduct a large-scale diploid screen for mutations that affect the formation of these organs. We will focus our future studies on mutations that affect the development of the liver. However, mutations that affect the formation of the pancreas and/or intestine will also be identified in our screen. We will set up a system to publish the phenotype of these mutations on the Web and make them available as soon as they are identified. In order to screen for mutations affecting the development of the liver, pancreas and intestine using this GFP transgenic line, we propose the following specific aims: 1) Characterize zebrafish liver development in detail. These studies will include further analysis of the GFP transgenic line as well as detailed examination of liver gene expression in wild-type and specific mutant backgrounds. 2) Screen for mutations that affect the formation of the liver, pancreas, and intestine. We will conduct a large- scale diploid screen using the GFP transgenic line. 3) Classify the liver mutations and begin to analyze several of them in detail. Complementation analysis, genetic mapping and molecular characterization will allow us to classify these mutations and select a few for further work. These molecular genetic studies of liver development will help provide the necessary context for further investigating the pathophysiology of malformations and malfunctions of the human liver, and may also facilitate differentiation-based strategies for treatment of a variety of diseased states.