To develop fully functional insulin-producing replacement cells for transpantation into diabetic patients, we need to identify the molecular pathways that initiate beta-cell formationand insulin production. Using genetically engineered mouse models, we have examined the role of Nkx6 class homeodomain transcription factors in beta-cell differentiation and function. During pancreas development, progenitor cells first make a choice between an exocrine and endocrine fate; endocrine progenitors subsequently differentiate into four distinct endocrine cell types. Our analysis of Nkx6.1 and Nkx6.2 single and double mutant mice suggests that Nkx6 transcription factors control multiple steps in pancreatic cell differentiation. Based on the observation that loss of Nkx6.1 in mice results in a specific defect in only the development of the insulin-producing beta-cells, it was initially thought that Nkx6 activity is selectively required for beta-cell development (Sander et al. 2000). Subsequent analysis of Nkx6.1/Nkx6.2 double mutant mice, however, revealed that Nkx6.2 partially compensates for Nkx6.1 function and masks a more general requirement for Nkx6 activity in the specification of the entire endocrine compartment (Henseleit et al. 2005). This suggests that Nkx6 transcription factors may function to drive differentiation of a common progenitor cell down an endocrine pathway and that in the absence of Nkx6 this cell type adopts an alternate fate. To test this hypothesis, we are now using genetic lineage tracing and rescue experiments to investigate whether Nkx6 factors first control the decision of a progenitor to become endocrine or exocrine and later instruct endocrine progenitors to differentiate into beta-cells. Since in vitro experiments have recently implicated Nkx6.1 as a potential proliferation factor of adult beta-cells (Schisler et al. 2005, PNAS), we are also investigating whether Nkx6.1functions as a positive regulator of beta-cell growth in vivo. For these experiments, the lab has developed genetic mouse models that now allow us to fully elucidate the specific temporal and spatial requirements for the different Nkx6 factors in the developing pancreas and in adult beta-cells. Subsets of these future projects entail collaborative efforts through funding from the NIH-Beta Cell Biology Consortium (BCBC; http://www.betacell.org/). As part of this consortium, we are currently developing mice, in which multiple progenitor populations are marked by expression of specific fluorochromes. These “multi-color” mice will be used for isolation and characterization of specific progenitors, as well as for isolation of ES-cells to monitor beta-cell differentiation in vitro.

The differentiation of tissue cells from stem/progenitor cells is called neogenesis and is the main mechanism of pancreatic endocrine and exocrine cell differentiation in the embryo. To date it is still unclear whether cells in the adult pancreas regenerate by neogenesis or whether replication of preexisting differentiated pancreatic cells is the only mechanism by which lost cells can be replaced. Progress in this area has been hampered by the lack of a true progenitor cell marker and a relative paucity in our knowledge about the cell-intrinsic determinants of progenitor cell maintenance. Members of the SOX transcription factor family have been implicated as markers of stem/progenitor cells in many tissues and have been shown to maintain cells in a stem-cell-like state and to inhibit cell differentiation. In our research we recently identified Sox9 as a novel selective marker of multipotent pancreatic progenitor cells throughout development of the pancreas. Furthermore, specific inactivation of Sox9 in the early pancreatic epithelium of mouse embryos revealed a role in pancreatic progenitor maintenance, partly through the control of Notch signal transduction (Seymour et al. 2007). In embryos deficient for Sox9, the pancreatic progenitor cell pool fails to expand as a consequence of reduced proliferation and precocious differentiation. Interestingly, in the adult pancreas expression of Sox9 exclusively persists in a subset of ductal cells; a cell type that is regarded as a potential reservoir of stem/progenitor cells in the adult pancreas. One of our main research goals is to test whether the ductal cells marked by this factor are a possible source of new beta-cells and whether similar to the embryo, Sox9 is also required for the expansion of these putative stem/progenitor cells in adult pancreas. It is our hope that these experiments will define a putative target cell and molecular pathway that could be therapeutically targeted to induce adult beta-cell regeneration.