Morpholino knockdown of xVEGFR-3 causes lymphatic defects. (A) Morphometric measurement of area 2 in the posterior trunk of xProx1-stained tadpoles at stage 35/36, showing dose-dependent inhibition of migration after xVEGFR-3 knockdown. The number of tadpoles is indicated within each bar. *P < .05 versus control (control morpholino) for pairwise dose analysis. The percentage of tadpoles exhibiting a lower value of the morphometric parameter than the average value in the control group (a measure of the penetrance of the phenotype) and the P value of the chi-square analysis comparing this percentage with the control group are indicated below the bar graphs. Note that the phenotype as well as its penetrance increased dose-dependently. Error bars represent SEM. (B,C) Whole-mount in situ hybridization for xProx1 of stage-42 control or xVEGFR-3 knockdown tadpoles (100 ng morpholino), showing only fragmentary formation of the ventral (VCLV) and dorsal (DCLV) caudal lymph vessels in the VEGFR-3^KD tadpoles (C) in contrast to completely formed VCLV and DCLV in the control (B). Arrows in panel C denote missing vessel segments. (D-I) Functionality of the lymphatic and blood vasculature in control and VEGFR-3^KD tadpoles. (D,F,H) Normal filling of the VCLV in control tadpole (stage 45) upon lymphangiography (D), with normal appearance of the vasculature by angiography (F) and no signs of edema (H). (E,G,I) In contrast, VEGFR-3^KD tadpoles at stage 45 showed dysfunctional lymph vessels upon lymphangiography, occasionally with reverse filling (E), and edema formation (I). Angiography revealed a normal overall architecture of the blood vasculature, however, the diffuse angiogram suggests vessel fragility (G). DA indicates dorsal aorta; DCLV, dorsal caudal lymph vessel; DLAV, dorsal longitudinal anastomosing vessel; ISV, intersomitic vessel; PCV, posterior cardinal vein; and VCLV, ventral caudal lymph vessel. Bars represent 500 μm (B,C), 100 μm (D-G), and 1 mm (H,I).