Project description:Hypoxia (low oxygen) and Notch signaling are two important regulators of vascular development, but how they interact in controlling the choice between arterial and venous fates for endothelial cells during vasculogenesis is less well understood. In this report, we show that hypoxia and Notch signaling intersect in promotion of arterial differentiation. Hypoxia upregulated expression of the Notch ligand Dll4 and increases Notch signaling, in a process requiring the vasoactive hormone adrenomedullin but not endogenous VEGF. Notch signaling also upregulated Dll4 expression, leading to a positive feedback loop sustaining Dll4 expression and Notch signaling. In addition, functional Notch signaling was required for hypoxia to upregulate the arterial marker genes Depp, connexin40 (Gja5), Cxcr4 and Hey1. In conclusion, the data reveal an intricate interaction between hypoxia and Notch signaling in the control of endothelial cell differentiation, including a hypoxia/adrenomedullin/Dll4 axis that initiates Notch signaling and a requirement for Notch signaling to effectuate hypoxiamediated induction of the arterial differentiation program.
Project description:Hypoxia (low oxygen) and Notch signaling are two important regulators of vascular development, but how they interact in controlling the choice between arterial and venous fates for endothelial cells during vasculogenesis is less well understood. In this report, we show that hypoxia and Notch signaling intersect in promotion of arterial differentiation. Hypoxia upregulated expression of the Notch ligand Dll4 and increases Notch signaling, in a process requiring the vasoactive hormone adrenomedullin but not endogenous VEGF. Notch signaling also upregulated Dll4 expression, leading to a positive feedback loop sustaining Dll4 expression and Notch signaling. In addition, functional Notch signaling was required for hypoxia to upregulate the arterial marker genes Depp, connexin40 (Gja5), Cxcr4 and Hey1. In conclusion, the data reveal an intricate interaction between hypoxia and Notch signaling in the control of endothelial cell differentiation, including a hypoxia/adrenomedullin/Dll4 axis that initiates Notch signaling and a requirement for Notch signaling to effectuate hypoxiamediated induction of the arterial differentiation program. 12 microarray samples consisting of >50,000 FACS sorted CD31+ cells purified from wild type mouse CCE ES cells that were differentiated into the endothelial lineages in 3 biological replicates. The ES cells were subjected to embryoid body formation over 4 days in hanging drop cultures, FACS sorted for Flk1 positive vascular progenitors cells and plated for a further 4 days in normoxia (21% oxygen) or hypoxia (1.5-2% oxygen) with or without 4 umol/l gamma-secretase inhibitor L-685.458.
Project description:Background: Interaction between key signaling mechanisms is important to generate the diversity in signaling output required for proper control of cellular differentiation and function, although the molecular manifestations of such cross-talk are only partially understood. Notch signaling and the cellular response to hypoxia intersect at different points in the signaling cascades, and in this report we analyze the consequences of this cross-talk at the transcriptome level. Results: Mouse ES cells were subjected to various combinations of hypoxia and/or activated Notch signaling, and the transcriptome changes could be grouped into different categories, reflecting various modes of hypoxia and Notch signaling integration. Two principal categories of novel Notch- and hypoxia-induced genes were identified: i) a larger set of genes induced by one pathway and not significantly affected by the activity status of the other pathway; and ii) a smaller set of genes co-regulated by Notch and hypoxia. In the latter category, we identified genes that were induced by hypoxia and the expression of which was enhanced by active Notch signaling. In addition, a number of genes were induced by Notch and hypoxia independently, and a final category of genes required simultaneous activation of Notch and hypoxia to be significantly induced. Several of the hypoxia- and Notch-induced genes were found to be upregulated in various forms of cancer. Conclusions: We identify novel Notch and hypoxia downstream genes and genes co-regulated by the two pathways, providing a molecular platform to better understand the intersection between the two signaling cascades in normal development and cancer.