Inhibition of Notch1 and Dll4 signaling impairs lymphatic development. (A) Lymphatic network in the P7 tail dermis isolated from neonates treated with PBS (control), αNotch1, αDll4, ALK1-Fc, and VEGFR3-Fc. In all treatments, LYVE1 staining was imaged at saturation to highlight the entire lymphatic network. The arrow indicates the multiringed structures that are interconnected by 1 or 2 (arrowhead) lymphatic vessels that are inhibited by Notch1/Dll4 blockade. Scale bar represents 200 μm. (B) LYVE1 and VEGFR3 costaining of the P7 tail dermis isolated from neonates treated with PBS (control), αNotch1, and αDll4. The arrows indicate the failed connection multiringed structures in αNotch1- or αDll4-treated pups, observed by both LYVE1 and VEGFR3 expression. Scale bar represents 200 μm. (C) Quantification of VEGFR3 staining in the tail dermis isolated from P7 neonates treated with PBS (control), αDll4, and ALK1Fc. n = 3, *P < .05. (D) LYVE1 staining of P7 ear tissue from neonates treated with PBS (control), αNotch1, αDll4, ALK1-Fc, and VEGFR3-Fc. Top panels are the inner surface and the lower panels are the outer surface of the ear. Scale bar represents 500 μm. (E) LYVE1 staining of P8 tail dermis isolated from neonates treated with PBS (control), αDll4, VEGFR3-Fc, or combination of αDll4 and VEGFR3-Fc treatments. Scale bar represents 200 μm. In panels A, B, and D, pups were injected with 10 mg/kg proteins on P2 and P5. In panel E. pups were injected with 10 mg/kg αDll4 on P3 and P5, VEGFR3-Fc on P7, or combination of both treatments.