Our long-term goals are to produce information relevant to the identification, purification, expansion and differentiation of multi- potential pancreatic stem cells, which may lead to material suitable for pancreas replacement therapy, and increase our understanding of, and eventually help to offset, the cellular defects in type II diabetes. Although some progress in these areas has come recently from studies of mice with inactivated genes that encode transcription factors expressed in the pancreas, our mechanistic understanding of endocrine/exocrine differentiation during embryogenesis, and maintenance of cell type and function in adults, is still rudimentary. We propose a focused set of experiments directed at filling some of these gaps. First, we will carry out a precise functional analysis of an essential pancreatic gene, pdx-1. Global pdx-1 inactivation blocks pancreas development, and we will now determine the role of pdx-1 in later stages of pancreatic cell differentiation, in the exocrine, beta and non-beta cell genes, indicating that pdx-1 transcriptional regulation is a major focus for the control of beta cell function. We have identified likely pdx-1 cis-regulatory elements that contain binding sites for transcription factors that include HNF3beta and the recently described MODY susceptibility factors. We will determine the role of these conserved cis-regulatory elements in vivo. Third, we will develop genetic lineage tracing techniques to characterized endocrine cell ontogeny.
Specific aims are: (1) to determine the effect of inactivating pdx-1 in selected endocrine and exocrine cells. (2) to determine in vivo the role of conserved cis-regulatory sequences in pdx-1, to begin to dissect the transcriptional regulatory networks controlling pancreas formation and function. (3) Develop genetic lineage tracing techniques for a rigorous understanding of pancreatic cell ontogeny, with special reference to the endocrine pathway.
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