A Membrane Associated mCherry Fluorescent Reporter Line for Studying Vascular Remodeling and Cardiac Function During Murine Embryonic Development

Abstract

The development of the cardiovascular system is a highly dynamic process dependent on multiple signaling pathways regulating proliferation, differentiation, migration, cell-cell and cell-matrix interactions. To characterize cell and tissue dynamics during the formation of the cardiovascular system in mice, we generated a novel transgenic mouse line, Tg(Flk1::myr-mCherry), in which endothelial cell membranes are brightly labeled with mCherry, a red fluorescent protein. Tg(Flk1::myr-mCherry) mice are viable, fertile, and do not exhibit any developmental abnormalities. High levels of mCherry are expressed in the embryonic endothelium and endocardium, and expression is also observed in capillaries in adult animals. Targeting of the fluorescent protein to the cell membrane allows for subcellular imaging and cell tracking. By acquiring confocal time lapses of live embryos cultured on the microscope stage, we demonstrate that the newly generated transgenic model beautifully highlights the sprouting behaviors of endothelial cells during vascular plexus formation. We have also used embryos from this line to imaging the endocardium in the beating embryonic mouse heart, showing that Tg(Flk1::myr-mCherry) mice are suitable for the characterization of cardio dynamics. Furthermore, when combined with the previously described Tg(Flk1::H2B-EYFP)

Dynamic responses of endothelial cells to changes in blood flow during vascular remodeling of the mouse yolk sac

“…Flk1-myr::mCherry Poché et al, 2009) and Flk1-H2B::eYFP (Fraser et al, 2005;Larina et al, 2009) (Huang et al, 2003;Lucitti et al, 2007). Embryos were genotyped using the following primers (5Ј-3Ј): mutant band, Mlc2a-mut-F (ACAGGGAATCACACCACCAT) and Mlc2a-mut-R (CGAACC -TGGTCGAAATCAG); wild-type band, Mlc2a-wt-F (GGCACGAT-CACTCAGTCAGA) and Mlc2a-wt-R (ATCCCTGTTCTGGTCAATGC).…”

“…We used transgenic mice that fluorescently label the EC membranes (Flk1-myr::mCherry) and nuclei (Flk1-H2B::eYFP) and that label primitive blood cells (ε-Globin-GFP) (Dyer et al, 2001;Fraser et al, 2005;Larina et al, 2009;Poché et al, 2009). Using time-lapse imaging of dynamic events, as well as the analysis of cell death and proliferation in fixed embryos, we have found that vessel diameter increases do not result from changes in EC proliferation or survival.…”

Quantitative imaging of cell dynamics in mouse embryos using light-sheet microscopy

“…To test the new hollow agarose cylinders in the light-sheet microscope, we imaged Flk1-myr::mCherry; Flk1-H2B::eYFP double-transgenic embryos (Fraser et al, 2005;Larina et al, 2009;Poché et al, 2009), in which endothelial cell (EC) membranes and nuclei were labeled with mCherry and yellow fluorescent protein (YFP), respectively, similar to our previous studies (Udan et al, 2013). For embryos between E8.5 and E9.5, we used agarose cylinders with a 4.7 mm outer diameter and a 2.5 mm inner diameter (see Materials and Methods).…”

Flk1-GFP BAC Tg Mice: An Animal Model for the Study of Blood Vessel Development

“…This enhancer was successfully used to drive mCherry expression in endothelial cells by other researchers [11,14]. However, deletion of this enhancer has no significant effect upon Flk1 expression in mesodermal progenitors at 7.5 dpc or in endothelial cells at 8.5 dpc [3].…”