Knockdown of synectin impairs early lymphatic development in zebrafish. In all panels, the head of the embryo faces left and dorsal is up. Scale bars represent 50 μm. DA indicates dorsal aorta; ISV, intersomitic vessel; and PCV, posterior cardinal vein. (A-B) Confocal images of 60-hpf control (A) and synectin^KD (B) Fli1:eGFP^y1 embryos revealing a reduced formation of the parachordal lymphangioblast (PL) string (arrows in A) upon synectin knockdown. Asterisks in panel B denote absence of PL cells. (C) Quantification of the PL cells in control and synectin^KDFli1:eGFP^y1 zebrafish embryos at 60 hpf. The percentages of embryos lacking all PL cells and displaying PL string formation over 10%-30%, 30%-90%, and 100% of its normal length are indicated per treatment group (see also supplemental Table 4). Formation of the PL was scored per somite in 10 consecutive somites between somites 5 and 15. (D-E) Whole-mount in situ hybridization of 48-hpf control (D) and synectin^KD (E) embryos for Tie-2, labeling all secondary sprouts (arrows) emerging from the PCV. In synectin^KD embryos the number of secondary sprouts was markedly reduced, by approximately 50%. Panels D′ and E′ are magnifications of panel D and E, respectively. In synectin^KD embryos somites lacking a secondary sprout are indicated with an asterisk. (F-G) Confocal images of 48-hpf Fli1:eGFP^y1xPLCγ1^y10 control (F) and synectin^KD(G) embryos showing a reduction of secondary sprouts upon synectin knockdown. Small arrows indicate unilateral secondary sprouts; long arrow denotes PL cells. (H) Quantification of the number of unilateral secondary sprouts in a 10-somite region of 48-hpf Fli1:eGFP^y1xPLCγ1^y10 embryos confirmed a significant reduction of nearly 50% in secondary sprouts budding from the PCV upon synectin knockdown. (I) Quantification of the fraction of venous ISVs, identified by their connection to the PCV upon confocal screening, in a 10-somite region in 4-dpf Fli1:eGFP^y1 embryos revealed a normal ratio of venous ISVs upon synectin knockdown. Error bars in panels H-I represent SEM; *P < .05 by univariate analysis.