Synectin knockdown impairs lymphatics in tadpoles. In all panels, the head of the tadpoles faces left and dorsal is up. Scale bars represent 100 μm in panels A-B and K-L; 50 μm in panels C-F; 150 μm in panels G-H; and 400 μm in panels I-J. DCLV indicates dorsal caudal lymph vessel; DLAV, dorsal anastomosing longitudinal vessel; PCV, posterior cardinal vein; and VCLV, ventral caudal lymph vessel. (A-F) Fluorescent analysis of blood and lymph vessels in stage 47 transgenic Flk1:eGFP tadpoles. Lymph vessels are labeled with TRITC-dextran that was intracardially injected at stage 45 and taken up by LECs after extravasation from the blood vessels. Hence, GFP⁺ BECs can be easily distinguished from doubly labeled (yellow) LECs. Areas in the trunk corresponding to the boxed areas in panels A or B are shown in panels C-F. In control tadpoles, both the DCLV (C) and the VCLV (E) developed normally. Injection of 30 ng of synectin morpholino resulted in a hypoplastic, disorganized, and discontinuous DCLV (D), while the VCLV (F) had a grossly normal appearance. (G-H) Lymphangiography of stage 45 Flk1:eGFP tadpoles, revealing dysfunction of the VCLV after synectin knockdown (H) in contrast to normal controls (G). Whereas in control injected embryos, the dye is drained normally in a rostral direction (white arrows in G), no dye uptake was observed in the VCLV in synectin^KD tadpoles. White asterisks indicate injection site of the fluorescent dye. (I-J) Bright-field pictures of live embryos at stage 45 (5 dpf), showing lymphedema (arrows) in a synectin^KD tadpole (J) compared with a control tadpole (I). (K-L) Whole mount Prox-1 in situ hybridization of stage 35/36 tadpoles, revealing the presence of fewer Prox-1+ LECs in the area ventrally to the dorsal margin of the endoderm (reflecting lymphatic lineage emergence) and in the area dorsally to this margin (reflecting LEC budding/migration) in the posterior trunk of synectin^KD-morphant tadpoles (L) compared control (K) tadpoles. Dotted line: dorsal margin of the endoderm. (M-N) Morphometric measurement revealed a dose-dependent decrease of the Prox-1⁺ area both ventrally (M) and dorsally (N) of the dorsal margin of the endoderm in the trunk of Prox-1–stained synectin^KD compared with control tadpoles at stage 35/36, indicating that lymphatic lineage development and migration is affected upon synectin knockdown. Values expressed relative to control. *P < .001 by multivariate analysis.