Investigating the role of NGN3 in Notch signaling repression during pancreatic endocrinogenesis - ABSTRACT Type 2 diabetes is a chronic condition characterized by abnormally high blood sugar levels that can result in stroke, kidney failure, and limb amputation. Dysfunction of pancreatic insulin-producing beta cells is a factor in the pathogenesis of diabetes. Transplantation of stem-cell derived beta cells represents a promising cellular therapy for patients with diabetes; however current protocols generate immature cells because they do not fully recapitulate the early molecular processes of human beta cell development. Our lab’s recent studies have demonstrated that cells lacking a functional NGN3 protein do not fully mature into PDX1+/ NKX6.1+ pancreatic progenitors capable of differentiating into all pancreatic lineages, implying that NGN3 has a critical role in facilitating early-stage human pancreatic development. To understand the role of NGN3 in early pancreatogenesis, I have performed combinatorial single cell transcriptome and chromatin analyses of functionally-null patient-specific induced pluripotent stem cell lines to interrogate the genetic and epigenetic landscape of pancreatic progenitor cells in the presence and absence of functional NGN3. Cell-cell interaction analysis indicated Notch signaling interactions between maturing pancreatic progenitors and other cell subtypes were stronger in the absence of functional NGN3. Notch signaling is known to regulate angiogenesis in the pancreas, and my data determined that pro-angiogenic factors are upregulated at the pancreatic progenitor stage. This preliminary data suggests that NGN3 has a previously unidentified role in inhibiting Notch signaling to facilitate progenitor cell maturation. I hypothesize that NGN3 represses Notch signaling to promote pancreatic progenitor cell maturation. This proposal will use differentiation assays, co-culture systems, and chromatin immunoprecipitation assays to: 1) determine if inhibition of NOTCH2/3 receptor signaling facilitates pancreatic progenitor cell maturation in the absence of NGN3 and 2) determine if NGN3 directly activates the expression of the endogenous Notch inhibitor NUMB. This set of experiments challenges the prevailing paradigm that NGN3 acts solely as a coordinator of endocrine-specific gene expression and may elucidate a novel role for it as facilitator of early pancreatic progenitor maturation. This fellowship training plan includes hands-on training in single cell analysis, molecular biology, and developmental biology. I will participate in seminars on cutting-edge research, scientific writing, and career development, and will present at national and international conferences. Children’s Hospital Los Angeles partners with the University of Southern California to be a premier teaching hospital. It provides state-of-the-art facilities, core services, and workshops through which I will be mentored by experts in their fields and can mentor numerous students. Overall, this training plan and supportive environment will equip me with the critical thinking and technical abilities needed to become a leading researcher in developmental biology and translational research.
