Angiogenesis and hematopoiesis are functionally related processes that generate the vessels and blood cells of the circulatory system. Recent data have demonstrated that oxygen deprivation (hypoxia), in addition to developmentally regulated signals, is a potent activator of angiogenesis and hematopoiesis. To investigate the molecular mechanisms whereby hypoxia regulates these processes, mutations have been generated in the murine Arnt gene, which encodes a bHLH-PAS transcription factor. ARNT protein regulates the expression of Epo, VEGF and other angiogenesis-related genes through interactions with its heterodimeric partner, HIF-1alpha. Arnt -/- ES cells fail to upregulate may target genes under hypoxic conditions, and Arnt-/- mutant embryos are arrested in development at embryonic day E9.5-10.5, apparently due to vascular abnormalities in the placenta, yolk sac and embryo itself. More recently, it has been shown that the Arnt -/- mutation also disrupts normal hematopoiesis in yolk sac blood islands. A second, highly related murine Arnt gene (Arnt 2) has recently been described. Although expressed in a strikingly different spatial pattern than Arnt 1 in embryonic and adult mice, it is possible that Arnt 2 may partly compensate for the loss of the Arnt protein in Arnt -/- mutant embryos. It is proposed to generate and analyze Arnt 2 -/- and Arnt -/-, Arnt 2-/- double mutant mouse strains, which will address many important questions regarding the role of both Arnt and Arnt2 in mediating hypoxia-induced angiogenesis and hematopoiesis. As hypoxic responses are more important in a variety of pathologies, including post-ischemic neovascularization and tumor cell apoptosis, the results of these experiments should prove valuable in developing clinical approaches in the future. At a basic level, they will provide important information oon hypoxia-induced transcriptional regulation that complements our rapidly expanding understanding of receptor/ ligand signalling in angiogenesis and hematopoiesis.
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