vegfc^um18 mutants display defects in vein and lymphatic vessel development. (A) Linkage map of markers on zebrafish chromosome 1 used in this study. (B) um18 mutation in vegfc exon 4. Box indicates the Gln202 to stop transition. (C) Vegfc domains and amino acid alignment near the um18 truncation. Hs, human; Mm, mouse; Dr, zebrafish. (D,E) (Left) Transmitted light images of embryos at 30 hpf. (Middle) Confocal micrographs of head blood vessels at 30 hpf. Primordial hindbrain channel (PHBC) is indicated by arrows. (Right) Confocal micrographs of trunk vessels at 5 dpf. Thoracic duct (TD) is indicated by arrowheads. Wild-type (D) and vegfc^um18 mutant (E) Tg(fli1a:egfp)^y1 siblings are shown. (F-H) Confocal micrographs of Tg(fli1a:egfp)^y1 vegfc^um18 mutant embryos co-injected with 25 pg tol2 transposase mRNA and 25 pg (F) pTol2-fli1ebs:egfp-2Amcherry, (G) pTol2-fli1ebs:vegfc^um18-2Amcherry or (H) pTol2-fli1ebs:vegfc-2Amcherry. Yellow arrowheads indicate arterial endothelial cells expressing mCherry. Bracket with asterisk indicates absence of TD. White arrows indicate TD rescue. (D-H) Lateral view, anterior to the left, dorsal up. (I) Percentage of embryos showing TD of the indicated genotype injected with the indicated constructs. Values are the average of three independent experiments. ^*P<0.05; N.S., not significant; error bars indicate s.e.m. DA, dorsal aorta; PCV, posterior cardinal vein. Scale bars: 250 μm in D,E left; 50 μm in D,E middle; 25 μm in D,E right, F-H.

vegfc^um18 enhances angiogenesis defects in kdrl^y17 mutant embryos. (A-D) Confocal micrographs of trunk blood vessels in Tg(fli1a:egfp)^y1 (A) wild-type, (B) vegfc^um18 mutant, (C) kdr^y17 mutant and (D) kdr^y17 vegfc^um18 double-mutant zebrafish embryos at 30 hpf. Arrows indicate intersomitic vessels (ISVs). Anterior is to the left, dorsal is up. (E) ISV length in embryos of the indicated genotype at 30 hpf. Values are the average from two clutches of embryos. ^*P<0.05; N.S., not significant; error bars indicate s.e.m. DA, dorsal aorta; PCV, posterior cardinal vein. Scale bar: 50 μm.

vegfc^um18 partially rescues loss of dll4. (A-F) Two-photon micrographs of trunk blood vessels in fixed Tg(fli1a:egfp)^y1 zebrafish embryos at 48 hpf immunostained for Fli1b. Anterior is to the left, dorsal is up. Yellow arrowheads indicate endothelial nuclei. Red arrows indicate ectopic vessel branches. Wild-type (A,B), vegfc^um18/+ (C,D) and vegfc^um18/um18 (E,F) embryos were injected with 15 ng control MO (A,C,E) or 15 ng Dll4 MO (B,D,F). (G,H) Quantification of (G) nuclei and (H) ectopic branches across three ISVs in MO-injected embryos of the indicated genotype. Values are the average of three experiments. ^*P<0.05; N.S., not significant; error bars indicate s.e.m. DA, dorsal aorta; PCV, posterior cardinal vein. Scale bar: 50 μm.

