Abstract 17270: Discovery and Characterization of a Novel Transcription Factor for Endothelial Cell Development and Lineage Specification
Background: We have developed bi-species heterokaryons [generated by fusion of mouse embryonic stem cells (mESC) and human endothelial cells (hEC)] as a model system for discovery of novel factors required for endothelial lineage. Our preliminary RNAseq data suggests that the determinants of endothelial phenotype in the hEC act on the mESC to recapitulate endothelial ontogeny. Furthermore, novel transcription factors in endothelial specification were implicated, such as POU domain-containing transcription factor (POU3F2), also called BRN2 or N-Oct3. This study assessed the role of POU3F2 in the endothelial cell differentiation and in the zebrafish vascular development.
Methods and Results: We used mESC to study differentiation towards endothelial lineage. EC differentiation was induced by culture of mESC with growth factors (VEGF, bFGF and BMP4). POU3F2 loss-of-function was induced by lentiviral shRNA in mESCs. FACS was used to analyse cell lineage. Tg(Fli1:eGFP) zebrafish embryos were used to analyse vascular development following caged morpholino (MO) knockdown of POU3F2. Injected caged morpholino was activated at 6 or 24 hour post fertilization by exposure of embryos to UV light. Real Time PCR and Western blotting were used to analyse gene and protein expression respectively. POU3F2 knockdown in mESCs reduced Flk1+CD144+ cell population during differentiation of mESCs. POU3F2 knockdown also reduced endothelial cell markers in mESC derived ECs, including Kdr, Cdh5, Nos3, Tie2 and Lmo2 and reduced EC tube formation in matrigel. In zebrafish embryos, micro-injection of MOs targeting POU3F2 reduced POU3F2 protein at 24 and 48 hpf. This was associated with an embryo phenotype characterized by severe vascular aberrations.
Conclusion: Our heterokaryon studies implicated the transcription factor POU3F2 in endothelial cell development. We validated the role of POU3F2 in the reprogramming of pluripotent stem cells to EC lineage. In addition, we provide data that POU3F2 is required for normal vascular development in the zebrafish.
Author Disclosures: G. Matrone: None. X. Tian: None. W. Wong: None. J.P. Cooke: None.
This research has received full or partial funding support from the American Heart Association
- © 2014 by American Heart Association, Inc.