Abstract
The intestinal epithelium is a specialized simple epithelium that lines the gut and performs primary functions of digestion, absorption and forms a barrier against luminal pathogens. It is organized in invaginations called crypts and finger-like protrusions called villi. The crypts harbor proliferating stem cells and their transit amplifying daughter cells,
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while villi are covered with terminally differentiated cell types with specialized functions. Malignant transformation of intestinal epithelium is almost invariably initiated by activating Wnt pathway mutations. As a common result, ?-catenin accumulates in the nucleus, and constitutively binds to the transcription factor TCF4, resulting in transcriptional activation of Wnt/TCF4 target genes. This in turn initiates transformation of intestinal epithelial cells. Physiologically, the Wnt pathway is essential for the maintenance of crypt progenitor proliferation. Because of the intimate connection between Wnt signaling and intestinal biology, we have attempted to unravel the TCF4 target gene program activated by this pathway in crypts and colorectal tumors. In chapter 2 and 3 we performed expression profiling studies of colorectal cancer cell lines carrying an inducible block of the Wnt signaling pathway. The differentially expressed, ?-catenin/TCF responsive, genes were compared with genes up-regulated in prospectively collected colorectal adenomas and/or carcinomas compared with normal mucosa from the same individuals. This resulted in the “intestinal Wnt signature”. The Wnt target genes identified through this approach can be either direct regulated by the TCF/ ?-catenin complex or through intermediate transcription factor(s). In chapter 4, we identify direct Wnt targets based on chromatin immunoprecipitation (ChIP)-coupled DNA microarray analysis (ChIP-on-chip). We immunoprecipitated chromatin-bound TCF4 from LS174T colorectal cancer cells, and identified the bound DNA sequences through hybridization on DNA microarrays. Testing of these TCF4-bound regions in luciferase-based reporter gene assays demonstrated that these DNA sequences often behave as Wnt-controlled enhancers or promoters. In chapter 5, we have determined a gene signature for intestinal stem cells. One of the genes was the Wnt target gene Ascl2. The Ascl2 gene encodes a bHLH transcription factor with an unusually restricted expression pattern, i.e. its expression is predominantly detected in extraembryonic tissues and in intestinal epithelium. Transgenic expression of the Ascl2 transcription factor throughout the intestinal epithelium induces crypt hyperplasia and de novo crypt formation on villi. Induced deletion of the Ascl2 gene in adult small intestine leads to disappearance of CBC stem cells within days. The combined results from these gain- and loss-of-function experiments imply that Ascl2 plays an essential role in the maintenance of adult intestinal stem cells. In chapter 6 and 7 we describe the generation of various new mouse models that will be used for further characterization of the intestinal epithelium. Two of the generated models will be used for characterization of intestinal stem cells. The others will be used to study the function of the Regenerating (Reg) gene family in the intestine.
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