Regionalization of the Vertebrate Endoderm

摘要 这项提议的总体目标是了解构成其基础的细胞和分子机制。 内分泌胰腺细胞类型的正常规格。我们的理由是，这些信息对 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，用于 糖尿病患者的移植治疗。我们的战略是利用斑马鱼作为一种强大的脊椎动物 模型研究正常胰腺的形成过程。 我们在之前工作的基础上提出了三个具体目标，并开发了一种新的细胞 我们开发的移植技术是为了测试生殖层单个基因的功能。我们的 来自前一个资金时期的数据表明，信号分子维甲酸(RA)是一种具有启发性的 中胚层来源的胰腺规范的正调控因子，而转录因子CDX4在 后内胚层作为胰腺规格的负性调节因子。在初步实验中，我们有 利用基因芯片分析确定RA信号的内皮靶点。有趣的是，这些目标包括 能够负向调节RA信号的分子，以及可能发挥作用的转录因子 RA下游，以指定胰腺细胞类型。在目标1中，我们将测试以下假设 Nr2f和Cyp26家族的RA信号负调控因子控制胰腺的规格和大小。这些 实验将利用药物抑制剂、斑马鱼突变分析、吗啉基因敲除 基因功能和生殖层特异性细胞移植。在目标2中，我们将测试RA-靶点的假设 编码Hox、Tcf2和HB9转录因子的基因在RA和CDX4下游发挥调节作用 胰腺的规范和定位。这些实验将利用功能的增益和损耗 结合生殖层特异性细胞移植的方法。最后，在目标3中，我们将探讨 胰腺规范中的转化生长因子？类骨形成蛋白信号。我们将使用携带热休克诱导剂的转基因鱼 BMP信号组件和生殖层特异性细胞移植，以测试何时何地BMP信号 功能。 拟议的实验将为RA信号通路的调控提供新的见解， 这对许多不同的发育和生理过程都是至关重要的。这项工作还将提供 与体外操作胰腺细胞类型相关的新信息。最后，我们将开始建立 RA下游的分子遗传网络，其功能是指定内分泌胰腺细胞类型。

Abstract The overall goal of this proposal is to understand the cellular and molecular mechanisms that underlie normal specification of endocrine pancreas cell types. 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 process of normal pancreas formation. We propose three Specific Aims, which build upon our previous work, and exploit a novel cell transplantation technique that we have developed to test in which germ-layer individual genes function. Our data from the previous funding period suggest that the signaling molecule Retinoic Acid (RA) is an instructive mesoderm-derived positive regulator of pancreas specification, while transcription factor Cdx4 functions in posterior endoderm as a negative regulator of pancreas specification. 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 likely to function downstream of RA to specify pancreatic cell-types. In Aim 1 we will test the hypothesis that members of the Nr2f and Cyp26 families of negative-regulators of RA-signaling control pancreas specification and size. 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-target genes encoding Hox, Tcf2 and Hb9 transcription factors function downstream of RA and Cdx4 to regulate pancreas specification and localization. These experiments will make use of gain and loss-of-function approaches coupled with germ-layer specific cell-transplantation. Finally, in Aim 3 we will explore the role of TGF¿ class BMP signals in pancreas specification. We will use transgenic fish carrying heat-shock inducible BMP signaling components, and germ-layer specific cell transplantation, to test when and where BMP signals function. The proposed experiments will provide new insights into the regulation of the RA signaling pathway, which is critical for many different developmental and physiological processes. The work will also provide novel information relevant to the manipulation of pancreas cell types in vitro. Finally, we will begin to establish the molecular-genetic network that functions downstream of RA to specify endocrine pancreas cell types.