Fig. 6

Knockdown of both vegfc and vegfd is required to prevent facial lymphatic development. (A-H) Stills from confocal time-lapse imaging of the facial lymphatic sprout in lyve1:egfp embryos from 1.5dpf to 1.9dpf (9:03h) injected with either vegfc MO-1 and control MO (A-D, supplementary material Movie 4) or vegfc MO-1 and vegfd MO (E-H, supplementary material Movie 5). Data are representative of three independent time-lapse experiments. The facial lymphatic sprout (red arrows) forms in vegfc MO-1 and control MO morphants but is not present in double-knockdown morphant embryos. The formation of the pectoral vein (green arrowheads) serves as a control to show that the embryos are developing normally. (I-P) Confocal images of the facial lymphatics in lyve1 embryos at 3dpf in control MO (I), vegfc MO-1 (J), vegfd MO (K), vegfc^hu5055+control MO (L), vegfc MO-1+vegfd MO (M), vegfc MO-1+vegfd MO+200pg vegfd mRNA (N), vegfc MO-1+vegfd MO+400pg vegfc mRNA (O) and vegfc^hu5055+vegfd MO (P). Silencing either vegfc or vegfd has no effect on early facial lymphatic development but double knockdown of both genes prevents the formation of the LFL at 3dpf (arrowheads). (Q) Quantitation of the length of the LFL vessel in 3dpf embryos (¹data reproduced from Fig. 1 for comparison). (R-U) Confocal images of the facial lymphatics in lyve1 embryos at 5dpf in control MO (R), vegfc^hu5055+control MO (S), vegfc MO-1+vegfd MO (T) and vegfc^hu5055+vegfd MO (U). Asterisks indicates the PHS. (V) The percentage formation of different facial lymphatic vessels at 5dpf. (¹data reproduced from Fig. 1 for comparison). For vegfc^hu5055 embryos, n=number of mutant embryos. Knockdown of both vegfc and vegfd results in a more severe defect in the development of the facial lymphatic network than knockdown of vegfc alone. CCV, common cardinal vein; PHS, primary head sinus; PCeV, posterior cerebral vein; LFL, lateral facial lymphatic; OLV, otolithic lymphatic vessel; MFL, medial facial lymphatic; LAA, lymphatic branchial arches. ***P<0.001, **P<0.01 by a Mann–Whitney test, unless indicated, significance was determined to control MO; error bars indicate s.d. Scale bars: 50μm.