Synectin modulates Vegfr3 signaling in lymphatic development in vivo. In all panels, the head of the embryo faces left and dorsal is up. Scale bars represent 50 μm in panels D-E and 100 μm in panels F-I. DA indicates dorsal aorta; ISV, intersomitic vessel; and PCV, posterior cardinal vein. (A) Quantification of parachordal lymphangioblast (PL) cell defects after injection of Syn^ATG1 (2.5 ng; syn^L-KD; N = 79), Vegfr3^ATG1 (1.25 ng; Vegfr3^L-KD; N = 62) or both (N = 50) at 60 hpf. Percentages of embryos displaying complete lack of PL cells, PL string over 10%-30% or 30%-90% of its normal length and a normal PL string are shown for each group. Compared with single knockdown groups, the PL formation was more severely impaired in double morphants (P < .001). (B) Quantification of TD defects after injection of Syn^ATG1 (2.5 ng; syn^L-KD; N = 101), Vegfr3^ATG1 (1.25 ng; Vegfr3^L-KD; N = 107) or both (N = 86) at 7 dpf. Percentages of embryos displaying complete lack of TD, TD formation over 10%-30% or 30%-90% of its normal length and a normal TD are represented for each treatment group. Compared with single knockdown groups, the TD was more severely impaired in double morphants (P < .001 vs synectin^L-KD; P < .05 vs Vegfr3^L-KD). (C) Quantification of the number of unilateral secondary sprouts in a 10-somite region of 48-hpf Fli1:eGFP^y1xPLCγ1^y10 embryos revealed that coknockdown of synectin and Vegfr3 significantly aggravated the secondary sprouting defects compared with single knockdown of either gene when using suboptimal doses of Syn^ATG1 (2.5 ng; syn^L-KD) and Vegfr3^ATG1 (2.5 ng; Vegfr3^L-KD); (N = 45, 37, 48, and 63 for control, Vegfr3^L-KD, synectin^L-KD, and coknockdown, respectively; *P < .05; **P < .01; ***P < .001). (D-E) Confocal images of 60-hpf control (D) and Nrp2a^KD (E) Fli1:eGFP^y1 embryos revealing impaired formation of the PL string (arrows) upon Nrp2a knockdown. Asterisks denote absence of PL cells. (F-I) Confocal images of GFP⁺ vessels in the trunk of 7-dpf Fli1:eGFP^y1 zebrafish embryos, showing formation of a normal TD in a control embryo (F,H) but not in a Nrp2a^KD embryo (G,I). Panels H and I represent close-up magnifications of the boxed areas in panels F and G; arrows denote TD, asterisks denote absence of TD. (J) Quantification of PL cells in control and Nrp2a^KDFli1:eGFP^y1 zebrafish embryos at 60 hpf. The percentages of embryos lacking PL cells and displaying PL string over 10%-30%, 30%-90%, and 100% of its normal length are indicated per treatment group. Formation of the PL string was scored per somite in 10 consecutive somites between somite 5 and 15 (N = 106, 152, and 60 for 0, 5, and 10 ng of Nrp2a^ATG1, respectively). (K) Quantification of TD in control and Nrp2a^KDFli1:eGFP^y1 zebrafish embryos at 7 dpf. The percentages of embryos lacking TD and displaying TD formation over 10%-30%, 30%-90%, and 100% of its normal length are indicated per treatment group. Formation of the TD was scored per somite in 10 consecutive somites between somite 5 and 15 (N = 99, 164, and 56 for 0, 5, and 10 ng of Nrp2a^ATG1, respectively). (L) Quantification of TD formation after injection of Nrp2a^ATG1 (5 ng; Nrp2a^L-KD; N = 74), Syn^ATG1 (2.5 ng; syn^L-KD; N = 98) or both (N = 41) revealed that coknockdown impaired lymphatic development more severely than single synectin^L-KD (P < .001) or Nrp2a^L-KD (P < .001).