Identification of Vascular Endothelial Growth Factor (VEGF) Receptor-2 (Flk-1) Promoter/Enhancer Sequences Sufficient for Angioblast and Endothelial Cell-Specific Transcription in Transgenic Mice

Abstract

The vascular endothelial growth factor (VEGF) receptor-2 (Flk-1) is the first endothelial receptor tyrosine kinase to be expressed in angioblast precursors, and its function is essential for the differentiation of endothelial cells and hematopoietic precursors. We have identified cis-acting regulatory elements of the murineFlk-1 gene that mediate endothelium-specific expression of a LacZ reporter gene in transgenic mice. Sequences within the 5′-flanking region of the Flk-1 gene, in combination with sequences located in the first intron, specifically targeted transgene expression to angioblasts and endothelial cells of transgenic mice. The intronic regulatory sequences functioned as an autonomous endothelium-specific enhancer. Sequences of the 5′-flanking region contributed to a strong, uniform, and reproducible transgene expression and were stimulated by the transcription factor HIF-2. The Flk-1 gene regulatory elements described in this study should allow the elucidation of the molecular mechanisms involved in endothelial cell differentiation and angiogenesis.

Using a histone yellow fluorescent protein fusion for tagging and tracking endothelial cells in ES cells and mice

“…The expression of the H2B-EYFP fusion was assessed in staged embryos ranging from late gastrulation/early headfold ($E7.5) to E17.5; the two transgenic lines gave indistinguishable patterns of expression. As anticipated from previous reports (Hirai et al, 2003;Kappel et al, 1999), the combination of Flk1 regulatory elements used for the Flk1::H2B-EYFP construct (Fig. 1) was not active in the extraembryonic mesoderm (yolk sac) prior to endothelial lineage specification.…”

“…Having established the subcellular localization and neutrality of the H2B-EYFP fusion in vitro, we isolated Flk1 BAC clones and generated a construct ( Fig. 1) containing previously characterized Flk1 promoter and intronic enhancer regions (Kappel et al, 1999;Ronicke et al, 1996). These sequences were demonstrated to drive expression of a linked reporter in endothelial cells, using immunostaining (Kappel et al, 1999), fluorescence activated cell sorting (FACS), or uptake of DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL) (Hirai et al, 2003).…”

“…Therefore, to understand in detail the dynamic events leading to vascular development in the mouse, it will be essential to image individual endothelial cells in live embryos. Transgenic mouse lines carrying lacZ reporters have been used by a number of investigators to examine blood vessel formation (e.g., see Kappel et al, 1999;Ronicke et al, 1996;Schlaeger et al, 1997). However, because identification of cells expressing the lacZ transgene requires tissue fixation, this analysis provides only a snapshot of a given developmental stage.…”

Evidence for novel fate of Flk1+ progenitor: Contribution to muscle lineage

“…It has been reported that deletion of part of the 5Ј UTR region of the Flk1 gene in the knock-in construct abolishes part of the endothelial expression in the yolk sac at embryonic day E8.5, although expression in the embryo was not affected (Kappel et al, 1999). However, in our hands this published work could not be reproduced.…”

VEGFR3gene structure, regulatory region, and sequence polymorphisms

“…Many tissue-specific gene regulatory elements are located within the first two introns, although such elements occasionally can be found at a great distance, even in the 3Ј parts of genes. Vegfr2 and Tie2 are examples of endothelial cell-specific genes whose activity is partly regulated by enhancers located in their first introns (31,32). Therefore, we are currently analyzing the large first introns of the mouse and human Vegfr3 genes to locate putative enhancer elements.…”