Many epithelial organs, such as the kidney, are composed largely of branching tubular structures. Our goal is to understand the biology of tubulogenesis in general and renal tubulogenesis in particular. Tubule formation is a poorly understood process involving multiple factors and receptors. Due to the complexity and transitory nature of organogenesis, it is exceedingly difficult to study tubulogenesis in vivo. We and others have successfully used two-dimensional (2D) in vitro cell culture (cells grown on plastic or permeable filters) to study growth factor dependent regulation of tubular epithelial cells. However, a growing body of evidence indicates that epithelial cells are differentially regulated depending upon the cellular microenvironment, arguing strongly for the study of tubulogenesis using an appropriate three-dimensional (3D) model, such as the well-studied assay involving growth of Madin-Darby canine kidney (MDCK) cells in a 3D collagen matrix until the cyst stage and induction of tubule formation with hepatocyte growth factor (HGF). We have used this system to identify proteins critical for tubulogenesis. Our underlying hypothesis is that gene expression changes following HGF induction in MDCK cells grown in 3D culture is more physiologic than gene expression changes in MDCK cells grown in 2D culture prior to stimulation with HGF, and, therefore, is relevant to renal tubulogenesis. To define novel genes and pathways involved in tubulogenesis of MDCK cells by an unbiased approach, we propose a system utilizing the 3D MDCK/HGF assay in combination with a newly available canine DNA microarray, which we recently described and validated (Aim 1.1). We will specifically determine how gene expression differs in renal tubular MDCK cells stimulated with HGF depending on the microenvironment (i.e. in 2D versus 3D culture) (Aim 1.2). The most promising candidate """"""""tubulogenes"""""""" will be selected based on mRNA fold change, pathway placement, and mechanistic plausibility (Aim 2.1). These candidate genes will be characterized during the different stages of in vitro renal tubule formation by localization of their protein products and mutational analysis, to define their role in tubulogenesis (Aim 2.2). Further studies will then be proposed that involve generation of in vivo models to test the role of the putative tubulogenes in renal development, with the ultimate goal of modulating these tubulogenes and pathways to repair defects in tubule formation. ? ?
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