Lymph vessel development in the posterior trunk in xVEGF-D knockdown tadpoles is impaired moderately. (A,B) Morphometric measurement of area 1 (LEC commitment; A) and area 2 (migration; B) in xProx1-stained tadpoles at the indicated stage after morpholino knockdown of xVEGF-D (75 ng morpholino). *P < .05 versus control. The number of tadpoles (n) is indicated. Error bars represent SEM. (C,D) Whole-mount xProx1 in situ hybridization of stage-37/38 tadpoles, revealing the presence of abundant xProx1⁺ LECs in area 1 but fewer cells in area 2 in the posterior trunk in VEGF-D^KD (D) than in control (C) tadpoles. Black dashed line indicates dorsal margin of the endoderm; yellow dashed line, floor plate of the neural tube. (E,F) Lymphangiography in stage-45 control (E) or VEGF-D^KD tadpoles (F), revealing normal filling of the VCLV. (G,H) Normal appearance of VEGF-D^KD tadpoles at stage 45 (H), comparable with control (G). (I,J) Angiography in stage-45 control (I) and VEGF-D^KD tadpoles (J), revealing normal blood vessel development. The insets in panels I and J represent larger magnifications, showing that the ISVs in the VEGF-D^KD tadpoles are intact and comparable with those of the control tadpole. DA indicates dorsal aorta; DLAV, dorsal longitudinal anastomosing vessel; ISV, intersomitic vessel; PCV, posterior cardinal vein; and VCLV, ventral caudal lymph vessel. Bars represent 250 μm (C-F), 1 mm (G,H), 100 μm (I,J) and 50 μm insets (I,J).