um18 mutation affects Vegfc secretion. (A) Western blot (W.B.) of Flt4 immunoprecipitates (I.P.) from cells treated with the indicated conditioned medium (CM), probed with antibodies against phosphotyrosine (pTyr) and zebrafish Flt4. (B) Quantification of ectopic subintestinal vessels (SIVs) in embryos injected with the indicated CM. Values are the average of three experiments. (C-E) Confocal micrographs of SIVs (arrowheads) in Tg(fli1a:egfp)^y1 embryos at 3.5 dpf following perivitelline injections with (C) Vegfc CM, (D) Vegfc^um18 CM or (E) mock CM. Arrows indicate ectopic SIVs. (F) Western blots probed with the indicated antibodies. Cell lysates and media from NIH3T3 cells transiently expressing human VEGFC or VEGFC^um18. Arrow indicates processed secreted form of wild-type VEGFC. (G) Quantitation of western blots in F. Values are the average of three experiments. A.U., arbitrary units. (H,I). Confocal image of xenografts in 4.5-dpf Tg(fli1a:egfp)^y1 embryos expressing (H) Vegfc-P2Amcherry or (I) Vegfc^um18-P2Amcherry (pseudocolored red). (J) Quantification of vascular density in xenografts from three experiments. (C-E,H,I) Anterior is to the left, dorsal is up. ^*P<0.05; N.S., not significant; error bars indicate s.e.m. Scale bars: 50 μm in C-E; 10 μm in H,I.

Endothelial cell-autonomous effects of Vegfc on angiogenesis. (A-C) Confocal micrographs of trunk blood vessels in Tg(fli1a:egfp)^y1 zebrafish embryos at 30 hpf. Anterior to the left, dorsal is up. Transgene expression (red fluorescence) is indicated in normal (arrow) or ectopically branched (arrowheads) ISVs. Embryos were co-injected with tol2 transposase mRNA and (A) pTol2-fliebs:egfp-2Acherry, (B) pTol2-fliebs:vegfc-2Acherry or (C) pTol2fliebs:vegfc^um18-2Acherry. (D) Quantification of ectopic ISV branching in cells expressing the fliebs:egfp-2Acherry (n=12 embryos) or fliebs:vegfc-2Acherry (n=18 embryos) transgene. Values indicate the proportion of embryos with Cherry-positive ISVs displaying ectopic branching. (E,F) Confocal micrographs of trunk blood vessels at 30 hpf of Tg(kdrl:rasmcherry)^s896 host embryos transplanted with Tg(fli1a:egfp)^y1 cells (green) from embryos injected with 5 ng (E) control MO or (F) Vegfc MO. Arrow indicates a stalk cell. (G,H) The proportion of embryos displaying donor cells in the indicated trunk vessel position. (G) Donor cells from embryos injected with the indicated MO. (H) Donor cells were from embryos derived from an incross of vegfc^um18 carriers. ^*P<0.05; N.S., not significant; error bars indicate s.e.m. Scale bars: 50 μm.

Transgenic haploid screen in Tg(fli1a:egfp)^y1 zebrafish to identify mutants affecting Vegfc/Flt4 signaling. (A) Tg(fli1a:egfp)^y1 males were treated with N-ethyl-N-nitrosourea (ENU) to induce mutations in the pre-meiotic germline and subsequently outcrossed to wild-type Tg(fli1a:egfp)^y1 females to establish F1 families. Eggs from individual F1 females were fertilized in vitro with UV-irradiated sperm from wild-type males to generate haploid embryos. (B) Epifluorescence images of cranial blood vessels in Tg(fli1a:egfp)^y1 haploid embryos. (Left) Normal primordial hindbrain channel (PHBC) formation (arrows) at 28 hpf. (Right) Putative mutant embryo (‘putant’), which failed to form a PHBC (arrowheads denote location where PHBC normally forms).

Decreased thoracic duct formation in vegfc^um18 heterozygous embryos. (A,B) Confocal microangiographs of trunk blood vessels in Tg(fli1a:egfp)^y1 larvae at 5 dpf in progeny from a um18 carrier incross. Microangiography dye is pseudocolored red. (A) Normal TD formation in a wild-type embryo. (B) Loss of TD in vegfc^um18/+ heterozygous embryo. The normal location of the thoracic duct (TD) is indicated by arrows. Lateral view, dorsal is up and anterior to the left. DA, dorsal aorta; PCV, posterior cardinal vein. (C) Quantification for presence of TD in embryos of the indicated genotype. Scale bar: 25 μm.

