MOLECULAR GENETICS OF PANCREAS DEVELOPMENT

描述（由申请人提供）：这项工作的长期目标是了解脊椎动物胚胎发生过程中胰腺发育的细胞和分子机制。在过去的2.5年中，我们已经进行了一个屏幕上的基因调控胰腺的发展，使用转基因斑马鱼线表达GFP的内胚层和内胚层衍生的器官。我们还开发了整体免疫染色技术，并利用新的转基因株系来表征这些突变体。重要的是，我们对斑马鱼的野生型胰腺发育进行了详细分析（Field等人，2003 b），该研究促进了使用斑马鱼系统来鉴定和/或测试与该过程有关的基因。具体来说，我们发现斑马鱼胰腺是由2个芽融合而成的，后1个芽仅包含胰岛细胞，前1个芽产生外分泌细胞和胰管。胰管又产生新的胰岛细胞。这种内分泌和外分泌谱系的初步分离应有助于它们的研究。我们建议继续进行研究，具体目标如下。1)分析我们筛选出的胰腺突变体。2)进一步表征6个突变体，包括详细的表型分析和受影响基因的分离。3)分析周围组织在胰腺发育和β细胞分化中的作用。我们将详细分析最初的胰岛细胞（形成后芽的细胞）的出现，目的是确定调节这一过程的信号。这些信息将有助于设计细胞培养方案，以将干细胞分化为β细胞。我们还将测试的假设，即信号从侧板中胚层控制前胰腺芽的发展，以及出现新的胰岛细胞从胰管。4)继续开发试剂和技术，以促进使用斑马鱼作为快速筛选胰腺发育和β细胞分化基因的工具。

DESCRIPTION (provided by applicant): The long-term goals of this work are to understand cellular and molecular mechanisms of pancreas development during vertebrate embryogenesis. Over the past 2.5 years, we have conducted a screen for genes regulating pancreas development using a transgenic zebrafish line expressing GFP in the endoderm and endoderm-derived organs. We have also developed wholemount immunostaining techniques and utilized novel transgenic lines to characterize these mutants. Importantly, we carried out a detailed analysis of wild-type pancreas development in zebrafish (Field et al., 2003b), a study that has facilitated the use of the zebrafish system to identify and/or test genes implicated in this process. Specifically, we found that the zebrafish pancreas is formed by the fusion of 2 buds, a posterior 1 that contains only islet cells and an anterior 1 that gives rise to the exocrine cells and the pancreatic duct. The pancreatic duct in turn, gives rise to new islet cells. This initial separation of endocrine and exocrine lineages should facilitate their investigation. We propose to continue our studies with the following specific aims. 1) Analyze the pancreas mutants identified and recovered from our screen. 2) Further characterize 6 mutants including detailed phenotypic analysis and isolation of the affected gene. 3) Analyze the role of the surrounding tissues in pancreas development and beta cell differentiation. We will analyze in detail the emergence of the initial islet cells (those that form the posterior bud) with the goals of identifying the signals that regulate this process. This information will be useful for designing cell culture protocols to differentiate stem cells into beta cells. We will also test the hypotheses that signals from the lateral plate mesoderm control the development of the anterior pancreatic bud, as well as the emergence of new islet cells from the pancreatic duct. 4) Continue to develop reagents and techniques to facilitate the use of the zebrafish as a rapid screening tool for genes regulating pancreas development and beta cell differentiation.