Chemical inhibition of xVEGFR-3 signaling with MAZ51 causes lymphatic defects. (A,B) Morphometric measurement of area 2a and 2b in the posterior trunk of xProx1-stained tadpoles at stage 35/36, showing dose-dependent inhibition of migration by MAZ51, reflected by the stalling of the cells in area 2a. *P < .05 versus control (DMSO). The number of tadpoles is indicated within each bar. Error bars represent SEM. (C-K) Functionality of the lymphatic and blood vasculature in control and MAZ51-treated tadpoles. Lymphangiography showed normal filling of the VCLV in DMSO-treated control tadpole (stage 45) (C) without signs of edema (E) and displaying normal intact blood vessels upon microscopic inspection (G) and angiography (I). In contrast, stage-45 tadpoles treated with 10 μM MAZ51 showed dysfunctional lymph vessels upon lymphangiography (D) and edema formation (F). The arrow in panel D indicates the distal site of VCLV filling. Angiography revealed blood vessel defects, including irregularly shaped DLAVs and malformed or absent intersomitic vessels (J,K), and leakage of the fluorescent dye (arrows in K) suggesting vessel fragility. Widespread hemorrhaging throughout tadpoles treated with 10 μM MAZ51 was also suggested by o-dianisidine staining of erythrocytes, showing numerous patches of cells outside of the blood vasculature (H; compare with control in G). DA indicates dorsal aorta; DLAV, dorsal longitudinal anastomosing vessel; ISV, intersomitic vessel; PCV, posterior cardinal vein; and VCLV, ventral caudal lymph vessel. Bars represent 500 μm (C,D,G,H), 1 mm (E,F), and 100 μm (I-K).