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期间胰腺祖细胞如何从未分化的内胚层产生。我们的理由是，这些信息将是非常宝贵的努力，分化成细胞和其他胰腺细胞类型的人干细胞在体外，用于移植治疗糖尿病患者。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最早阶段。我们提出了三个具体的目标，建立在我们以前的工作，并利用一种新的细胞移植技术，使我们能够测试生殖层特异性基因功能。在上一个资助期发表的工作中，我们使用这种方法确定信号分子视黄酸（RA）是胰腺发育的指导性中胚层来源的正调节因子，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是胰腺发育的负调节因子，其在后内胚层内起作用以设置胰腺的后界限。在初步的实验中，我们已经使用微阵列分析，以确定内胚层目标的RA信号。有趣的是，这些目标包括能够负调节RA信号传导的分子，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。在目的1中，我们将测试的假设，Cyp26和Nr2f负调节RA信号传导功能，以调节区域化的内胚层前肠通过控制RA信号传导域的精确大小。这些实验将利用药理学抑制剂，斑马鱼突变分析，吗啉基因功能敲低，和生殖层特异性细胞移植。在目的2中，我们将测试的假设，RA和Cdx4积极调节内胚层Hox转录因子的表达，功能，以促进或阻止胰腺的发展，分别。我们将确定RA和Cdx4调节的Hox基因，然后使用获得和丧失功能的方法，再加上生殖层特异性细胞移植，以测试它们在内胚层区域化中的作用。在目标3中，我们将研究先前涉及早期胰腺发育的两个额外的候选RA靶点的作用：Hb9和Tcf2转录因子。我们将使用突变体分析和吗啉敲低来检验这些转录因子指定胰腺内分泌细胞类型的不同子集的假设。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。所提出的实验将提供新的信息有关的胰腺细胞类型在体外的操作。此外，这项工作将为RA信号通路的调节提供新的见解，这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如何形成。我们的理由是，增加对这一过程的复杂性的理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是非常宝贵的。

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.