Title: The role of Notch signaling in hemogenic endothelial cell development
Description: Notch signaling plays important roles in development of embryos by participating in cell-fate decisions and differentiation of many different cell types, including endothelial cells and hematopoietic cells. Hematopoiesis in the embryo occurs in two phases; a transient primitive phase and a definitive phase which generates hematopoietic stem cells (HSCs) that constitute the whole blood system. Notch has been known to be specifically required for definitive hematopoiesis and proper endothelial cell development. In studying mouse embryonic stem cell (mESC) differentiation, we generated an ESC line that expresses the active form of Notch1 (ICN1) after induction with doxycycline. During embryoid body (EB) differentiation, ICN1 induction increased hematopoietic differentiation with an increase of CD41 - VE-cadherin + α4-integrin + CD45 - hemogenic endothelial population, which showed hematopoietic and endothelial cell outgrowth in the subsequent culture after sorting. Gene expression analysis showed an upregulation of Foxc2 in this population after ICN1 induction. Genetic studies in the zebrafish showed that Foxc2 and its orthologs are downstream targets of Notch signaling in hemogenic endothelial cell development, and the analysis of Foxc2-/- mouse embryos further confirmed the requirement of Foxc2 in definitive hematopoiesis. In human embryonic stem cell (hESC) differentiation, Notch ligand treatment promoted hematopoietic differentiation with an increase of VE-cadherini low α4-integrin+ population. These results indicate that upregulating Notch signaling during ES cell differentiation may promote definitive hematopoiesis through hemogenic endothelial cells.

In summary, in the zebrafish, mouse, and human, data collected here suggest that Notch signaling plays an important role in hemogenic endothelial cell development. mEBs provided the platform to capture the gene expression profile of hemogenic endothelial cells, which lead to the further analysis of Foxc2 in zebrafish and mouse embryos. Ligand treatment during hEB differentiation showed similar results observed in mEB differentiation without genetic modification, suggesting evolutionary conservation of the Notch pathway and its effect on blood development. Further characterization of the emergence of hemogenic endothelial cells during embryo development may help better understand the guided differentiation of HSCs from pluripotent stem cells and open new clinical opportunities.