EXCEED THE SPACE PROVIDED. The epithelium of mammalian small intestine has a complex, but highly defined architecture. The primary components of this architecture are the crypt and villus. Stem cells in the crypt are precursors of all four major epithelial cell types. After cell division and initiation of differentiation, immature cells begin an interesting bipolar migration. Enterocytes, goblet cells, and enteroendocrine cells migrate up along the villus, while Paneth cells migrate down into the crypt. The process of differentiation into each epithelial cell types involves activation of a specific program of gene expression characteristic of that cell type. Variations in gene expression are also observed for each epithelial cell type along the crypt-villusmigration axis. In addition, there are large variations in gene expression along two other axes in the small intestine, the cephalocaudal axis from duodenum to ileum and the developmental time axis. During development, there are changes in gene expression corresponding to both establishment of the intestinal architecture and to activation metabolic function. Once the adult pattern of gene expression is established along the cephalocaudal axis, it is maintained despite a continuous and rapid turnover of epithelial cells throughout life. The network of regulatory factors and elements that specify and determine gene expression along the various intestinal axes is poorly understood. A basic understanding of normal intestinal development and function at the molecular level is a critical foundation necessary for investigating functional or developmental changes in intestine related to disease states or physical damage. This project utilizes the adenosine deaminase (ADA) gene as a model system for investigating the network regulating gene expression along various axes in the small intestine. High-level ADA expression is limited to duodenum and within the duodenum it is limited to villus enterocytes, being activated at the crypt-villusjunction. Recent studies with transgenic mice have identified an enhancer responsible for this pattern of intestinal ADA expression. Two temporal control segments have been identified that determine the developmental time at which the enhancer is activated. This application proposes the following specific aims: to characterize the functional roles of elements and transcription factors identified for the duodenal enhancer (especially PDX-1), to identify roles for cofactors in enhancer function, and to characterize the temporal regulatory elements and their role in the enhancer function.
