EXPRESSION AND FUNCTION OF THE GUANYLIN LIGAND FAMILY

本申请的总体目标是：1）使用成对配体鸟苷素和尿鸟苷素的表达模式来理解有助于肠上皮细胞中转录调节的因子，和2）通过产生和研究转基因动物模型来鉴定这些配体的特异性生理和病理生理功能。 由于鸟苷素和尿鸟苷素的表达的定义的空间模式，它们的高水平的肠表达，以及我们目前拥有的工具，这些配对的基因代表了一个独特的机会，以确定基因表达的机制，特别是在结肠和小肠隐窝。 体外技术，包括瞬时转染测定、DNase 1超敏反应和足迹法将用于指导基因表达的体内研究。 表达与荧光素酶报告基因连接的鸟苷素和尿鸟苷素启动子元件的各个部分的转基因小鼠将用于定义指导组织特异性表达的序列。 为了实现我们的第二个目标，我们将创建鸟苷素和尿鸟苷素敲除小鼠。 我们最初通过同源重组进行基因靶向的努力已经产生了围产期致死和/或胚胎致死表型。 为了确定导致这种表型的机制，我们将鉴定鸟苷素和尿鸟苷素在发育中的小鼠胚胎、胚状体、胎盘和卵黄囊中的表达位点;为此，我们将使用RT-PCR、整体包埋和组织切片原位杂交。 这将鉴定鸟苷素或尿鸟苷素是否在发育中的肠中以及在发育中的小鼠的肠外的关键器官中表达。基于这些研究的结果和解释，我们将寻求一种策略，通过利用体外或体内条件靶向方法实现鸟苷素的组织特异性消融。 这将产生肠中缺乏鸟苷素和/或尿鸟苷素的转基因小鼠。 为了辨别鸟苷素和尿鸟苷素在体内的作用，我们将使用生物电、离子通量和原位结扎环测量来测量基础和刺激的肠分泌。 基于所观察到的鸟苷素的生理作用，我们将研究鸟苷素和尿鸟苷素损失对盐耐受性、碳酸氢盐缓冲、急性期损伤、肠运动和肠腺瘤形成的影响。 我们将确定鸟苷素或尿鸟苷素受体是否在这些小鼠中上调，以及鸟苷酸环化酶C（GC-C）以外的受体是否在任何已鉴定的表型中发挥作用。 如果鸟苷素或尿鸟苷素失活导致“无基础表型”，我们将寻找允许这种“正常”表型的补偿或冗余机制。

The overall objectives of this application are: 1) to use the expression patterns of the paired ligands guanylin and uroguanylin to understand the factors which contribute to transcriptional regulation in intestinal epithelial cells and 2) to identify the specific physiologic and pathophysiologic functions of these ligands by generating and studying transgenic animal models. Because of the defined spatial patterns of expression of guanylin and uroguanylin, their high levels of intestinal expression, and the tools which we currently possess, these paired genes represent a unique opportunity to identify mechanisms of gene expression, especially in the colon and in the small intestinal crypts. In vitro techniques, including transient transfection assays, DNasel hypersensitivity and footprinting will be used to guide in vivo studies of gene expression. Transgenic mice expressing various portions of the guanylin and uroguanylin promoter elements linked to the luciferase reporter gene will be used to define sequences that direct tissue specific expression. In order to accomplish our second objective we will create a guanylin and a uroguanylin knockout mouse. Our initial efforts at gene targeting by homologous recombination have produced a perinatal-lethal and/or embryo-lethal phenotype. In order to determine the mechanism(s) responsible for this phenotype, we will identify the sites of expression of guanylin and uroguanylin in the developing mouse embryo, embryoid bodies, placenta and yolk sac; to do this we will use RT-PCR, whole mount and tissue slice in situ hybridization. This will identify whether guanylin or uroguanylin is expressed in the developing intestine as well as in critical organs outside the intestine in the developing mouse. Based on the results and interpretation of these studies, we will pursue a strategy to effect tissue-specific ablation of guanylin by taking advantage of an in vitro or an in vivo conditional targeting approach. This will generate transgenic mice lacking guanylin and/or uroguanylin in the intestine. To discern the role of guanylin and uroguanylin in vivo, we will measure basal and stimulated intestinal secretion using bioelectric, ion flux and in situ ligated loop measurements. Based on the observed physiologic actions of guanylin, we will investigate the effect of guanylin and uroguanylin loss on salt tolerance, bicarbonate buffering, an acute phase injury, intestinal motility and intestinal adenoma formation. We will determine whether guanylin or uroguanylin receptors are upregulated in these mice and whether receptors other than guanylyl cyclase C (GC-C), play a role in any identified phenotype. Should guanylin or uroguanylin inactivation result in "no basal phenotype," we will search for compensatory or redundant mechanisms which permit this "normal" phenotype.