This proposal integrates the expertise of scientists working in complementary model systems of pancreatic developmental biology. The experimental plan presented will use both human fetal and adult pancreatic cell populations (PI's laboratory), as well as murine models for the in vivo dynamic study of connexins function as putative regulators of islet cell growth and differentiation (Dr. Meda's laboratory). The integrated interaction of these two research initiatives provides a unique opportunity to exchange data and reagents for the translation of knowledge gathered from animal studies into the biology of human islet cells. Preliminary studies from both our laboratory and Dr. Meda's demonstrate that specific """"""""connexins"""""""", the individual monomeric proteins building up gap junction channels, characterize distinct populations of pancreatic epithelial cells in the human and murine pancreas. Thus, while Cx32 appears expressed in most pancreatic undifferentiated epithelial cells, Cx36 is mainly restricted to developing beta-cells. Conversely, Cx43 highlights a discrete population of cells associated with developing islet clusters and/or emerging from the ductal epithelium. Our data also demonstrate that expression of Cx36 is developmentally regulated during (beta-cell ontogeny, with the highest levels detected in mature (glucose-responsive) islet beta-cells. Functional gene transfer studies using Lentiviral vectors show that transduction of Cx32 in fetal pancreatic cells causes a significant down-regulation of insulin and glucagon gene expression, whereas transduction of Cx36 causes an increased insulin gene expression. These data provide direct proof of principle for a role of these connexins in pancreatic cell growth and differentiation. The central hypothesis of this proposal is that specific connexin isoforms, each assembling into specific gap junction channels with distinct gating capabilities, may contribute unique signaling functions to distinct developmental stages of islet cell ontogeny and in adult beta -cell homeostasis. Hence, our objective is to test whether Cx32, Cx36, and/or Cx43 can be used as molecular tools to promote beta-cell development from populations of undifferentiated pancreatic progenitor cells, and/or trigger a growth pathway in fully mature adult beta -cells. To perform these studies, we have generated a panel of Lentiviral vectors carrying the coding sequence of select connexins controlled by either a CMV promoter, or by tissue specific promoters such as the PDX-1, ngn3, and the Insulin promoter. In parallel experiments, we will generate Cre/7ox murine models to dissect the functional roles of Cx36 and Cx43 in vivo at distinct developmental stages of the islet cell lineage.
Our Specific Aims are: 1) To study the function of Cx32, Cx36, and Cx43 in human fetal pancreatic cell populations, by inhibitory peptides; 2) To study the developmental effects of transient and stable gene transfer of Cx32, Cx36, and Cx43 in human fetal pancreatic cell populations; 3) To test the ability of Cx32, Cx36, and Cx43 to stimulate either cell growth of mature islet cells, or differentiation of ductal cells from the human adult pancreas; and 4) To investigate the developmental effects of in vivo inactivation of Cx36 and Cx43 using Cre/lox murine models.
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