The long-term objective of this application is to elucidate the molecular mechanism(s) underlying specification of intestinal cell identity. We have identified a previously unrecognized patterning step that takes place at embryonic day 16.5 in the mouse. This step involves the upregulation of >1,000 genes in the intestinal epithelium, a process that creates the distinct epithelial border that defines intestinal vs. stomach identity. We call this process intestinalization. Our evidence also implicates the transcription factor Tcfec as a potential regulator of intestinalization. Thus, the Specific Aims of this application are to: 1) determine if Tcfec is an activator in intestinal epithelium, 2) characterize the downstream pathways affected by Tcfec during intestinal epithelial cell specification, and 3) analyze the contribution of a conserved non-coding element (CNE) near the Tcfec gene to the temporal regulation of its expression in intestinal epithelium.
For Aim 1, we will clone intestinal forms of Tcfec by RLM-RACE and determine their transcriptional activity using cell-based, luciferase reporter assays.
For Aim 2, Tcfec expression will be manipulated in Caco-2 cells, an in vitro system that recapitulates intestinal epithelial cell differentiation. We will also establish several mouse models to test the requirement for Tcfec in the intestine, as well as the effect of Tcfec misexpression in the stomach.
For Aim 3, we will generate transgenic reporter mice to ascertain the pattern of expression directed by the Tcfec CNE. The requirement of specific transcription factor binding sites will then be tested by mutagenesis. In addition to their contribution to the understanding of intestinalization per se, these studies may well be relevant to the pathologic state known as intestinal metaplasia of the stomach, in which cells within the stomach take on intestinal character. Since intestinal metaplasia is thought to represent an early step in the development of some forms of gastric cancer, our findings could eventually impact diagnosis or early treatment of this often fatal cancer. ? ? ?
