xVEGF-D and xVEGF-C coregulate lymph vessel development in the posterior trunk. (A-D) Whole-mount xProx1 in situ hybridization of stage-37/38 tadpoles. Single or simultaneous knockdown of xVEGF-D (50 ng morpholino) or of xVEGF-C (35 ng morpholino) did not affect lymphatic commitment (area 1). In contrast, the numbers and distance of migrating cells (area 2) in single xVEGF-D or xVEGF-C knockdowns were, respectively, normal (B) and partially reduced (C), but further reduced significantly in xVEGF-C/D double knockdowns (D). Black dashed line indicates dorsal margin of the endoderm. (E) Morphometric measurement of area 2 in xProx1-stained tadpoles at stage 37/38 using single and combined morpholino injection at the indicated concentrations. *P < .05 versus control. The number of tadpoles (n) is indicated. Error bars represent SEM. (F,G) Representative picture of lymphangiography of VEGF-C/D^KD tadpoles showing failure of the DCLV to drain injected dye (G) as opposed to normal uptake and drainage in control tadpoles (F). The arrow in panel G indicates the distal site of DCLV filling. (H,I) Significant lymphedema in VEGF-C/D^KD tadpoles (35 ng xVEGF-C/50 ng xVEGF-D morpholino) at stage 45 (I), in contrast to normal appearance of the controls (H). (J,K) Angiography of control (J) and VEGF-C/D^KD tadpoles (K) showing normal appearance of the vasculature in a VEGF-C/D^KD tadpole that displayed lymph vessel dysfunction (G). DA indicates dorsal aorta; DCLV, dorsal caudal lymph vessel; DLAV, dorsal longitudinal anastomosing vessel; ISV, intersomitic vessel; MO, morpholino; and PCV, posterior cardinal vein. Bars represent 250 μm (A-D), 100 μm (F,G,J,K), and 1 mm (H,I).