Regionalization of the Vertebrate Endoderm

描述（由申请人提供）：本提案的总体目标是了解胚胎发育过程中未分化的内胚层如何产生胰腺祖细胞。我们的基本原理是，这一信息将对在体外将人类干细胞分化为细胞和其他胰腺细胞类型，用于糖尿病患者的移植治疗的努力具有宝贵的价值。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的最早步骤。我们提出了三个具体目标，这建立在我们以前的工作，并利用一种新的细胞移植技术，使我们能够测试细菌层特异性基因功能。在之前的资助期内发表的工作中，我们使用这种方法确定了信号分子维甲酸（RA）是胰腺发育的指导性中胚层源性正调节因子，它直接作用于内胚层以指定前肠区域。相比之下，我们发现转录因子Cdx4是胰腺发育的负调控因子，它在后内胚层内起作用，设定胰腺的后边界。在初步实验中，我们使用微阵列分析来识别RA信号的内胚层靶点。有趣的是，这些靶点包括能够负调控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Aim 1中，我们将验证RA信号的Cyp26和Nr2f负调节因子通过控制RA信号域的精确大小来调节内胚层到前肠的区域化的假设。这些实验将利用药物抑制剂、斑马鱼突变体分析、基因功能的morpholinos敲低和生殖层特异性细胞移植。在Aim 2中，我们将验证RA和Cdx4分别正调控内胚层Hox转录因子表达的假设，这些转录因子具有促进或阻断胰腺发育的功能。我们将鉴定RA和Cdx4调控的Hox基因，然后使用获得和丧失功能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Aim 3中，我们将研究两个额外的候选ra靶点在早期胰腺发育中的作用：Hb9和Tcf2转录因子。我们将使用突变分析和morpholino-knockdown来检验这些转录因子指定胰腺内分泌细胞类型不同亚群的假设。最后，我们将研究Hox基因、Hb9和Tcf2如何在RA下游的转录因子网络中相互作用，以指定胰腺细胞类型。提出的实验将提供新的信息相关的操作胰腺细胞类型在体外。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：本提案的长期目标是通过细胞移植治疗糖尿病。我们的总体策略是利用斑马鱼，一种小型的淡水水族馆物种，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过程中是如何形成的。我们的基本原理是，增加对这一过程复杂性的理解，将证明对在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的发展是无价的。

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.