Fig. 3

Vegfd contributes to thoracic duct development in the zebrafish trunk. (A-F) Confocal images of Tg(fli1a:egfp) at 5 dpf showing formation of the TD (arrows) is normal in vegfd^uq9b mutants (A,B). The phenotype for the formation of the TD is variable in vegfc^hu5055 mutants, ranging from a complete loss (C) to very mild loss (D). The formation of the TD is reduced (asterisk) in vegfc^hu5055 mutants that were heterozygous (E) or mutant (F) for vegfd^uq9bh. (G-I) Quantification of TD extent across nine somites in vegfd^uq9bh mutant embryos in vegfc^hu5055 wild-type (G), heterozygous (H) and mutant (I) backgrounds. There was significantly less TD formed in vegfd^uq9bh mutant embryos (n=42) when compared with vegfd^uq9bh heterozygous (n=112) and wild-type (n=63) embryos in a vegfc^hu5055 heterozygous background (H); and in vegfd^uq9bh mutant embryos (n=25) when compared with vegfd^uq9bh heterozygous (n=56) and wild-type (n=33) embryos in a vegfc^hu5055 homozygous mutant background (I). Data represent mean±s.e.m.; *P<0.05 from Kruskal–Wallis test from three independent clutches of embryos. (J-K′) Confocal images of TD formation in 5 dpf Tg(fli1a:negfp),Tg(-5.2lyve1b:dsRed) embryos showing that fragments of TD form in vegfc^hu5055 mutant/vegfd^uq9bh wild-type embryos (J,J′), but are absent in vegfc^hu5055 /vegfd^uq9bh double-mutant embryos (K,K′). (L) Quantification of experiment in J-K′. The number of nuclei present in the TD of vegfd^uq9bh mutant (n=14) embryos was significantly lower when compared with vegfd^uq9bh heterozygous (n=21) and vegfd^uq9bh wild-type (n=13) embryos in a vegfc^hu5055 mutant background. Data represent mean±s.e.m.; **P<0.01, from one-way ANOVA from three independent clutches of embryos. (M,N) Confocal images of TD formation in 7 dpf Tg(-5.2lyve1b:dsRed) embryos (yellow arrows) in a flt4^hu4602 heterozygous background. vegfd^uq9bh mutants (N) have reduced TD formation compared with vegfd^uq9bh wild-type embryos (M). (O) Quantification of experiment in M,N. There was significantly less TD formed in vegfd^uq9bh mutant embryos (n=33) when compared with vegfd^uq9bh heterozygous (n=19) and wild-type embryos (n=27) in a flt4^hu4602 heterozygous background. **P<0.01, *P<0.05 from Kruskal–Wallis test from three independent clutches of embryos.