The ALK1 pathway is directly involved in lymphatic vessel development. (A) Analysis of lymphatic and vascular development at P6, after treatment with ALK1Fc, ACVR2BFc, BMPR2Fc, or VEGFR3Fc (10 mg/kg, P1 and P3). Lymphatic development is visualized by LYVE1 (green) staining in the tail dermis. Vascular development is visualized by isolectin-B4 (green) and SMA (red) staining of the retina. Scale bar represents 250 μm. (B) Inhibition of rhBMP9 induced up-regulation of Smad6 expression by the ALK1 immunized serum (α-ALK1-s) or affinity purified antibody (α-ALK1-a), whereas preimmune serum (Pre) has no effect. Red boxes represent each data point (n = 3). (C) Inhibition of rhBMP9 binding to ALK1 protein by treatment with the ALK1 immunized serum (α-ALK1-s) or affinity purified antibody (α-ALK1-a), whereas preimmune serum (Pre) has no effect. Red boxes represent each data point (n = 2). (D) ALK1-neutralizing antibody binds mouse ALK1 but not ALK2, 3, 4, 5, 6, or 7. (E) Analysis of lymphatic development at P6 after treatment with the ALK1 neutralizing antibody (10 mg/kg, P1 and P3) in the tail dermis. (F) Analysis of lymphatic development at P6 after treatment with the ALK1-neutralizing antibody (10 mg/kg, P1 and P3) in the ear. Lymphatic development is visualized by LYVE1 (green) staining in the tail dermis and ear. Scale bar represents 250 μm.