Fig. 5. VEGF is not required for Npn1-mediated cleft formation in mice. (A) Whole-mount immunohistochemistry showed that VEGF and its receptors, Flt1 and Flk1, did not express in the epithelial buds of the SMG. By contrast, Fgfr2 was expressed in the SMG epithelial buds as previously reported. (B) Neither exogenous VEGF nor VEGF antibody (VEGF Ab) affected normal branching morphogenesis of the SMG cultured ex vivo. The bar graph summarizes five independent experiments. (C) VEGF had no effects on the branching activities promoted by Sema3A. The bar graph summarizes four independent experiments. (D) VEGF could not change effects caused by application of Npn1-neutralizing antibody at sub-optimal concentration (2 µg/ml) in the SMG ex vivo cultures. The bar graph summarizes five independent experiments. Scale bars: 100 µm.