Nkx2.2 regulated target genes and downstream molecular mechanisms necessary for pancreas cell type specification

The prevalence of all forms of diabetes mellitus continues to rise across the globe. Advances in islet transplantation have encouraged efforts to overcome the shortage of pancreas donor tissue needed for the ever-increasing patient population. Recent hES cell differentiation protocols have successfully induced pancreatic progenitor and islet cell type specification characterized by transcription factor expression profiles. A significant impediment to extend the technology for large-scale therapeutic use is the lack of efficient and highly specific differentiation protocols. Current procedures harness the knowledge gained through extensive studies of extracellular signaling pathways important for pancreas morphogenesis during embryogenesis. Multiple signaling pathways are induced at critical developmental stages to promote transcriptional programs important for endodermal and pancreas cell specification. Nkx2.2 is one of the critical homeodomain transcription factors that is essential for endocrine pancreas cell specification. Endocrine compartments of Nkx2.2-/- mice lack beta cells, have reduced alpha and PP cells, and instead contain increased numbers of epsilon cells. To elucidate a molecular mechanism for Nkx2.2, we first compared gene expression differences between wildtype and Nkx2.2-/- pancreata at the onset of islet differentiation within the pancreas. This analysis determined that Nkx2.2 is cooperating in an early transcription factor module comprised of Ngn3, Myt1, NeuroD1 and MafB, which are important for endocrine progenitor cell differentiation. Through comprehensive NeuroD1 promoter studies, we have determined that Nkx2.2 activates NeuroD1 expression through indirect and direct regulatory mechanisms. In addition, we determined Nkx2.2 cooperates with Ngn3 to activate NeuroD1 expression, providing further evidence of a role for Nkx2.2 in a progenitor transcription factor module.;Another notable finding of this is study is the identification of the tetraspanin-like protein, Tm4sf4, which is highly upregulated in the absence of Nkx2.2. Tm4sf4 is expressed in ductal progenitors, and functions to inhibit alpha and beta cell specification and/or formation. To date, most of the events that function downstream of transcription factor mechanisms responsible for cell specification are unknown. With the discovery of Tm4sf4, we are proposing a model whereby Nkx2.2 represses Tm4sf4, which allows for endocrine progenitor cell migration and differentiation. Together, our studies have provided the first mechanistic insight into Nkx2.2 regulation of early pancreas progenitor specification. The downstream factors and mechanisms regulated by Nkx2.2 highlight the existence of tightly controlled processes that regulate pancreas cell fate determination. Our preliminary studies suggest that down-regulation of the transmembrane protein, Tm4sf4, should allow improvement of hES cell differentiation protocols and may allow targeted drug design to induce differentiation of quiescent adult pancreas progenitors. Combined, our results will benefit pancreas development research and provoke new approaches to diabetes therapy.