The proepicardium (PE) is an embryonic tissue that gives rise to multipotent vascular progenitors. Most notably the PE gives rise to the epicardium, cardiac fibroblasts, myocardium, and coronary vessels including both vascular smooth muscle and vascular endothelium. Much attention has been given to epicardial-derived cells that show the capacity to differentiate into a wide variety of vascular progenitors including cardiomyocytes. However, it is the PE itself that possesses the greatest potential as a source of multipotent vascular progenitors. We show here a simple method to manually isolate mouse PE at the ninth day of mouse embryonic development and culture highly pure PE tissue in serum-free conditions. This PE culture method allows for the ex vivo analysis of specific growth factors on PE and epicardial development with greater efficiency and precision than existing epicardial culture methods.

Polarization of node cells along the anterior-posterior axis of mouse embryos is responsible for left-right symmetry breaking. How node cells become polarized has remained unknown, however. Wnt5a and Wnt5b are expressed posteriorly relative to the node, whereas genes for Sfrp inhibitors of Wnt signaling are expressed anteriorly. Here we show that polarization of node cells is impaired in Wnt5a^-/-Wnt5b^-/- and Sfrp mutant embryos, and also in the presence of a uniform distribution of Wnt5a or Sfrp1, suggesting that Wnt5 and Sfrp proteins act as instructive signals in this process. The absence of planar cell polarity (PCP) core proteins Prickle1 and Prickle2 in individual cells or local forced expression of Wnt5a perturbed polarization of neighboring wild-type cells. Our results suggest that opposing gradients of Wnt5a and Wnt5b and of their Sfrp inhibitors, together with intercellular signaling via PCP proteins, polarize node cells along the anterior-posterior axis for breaking of left-right symmetry.