Differentiation of Embryonic Stem Cells Conditionally Expressing Neurogenin 3

¹ University of Wisconsin-Madison School of Medicine and Public Health, Department of Surgery, Madison, Wisconsin; William S. Middleton Memorial Veterans Administration Hospital, Madison, Wisconsin; Reproductive Medicine Associates of New Jersey, Morristown, New Jersey
² University of Wisconsin-Madison School of Medicine and Public Health, Department of Surgery, Madison, Wisconsin; William S. Middleton Memorial Veterans Administration Hospital, Madison, Wisconsin
³ University of Minnesota, Departments of Veterinary and Biomedical Sciences and Microbiology, St. Paul, Minnesota
⁴ University of Wisconsin-Madison School of Medicine and Public Health, Department of Surgery, Madison, Wisconsin; William S. Middleton Memorial Veterans Administration Hospital, Madison, Wisconsin; WiCell Research Institute, Madison, Wisconsin

^* To whom correspondence should be addressed. E-mail: jon{at}surgery.wisc.edu.

	Abstract

Expression of the proendocrine gene neurogenin 3 (Ngn3) is required for the development of pancreatic islets. In order to better characterize the molecular events regulated by Ngn3 during development, we have determined the expression profiles of murine embryonic stem cells (mESCs) uniformly induced to overexpress Ngn3. A mESC line was created in order to induce Ngn3 by adding doxycycline (Dox) to the culture medium. Genome-wide microarray analysis was performed to identify genes regulated by Ngn3 in a variety of contexts including undifferentiated ESCs and differentiating embryoid bodies (EBs). Genes regulated by Ngn3 in a context-independent manner were identified and analyzed using systematic gene ontology tools. This analysis revealed Notch signaling as the most significantly regulated signaling pathway (p=0.009). This result is consistent with the hypothesis that Ngn3 expression makes the cell competent for Notch signaling to be activated and conversely, more sensitive to Notch signaling inhibition. Indeed, EBs induced to express Ngn3 were significantly more sensitive to -secretase inhibitor-mediated Notch signaling inhibition (p<0.0001) when compared to uninduced EBs. Moreover, we find that Ngn3 induction in differentiating ESCs results in significant increases in insulin, glucagon, and somatostatin expression.

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