The cellular origin and identity of the signals that regulate induction of Insulin-producing pancreatic beta-cell in vivo are not clearly defined. Recent efforts have been aimed at generating functional pancreatic beta-cells from embryonic stem (ES) cell cultures for use in transplantation therapy of diabetes mellitus. A complete mechanistic understanding of how pancreatic beta-cells are induced in vivo will help achieve this goal. During my thesis work, I studied the mechanisms underlying pancreatic beta-cell induction, using the zebrafish as a model system.

First, contrary to the general assumption that mesodermal cells are mostly responsible for inducing pancreatic beta-cells, I found that interactions between endodermal cells are essential for pancreatic beta-cell induction. Through cell lineage analyses, I showed that when the zebrafish endoderm forms a sheet, pancreatic beta-cell precursors lie closest to the midline, the site of Hedgehog (Hh) ligand production. Cell transplantation experiments revealed that Smoothened function, which is activated by Hh ligands and required for the induction of pancreatic beta-cells, is not required in beta-cell precursors. However, the requirement for Smoothened lies in the cells directly adjacent to the beta-cell precursors, which ultimately give rise to the exocrine pancreas and intestine. Thus, pancreatic beta-cell induction requires Smoothened function cell non-autonomously to allow subsequent intra-endodermal interactions.

Second, intrigued by the results showing potential differences between the medial vs lateral endodermal cells, I subsequently performed single cell lineage tracing experiments and the resulting data led me to propose that the endodermal sheet undergoes medio-lateral patterning, a concept that had not been appreciated before, especially as endodermal organs line up along the anterior-posterior axis. Loss- and gain-of-function analyses showed that Bmp2b, which is expressed in the lateral plate mesoderm, signals through Alk8 to induce lateral endodermal cells to become liver. When Bmp2b was overexpressed, medially located endodermal cells, fated to become pancreas and intestine, contributed to the liver. These data provide in vivo evidence for the existence of bipotential hepatopancreatic progenitors, and indicate that their fate is regulated by the medio-lateral patterning of the endodermal sheet, a process controlled by Bmp2b.

Finally, I showed that Bmp signaling restricts the ability of endodermal cells to differentiate into pancreatic endocrine cells, including Insulin-secreting beta-cells. Detailed analysis of mosaic embryos reveals that activation of Bmp signaling in endodermal cells blocks induction of pancreatic beta-cells from both dorsal and ventral pancreatic progenitors. Conversely, suppression of Bmp signaling and knock-down of the type I Bmp receptor Alk8 (also called Alk2 and AcvR1) in the endoderm results in the ectopic formation of pancreatic beta-cells. Further data suggest that Bmp signaling mediates its inhibitory effect by inducing Id2, which in turn inhibits the formation of pancreatic endocrine cells. Thus, Bmp signaling needs to be specifically suppressed in the pancreatic progenitors to allow their differentiation into beta-cells.