The long-term goal of this proposal is to be able to differentiate into pancreatic beta-cells, a fist step towards a potential cure of Type 1 diabetes. Understanding the molecular requirements for an embryonic cell to enter the endodermal lineage is an essential step towards this goal. Our previous genetic work in zebrafish has led to the identification of three transcriptional regulators of endoderm formation, namely Casanova (Ca)< Sox-related protein, Bonnie and Clyde (Bon), a Mix-type homeodomain protein and Gata5, a zinc finger protein. Our data further indicate that Cas is the main regulator of endoderm. We hypothesize that forced Cas expression in combination with the activation of the Nodal signaling pathway may be sufficient to activate the endodermal program in all embryonic cells. In order to test this hypothesis, we propose the following specific aims: 1) Further analyze the sufficiency of Cas, Bon and Gata5 in endoderm formation. These factors will be tested alone and in combination with the activation of the Nodal signaling pathway. 2) Identify functional mouse orthologues of Cas and bon as the mouse genes may be more efficient to convert mouse embryonic cells into endoderm. 3) Test the hypothesis that Cas (or Bon or Gata5) alone, or in combination with the activation of the Nodal signaling pathway, is sufficient to drive mouse embryonic stem (ES) cells towards an endodermal fate. 4) Test the functionality of various mouse endodermal promoters in zebrafish to establish tools to test candidates for the n3ext step of the process, namely genes that will be able to drive endoderm into pancreatic beta-cells.
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