The long term goals of this study are to determine the molecular mechanisms that specify the development and differentiation of pancreatic endocrine tissue. Currently, there is a limited understanding of pancreatic cell differentiation and the origin of islet cell lineages. As in many other developmental systems, the molecular mechanisms that direct the development and differentiation of the pancreas are likely to be established by an integrated network of regulatory factors. In the past few years, major progress has been achieved in identifying transcription factors important for islet development. In particular, Nkx2.2 has been shown to be critical for the development of three of the four endocrine cell types, and is essential for the differentiation of all insulin-producing beta cells. We hypothesize that the activity of Nkx2.2 is regulated by interactions with other factors. To test this hypothesis, we used the yeast two-hybrid system to identify proteins that interact with Nkx2.2. These studies led to the identification of Gata6 as a potential Nkx2.2 interacting factor in the pancreas. Consistent with this interaction, we have determined that Gata6 is expressed in the region of the foregut epithelium that gives rise to pancreas and later becomes restricted to the endocrine cells of the islet. In this R21 exploratory grant we propose the following specific aims to determine the molecular nature of the interaction between Gata6 and Nkx2.2 and the functional significance of their interaction in pancreatic islet development: 1. Define the gene expression pattern of Gata6 in the developing islet. We have determined Gata6 is expressed in the pancreatic epithelium and then restricted to endocrine cells. We will determine precisely when and where Gata6 is expressed in the embryonic pancreas and compare its expression pattern to known islet cell type markers. We will also precisely determine its coexpression with Nkx2.2 in the islet. 2. Confirm the physical interaction between Gata6 and Nkx2.2 in the islet. Yeast two-hybrid analysis, in vitro pull down assays and co-immunoprecipitation assays in islet and non-islet cell lines will be performed to confirm the protein-protein interaction and delineate the domains of each protein that are needed for the interaction. 3. Determine whether Nkx2.2 and Gata6 cooperatively interact to regulate islet cell type specific genes that carry adjacent Nkx2.2 and Gata6 binding sites in their upstream promoter regions; these include glucokinase and Pax4. In addition, we will identify other islet genes that are regulated by Nkx2.2 and/or Gata6.
