Regionalization of the Vertebrate Endoderm

描述(申请人提供)：本方案的总体目标是了解在胚胎发育过程中未分化的内胚层是如何产生胰腺前体细胞的。我们的基本原理是，这些信息对于在体外将人类干细胞分化为细胞和其他类型的胰腺细胞的努力将是无价的，用于糖尿病患者的移植治疗。我们的策略是利用斑马鱼作为一个强大的脊椎动物模型来研究正常胰腺发育过程中的早期步骤。我们提出了三个特定的目标，这三个目标建立在我们之前的工作基础上，并开发了一种新的细胞移植技术，使我们能够测试生殖层特异性基因功能。在前一个资助时期发表的研究中，我们使用这种方法来确定信号分子维甲酸(RA)是一种有益的中胚层衍生的胰腺发育的正向调节因子，它直接作用于内胚层来指定前肠区域。相反，我们发现转录因子CDX4是胰腺发育的负调控因子，它在后内胚层发挥作用，设定胰腺的后极限。在初步实验中，我们使用微阵列分析来识别RA信号的内皮靶。有趣的是，这些靶点包括能够负向调节RA信号的分子，以及转录因子，包括Hox基因产物，可能在RA下游发挥作用，以指定胰腺祖细胞。在目标1中，我们将检验这一假设，即Cyp26和Nr2f是RA信号的负调控因子，通过控制RA信号域的精确大小来调节内胚层到前肠的区域化。这些实验将利用药物抑制剂、斑马鱼突变分析、基因功能的吗啉敲除和生殖层特定细胞移植。在目标2中，我们将检验这一假设，即RA和CDX4正向调节内皮HOX转录因子的表达，这些转录因子分别起促进或阻止胰腺发育的作用。我们将确定RA和CDX4调节的HOX基因，然后使用获得和功能丧失的方法结合生殖层特异性细胞移植来测试它们在内胚层区域化中的作用。在目标3中，我们将研究另外两个先前与胰腺发育有关的候选RA靶标的作用：HB9和Tcf2转录因子。我们将使用突变分析和吗啉基因敲除来检验这些转录因子指定不同的内分泌胰腺细胞亚群的假设。最后，我们将研究Hox基因、HB9和Tcf2如何在转录因子网络中相互作用，该网络在RA下游发挥作用，以确定胰腺细胞类型。拟议的实验将提供与体外操作胰腺细胞类型相关的新信息。此外，这项工作将为RA信号通路的调控提供新的见解，RA信号通路对许多不同的发育和生理过程至关重要。公共卫生相关性：该提案的长期目标是为通过细胞移植治愈糖尿病的努力提供信息。我们的总体策略是利用斑马鱼，一种小型淡水水族馆物种，作为一个方便的模型系统来研究脊椎动物胰腺在正常胚胎发育期间是如何形成的。我们的理论基础是，对这一过程的复杂性的进一步了解将证明对体外将人类干细胞分化为胰腺细胞和其他内分泌细胞类型的方法学的发展具有非常重要的价值。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to understand how pancreatic progenitors are produced from undifferentiated endoderm during embryonic development. Our rationale is that this information will be invaluable to efforts to differentiate human stem cells into -cells and other pancreatic cell types in vitro, for use in transplantation therapies for diabetic patients. Our strategy is to exploit the zebrafish as a powerful vertebrate model to study the earliest steps in the process of normal pancreas development. We propose three Specific Aims, which build upon our previous work, and exploit a novel cell transplantation technique that allows us to test germ-layer specific gene function. In work published during the previous funding period, we used this approach to establish that the signaling molecule Retinoic Acid (RA) is an instructive mesoderm-derived positive regulator of pancreas development, which acts directly on endoderm to specify the foregut region. By contrast, we showed that transcription factor Cdx4 is a negative regulator of pancreas development, which functions within posterior endoderm to set the posterior limit of the pancreas. In preliminary experiments we have used microarray analysis to identify endodermal targets of RA signaling. Intriguingly, these targets include molecules able to negatively-regulate RA signaling, as well as transcription factors, including Hox gene products, likely to function downstream of RA to specify pancreatic progenitors. In Aim 1 we will test the hypothesis that Cyp26 and Nr2f negative-regulators of RA-signaling function to regulate regionalization of endoderm to foregut by controlling the precise size of the RA signaling domain. These experiments will make use of pharmacological inhibitors, zebrafish mutant analyses, morpholino-knockdown of gene function, and germ-layer specific cell transplantation. In Aim 2 we will test the hypothesis that RA and Cdx4 positively regulate expression of endodermal Hox transcription factors that function to promote or block pancreas development, respectively. We will identify RA and Cdx4 regulated Hox genes, then use gain and loss-of-function approaches coupled with germ-layer specific cell transplantation to test their roles in endoderm regionalization. In Aim 3 we will investigate the roles of two additional candidate RA-targets previously implicated in early pancreas development: the Hb9 and Tcf2 transcription factors. We will use mutant analysis and morpholino-knockdown to test the hypothesis that these transcription factors specify distinct subsets of endocrine pancreas cell types. Finally, we will investigate how Hox genes, Hb9 and Tcf2 interact within the transcription factor network that functions downstream of RA to specify pancreatic cell types. The proposed experiments will provide novel information relevant to the manipulation of pancreas cell types in vitro. In addition, this work will provide new insights into the regulation of the RA signaling pathway, which is critical for many different developmental and physiological processes. PUBLIC HEALTH RELEVANCE: The long-term goal of this proposal is to inform efforts to cure diabetes by cell-transplantation. Our general strategy is to make use of the zebrafish, a small fresh-water aquarium species, as a convenient model system to study how the vertebrate pancreas forms during normal embryonic development. Our rationale is that an increased understanding of the intricacies of this process will prove invaluable to the development of methodology to differentiate human stem cells into pancreatic ¿-cells and other endocrine cell types in vitro.