EXPRESSION AND FUNCTION OF THE GUANYLIN LIGAND FAMILY

本应用的总体目标是：1)利用成对配体观音林和尿观音林的表达模式来了解肠上皮细胞中转录调控的因素；2)通过建立和研究转基因动物模型来鉴定这些配体的特定生理和病理生理功能。由于观音林和尿观音林明确的空间表达模式，它们在肠道中的高水平表达，以及我们目前拥有的工具，这些配对基因代表了一个独特的机会来识别基因表达机制，特别是在结肠和小肠隐窝中。体外技术，包括瞬时转染试验，DNasel超敏反应和足迹将用于指导基因表达的体内研究。转基因小鼠表达与荧光素酶报告基因相关的观音和尿观音启动子元件的不同部分，将用于确定直接组织特异性表达的序列。为了实现我们的第二个目标，我们将创建一个观音和一个尿观音敲除小鼠。我们通过同源重组对基因靶向的初步研究已经产生了围产期致死和/或胚胎致死表型。为了确定这种表型的机制，我们将在发育中的小鼠胚胎、胚状体、胎盘和卵黄囊中鉴定观音碱和尿观音碱的表达位点；为了做到这一点，我们将使用RT-PCR，全载和组织切片原位杂交。这将确定观音或尿观音是否在发育中的肠道以及发育中的小鼠肠道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，利用体外或体内条件靶向方法来影响组织特异性消融观音林。这将产生肠道中缺乏观音碱和/或尿观音碱的转基因小鼠。为了了解观音林和尿观音林在体内的作用，我们将使用生物电、离子通量和原位结环测量来测量基础和刺激肠道分泌。在观察到观音林生理作用的基础上，我们将探讨观音林和尿观音林损失对盐耐受性、碳酸氢盐缓冲、急性期损伤、肠道运动和肠腺瘤形成的影响。我们将确定在这些小鼠中观音苷或尿观音苷受体是否上调，以及除观音苷环化酶C （GC-C）外的受体是否在任何已确定的表型中发挥作用。如果观音或尿观音失活导致“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.