HoxA3 Controls Notch Pathway to Repress Blood Development

Hematopoietic stem cells (HSC) are generated from a specialized subset of endothelial cells, hemogenic endothelium. Previous studies performed by our group showed that HoxA3 restrains the cell at the hemogenic endothelium stage, inhibiting further differentiation toward blood by direct repression of Runx1. Building on our previous work, we show here that overexpression of HoxA3 affects the Notch pathway. Upon HoxA3 upregulation in endothelial cells, Jag1 is induced, Mfng (Manic) and Lfng (Lunatic) fringes are downregulated, and there is a trend towards Notch target gene repression. These data suggest that in the presence of HoxA3, endothelial cells are blocked from receiving Notch signal through ligand cis-inhibition with resulting blood inhibition. In order to test this hypothesis, we evaluated the effect of activation or inhibition of the Notch pathway during blood development. We show here that the number of blood progenitors originating from the hemogenic endothelium is decreased when the Notch pathway is inhibited. Conversely, induction of the pathway by upregulation of NICD (Notch1 Intra Cellular Domain) promotes an increase in the number of blood progenitors originating from hemogenic endothelium. Furthermore, inhibition of the pathway when HoxA3 is upregulated has little or no effect in blood while induction of the pathway in HoxA3 presence in part promotes blood development. Taken together, these results demonstrate that Notch pathway is both sufficient and essential for blood development. Specifically HoxA3 inhibits Notch signal reception in two ways: 1) HoxA3 increases Jag1 ligand expression that acts in cis-inhibition; 2) represses Manic and Lunatic fringes both necessary to increase the affinity of Notch receptors for the Delta ligands. When this blockage is bypassed by NICD upregulation, blood is formed, demonstrating that HoxA3-dependent Notch inhibition results in blood suppression.