Vegfc^um18 mutants display primary defects in lymphatic progenitor and vein sprouting. (A) Diagram describing lymphatic vessel formation in zebrafish. (1) Dorsal sprouting of parachordal lymphangioblasts (PLs) from the posterior cardinal vein (PCV) at 1.5-2 dpf. (2) Ventral migration of PLs along the horizontal myoseptum. (3) Ventral migration of PLs to a position between the dorsal aorta and posterior cardinal vein to form the TD. (B-D) Confocal microangiographs of trunk blood vessels in Tg(fli1a:egfp)^y1 embryos at 2 dpf. Anterior is to the left, dorsal is up. White arrows indicate position where PLs normally form. (B) Wild-type embryo. (C) vegfc^um18/+ heterozygous embryo. (D) vegfc^um18 homozygous mutant embryo. (E) Quantification of PL formation. Values indicate the percentage of embryos of the indicated genotype that display either presence or absence of PLs. (F,G) Confocal microangiographs of trunk blood vessels at 3 dpf. Anterior is to the left, dorsal is up. DA, dorsal aorta; PCV, posterior cardinal vein; SegA, segmental artery; SegV, segmental vein. White arrowheads indicate SegA. (F) Wild-type sibling embryo. (G) Homozygous vegfc^um18 mutant embryo. (H) Quantification of percentage artery and vein connections in wild-type and vegfc^um18 mutant embryos. Mutants were identified as those lacking a PHBC at 30 hpf. At 3 dpf, embryos of both phenotypic classes were scored for the connection of intersegmental vessel to either DA or PCV based on blood flow. The values shown are based on the average of three independent experiments. Scale bar: 25 μm.

A furin-resistant form of Vegfc (Vegfc^ss) is secreted and can partially rescue lymphatic development in vegfc^um18 mutant embryos. (A) Vegfc structure showing location of the furin cleavage domain and the conserved amino acid sequences from zebrafish (Dr), human (Hs), mouse (Mm) and rat (Rn) Vegfc proteins. The amino acid substitutions to prevent furin cleavage are shown. (B) The proportion of embryos with indicated genotypes that displayed TD formation following injection of an endothelial driven egfp- 2Acherry or vegfc^ss-2Acherry transgene. (C) Zebrafish Vegfc structure showing location of the hemagglutinin (HA) epitope tag at the C-terminus used to detect expression of furin-resistant Vegfc (Vegfc^ss). Western blot shows expression of wild-type zebrafish Vegfc or Vegfc^ss in cell lysates or extracellular media from transiently transfected NIH3T3 cells. In cell lysates, full-length Vegfc can be detected in both cases. Interestingly, we can detect a small amount of full-length wild-type Vegfc in the medium, along with the C-terminal domain that has been cleaved. Furthermore, we are able to detect furin-resistant Vegfc^ss in the medium, the vast majority of which remains unprocessed.

Developmental angiogenesis defects in vegfc-deficient mouse and zebrafish embryos. (A,B) Confocal micrographs of mouse embryos of the indicated genotype at E9.5 stained with endomucin to visualize endothelial cells. (A) Cranial vessels (left) and quantification of branching points (right). (B) ISVs (left) and quantification of added ISV length, between somites 14-16 (right). n=3. (C-F) Confocal micrographs of Tg(fli1a:egfp)^y1 wild-type zebrafish embryos at 30 hpf injected with (C) 5 ng control, (D) 2.5 ng Vegfc or (E) 5 ng Vegfc MO. Lateral views, dorsal is up, anterior to the left. (F) Quantification of ISV length in MO-injected Tg(fli1a:egfp)^y1 embryos. *P<0.05; N.S., not significant. Scale bars: 100 μm in A,B; 25 μm in C-E.