Abstract
Previous reports regarding the genetic hierarchy between Ets related protein 71 (Er71/Etv2) and Flk1 is unclear. In the present study, we pursued a genetic approach to define the molecular cascade between Etv2 and Flk1. Using a transgenic Etv2-EYFP reporter mouse, we examined the expression pattern of Etv2 relative to Flk1 in the early conceptus. Etv2-EYFP was expressed in subset of Flk1 positive cells during primitive streak stages, suggesting that Flk1 is upstream of Etv2 during gastrulation. Analysis of reporter gene expression in Flk1 and Etv2 mutant mice further supports the hypothesis that Flk1 is necessary for Etv2 expression. The frequency of cells expressing Flk1 in Etv2 mutants is only modestly altered (21% decrease), whereas expression of the Etv2-EYFP transgenic reporter was severely reduced in the Flk1 null background. We further demonstrate using transcriptional assays that, in the presence of Flk1, the Etv2 promoter is activated by VEGF, the Flk1 ligand. Pharmacological inhibition studies demonstrate that VEGF mediated activation is dependent on p38 MAPK, which activates Creb. We identify the VEGF response element in the Etv2 promoter and demonstrate that Creb binds to this motif by EMSA and ChIP assays. In summary, we provide new evidence that VEGF activates Etv2 by signaling through Flk1, which activates Creb through the p38 MAPK signaling cascade.
Highlights
Homozygous gene disruption of Etv2, an Ets DNA binding domain containing transcription factor, results in nonviable embryos at a midgestational age (E9.5)
In collaboration with Etv2 and Flk1, vascular endothelial growth factor (VEGF) is essential for vasculogenesis as embryos deficient for a single VEGF allele are nonviable at a midgestational age (E10.5) and have severe vascular defects [8,9]
We provide data that the Flk1 receptor is activated by its ligand VEGF and activates the p38 MAPK signaling cascade, which, in turn, activates CRE binding proteins (Creb) to induce expression of Etv2
Summary
Homozygous gene disruption of Etv, an Ets DNA binding domain containing transcription factor ( known as Er71/ Etsrp71), results in nonviable embryos at a midgestational age (E9.5). While the Flk knockout embryos retain Flk1-LacZ expressing cells, the endothelial and hematopoietic lineages are lost, suggesting that Flk1+mesoderm is specified but does not further differentiate to form vessels or blood. Flk is a cell surface receptor that is known to induce signaling cascades in response to vascular endothelial growth factor (VEGF). VEGF/Flk signaling is known to activate p38 MAPK, ERK1/2 MAPK, PKC, and other signaling cascades to induce phosphorylation of target proteins, including cellular enzymes and transcription factors [5,6,7]. In collaboration with Etv and Flk, VEGF is essential for vasculogenesis as embryos deficient for a single VEGF allele are nonviable at a midgestational age (E10.5) and have severe vascular defects [8,9]
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