MOLECULAR GENETICS OF PANCREAS DEVELOPMENT

描述(申请人提供)：这项工作的长期目标是了解脊椎动物胚胎发育过程中胰腺发育的细胞和分子机制。在过去的2.5年里，我们使用在内胚层和内胚层衍生器官中表达GFP的转基因斑马鱼品系进行了胰腺发育调控基因的筛选。我们还开发了全蒙免疫染色技术，并利用新的转基因品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的分析(菲尔德等人，2003b)，这项研究促进了使用斑马鱼系统来识别和/或测试与这一过程有关的基因。具体地说，我们发现斑马鱼的胰腺是由2个芽融合而成的，后1个芽只含有胰岛细胞，前1个芽产生外分泌细胞和胰管。胰管又会产生新的胰岛细胞。这种内分泌和外分泌谱系的初步分离应该有助于他们的研究。我们建议继续我们的研究，具体目标如下。1)分析从我们的筛选中鉴定和回收的胰腺突变。2)进一步鉴定了6个突变体，包括详细的表型分析和致病基因的分离。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，将干细胞分化为β细胞。我们还将测试来自侧板中胚层的信号控制胰腺前芽的发育以及来自胰管的新的胰岛细胞出现的假设。4)继续开发试剂和技术，以促进将斑马鱼用作调节胰腺发育和β细胞分化的基因的快速筛选工具。

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.