Fig. 1

vegfd regulates facial lymphangiogenesis. (A) Forward (red) and reverse (blue) Talens used to target the vegfd locus induced a 7 bp deletion designated as the uq9bh allele. (B) The uq9bh allele results in a premature stop codon at amino acid 65, prior to the VEGF homology domain. (C,D) Confocal images of Tg(-5.2lyve1b:dsRed);Tg(flt1:yfp) wild-type (C, lyve1b:dsRed only; C′) and vegfd^uq9bh mutant embryos (D, lyve1b:dsRed only; D′) at 5 dpf. (E-H) vegfd^uq9bh adult mutants are viable and have no obvious defects (G,H) when compared with siblings (E,F). (J-N′) Confocal images showing the facial region of 5 dpf Tg(fli1a:negfp);Tg(-5.2lyve1b:dsRed) transgenic embryos produced by in-crossing vegfd^uq9bh, vegfc^hu5055 double heterozygotes. Arrows indicate the presence of normal medial facial lymphatics (MFL), lateral facial lymphatics (LFL) and otolithic lymphatic vessel (OLV). Asterisks indicate the reduction of LEC number in these vessels. (O-Q) vegfd^uq9bh mutant embryos have a reduced number of facial lymphatic endothelial cells (LECs) when quantified in 5 dpf Tg(fli1a:negfp):Tg(-5.2lyve1b:dsRed) embryos. (O) Significant differences in LFL LEC numbers were observed when vegfd^uq9bh mutants (n=5) were compared with vegfd^uq9bh heterozygous (n=12) or wild-type (n=7) embryos in a vegfc^hu5055 wild-type background; and when vegfd^uq9bh mutants (n=7) were compared with vegfd^uq9bh heterozygous (n=18) or wild-type (n=6) embryos in vegfc^hu5055 mutants. (P) Significant differences in MFL LEC numbers were observed when comparing vegfd^uq9bh mutants (n=6) with vegfd^uq9bh wild-type (n=7) embryos in a vegfc^hu5055 wild-type background; when comparing vegfd^uq9bh mutant (n=10) or heterozygous embryos (n=18) with vegfd^uq9bh wild type (n=6) and embryos in a vegfc^hu5055 heterozygous background; and when vegfd^uq9bh mutants (n=9) were compared with vegfd^uq9bh heterozygous (n=18) and wild-type (n=6) embryos in a vegfc^hu5055 mutant background. (Q) There was no interaction between vegfc and vegfd in the formation of the otolithic lymphatic vessel. Data represent mean±s.e.m.; ***P<0.001, ** P<0.01, *P<0.05 from one-way ANOVA from three independent clutches of embryos.