Lefty2 and Pitx2 analyzed after administration of FK506. (a) Western blotting of Lefty2. Bands from FK506-treated fetuses were slightly thinner compared with those from saline-treated fetuses. Lefty2 expression was decreased by about 20% in FK506-treated fetuses relative to saline-treated fetuses. (b) Western blotting of Pitx2. The Pitx2 band from FK506-treated fetuses was strong, and quantification indicated that pitx2 protein expression was about 50% greater than that in saline-treated fetuses. ((c) and (d)) Pitx2 immunohistochemistry on a section from saline-treated fetuses (c) and FK506-treated fetuses (d). After injection of 1.5 mg/kg of FK506 into amniotic fluid, the expression of Pitx2 extended to the right side of the embryo. The arrow indicates the front edge of ectopic expression on the right side.