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.
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