The Role of Methionine Aminopeptidase II in Definitive Hematopoiesis in Zebrafish Embryos

Abstract

Objective: Methionine aminopeptidase 2 (Metap2) is the target of anti-angiogenic compound fumagillin and has been investigated for its expression in cancers and its antiproliferative effects on endothelial cells. It is a protease which removes N-terminal methionine from newly synthesized peptides and also the eIF- 2 associated 67 kDa protein (p67), which prevents phosphorylation of eIFα and ERK1/2, fine-tuning cellular protein synthesis and proliferation. However, the role of Metap2 during embryonic development remained unclear. Here, we used zebrafish model to investigate the role of metap2 during embryonic development, with particular reference to definitive hematopoiesis. Methods and Results: We injected morpholino targeting the splice-junction of metap2 into zebrafish embryos (referred as metap2MO embryos). Molecular targeting was confirmed by RT-PCR. Modulation of metap2 activity was shown by shifting of the gapdh isoelectric point. The metap2MO embryos exhibited significant upregulation of definitive hematopoietic genes (cmyb and runx1) as shown by both in-situ hybridization and real-time PCR at 36 hour-postfertilization. The phenotype could be rescued by co-injecting the embryos with metap2 mRNA and re-capitulated by treating the embryos with fumagillin. Mechanistically, metap2MO embryos shows ectopic erk1/2 phosphorylation and the hematopoietic phenotypes could be rescued by treating embryos with ERK inhibitors. The metap2MO embryos also exhibited reduced camodulin kinase 2 (camk2) activity and the hematopoietic phenotypes could also be rescued by camk2 mRNA and recapitulated by a CaMK2 inhibitor. The canonical Wnt pathway, characterized by