Abstract 17036: Flk1 Mediated Activation of ER71 and Specification of Cardiovascular Lineages
We have previously demonstrated that Ets Related Protein 71 (ER71) is essential for primitive hematopoiesis and development of the endothelial/endocardial lineages. Lack of ER71 results in early (E8.0-E9.0) embryonic lethality. We generated an ER71-EYFP transgenic reporter mouse model using FACS and transcriptome analysis and determined that hematopoieitic and endothelial/endocardial molecular programs are restricted to the FACS sorted ER71-EYFP+ population. Using immunohistochemical techniques, we observed coexpression of EYFP (representing ER71 expression) and Flk1 between E7.0 and E9.5. However, Flk1 expression appears to precede ER71 expression in the primitive streak. Based on our observation that ER71 mutants phenocopy Flk1 null embryos at E8.0, we undertook a genetic analysis to determine whether Flk1 was upstream of ER71. We analyzed Flk1 expression in the ER71 mutant background by utilizing the Flk1-LacZ allele, FACS analysis, and qPCR. While Flk1-LacZ shows an aberrant spatial expression pattern, the frequency of cells expressing Flk1 is unchanged at E7.75 and is decreased modestly (two-fold) at E8.0. This demonstrates that Flk1 is not completely dependent on ER71 expression. In contrast, crossing the ER71-EYFP transgenic reporter allele into the Flk1 null background revealed that ER71 expression at E7.75 and E8.0 is dependent on Flk1. Furthermore, we have identified an evolutionarily conserved NFAT binding site in the essential ER71 promoter. NFAT is a family of transcription factors known to mediate a VEGF signalling cascade that involves the Flk1 receptor and Calcineurin. We have used ChIP and EMSA to verify that NFAT directly binds the ER71 promoter. Using transcriptional assays, we further demonstrate that ER71 is regulated by NFAT and Calcineurin. Collectively, these studies support the hypothesis that a Flk1-ER71 network specifies and coordinates the endothelial/endocardial lineages during cardiovascular development.
- Endothelial progenitor cell
- Gene mutations
- Molecular biology
- Vascular development
- Biology, developmental
- © 2011 by American Heart Association, Inc.