The directed differentiation of stem, progenitor, or surrogate cells towards the pancreatic beta cell fate will require a detailed knowledge of how the program is normally triggered in vivo. Such knowledge could assess the fidelity of induced programs, and the quality/robustness of the final cell fate-both critical issues for cellular therapies for diabetes whether transplantation or regeneration based. The firmest information in this respect comes from genetic interference in model organisms, but very few studies have mapped the precise effect on cell lineage pathways. Our work is focused on obtaining a high-resolution characterization of the orchestrated interplay between transcription factors and intercellular signals during pancreas organogenesis. A stable foundation for long-term gains will be made by placing newly emergent transcription factors and signaling molecules into a functional framework in which well-understood genes act as """"""""fixed"""""""" reference points. Our previous studies rationalize the homeobox pdx1 and bHLH ptf1a genes as compelling choices in this context, based upon their dramatic null phenotypes and role in both progenitor and differentiated cell lineages. They thus both afford opportunities for making many tools to dissect the function of other genes in mice. Despite much work on pdx1, and less on ptf1a, neither has been placed precisely in the transcriptional regulator network controlling endocrine specification and differentiation. To fill these gaps, and to pioneer analyses for known and emergent regulatory genes, we will: (1) Characterize endocrine differentiation when pdx1 is reduced or inactivated in specific progenitor classes or differentiated beta cells, analyzing cell lineage pathways, proliferation and maintenance of mature beta cells. (2) Create loxed cassette acceptor alleles of pdx1 via bacterial artificial chromosome engineering to allow the flexible, rapid insertion of markers and minigenes (e.g., recombinases, transcription factors, signaling molecules). We will test the effects of deleting pdx1 cis-regulatory motifs for transcription factors thought to be essential upstream regulators (with a later similar focus on ptf1a). (3) Create inducible ptf1a-CreERTm and floxed ptf1a alleles to connect the gene functionally to progenitor and committed cell behavior. Our function-based gene discovery program in zebrafish (separately funded) will define new loci that respond to or regulate pdx1 and ptf1a, and these will be imported into our analysis. Accurate mapping of cell lineages after gene disruption will hone our ideas on which transcription factors and signaling molecules to express, at what level (titer), and in what cell types, to trigger full beta cell differentiation.
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