Splice site mutation c.325+5G>A in vegfc abolishes the effect of ectopic expression on excessive vessel sprouting. (A) Sequence of the human and zebrafish VEGFC/vegfc orthologs. Alignment of zebrafish and human exon 2 and intron 2–3 highlights the mutated site c.361+5G, which is equivalent to zebrafish c.325+5G. (B) Diagram of constructs used for forced expression of either vegfc-intron2–3 wt or vegfc-intron2–3 c.325+5G>A together with tagRFP in the floorplate, which consists of a 5′ and 3′ Tol2 element, the -2.2Shh promoter region, exon 1–2 of the cDNA of vegfc (Ex1–2), vegfc-intron 2–3 (Int 2) of vegfc (wt or c.325+5G>A), exon 3–7 of vegfc (Ex3–7), an IRES tagRFP cassette, and the activating region Ar-B driving expression in the floorplate. (C) Analysis of vegfc-intron2–3 wt and vegfc-intron2–3 c.325+5G>A overexpression in the floorplate using the transgenic line Tg(flt4^BAC:mCitrine)^hu7135 at 56 hpf. Overexpression of vegfc-intron 2–3 wt led to lymphovenous hypersprouting while forced expression of vegfc-intron2–3 c.325+5G>A had no effect on vessel growth. (D) Quantification of the YFP positive area of vessels surrounding the site of tagRFP expression showed an increased vessel density in zebrafish overexpressing vegfc-intron 2–3 wt but not in zebrafish overexpressing the vegfc-intron 2–3 c.325+5G>A variant. Values are presented as means ± standard error of mean values (SEM). *** p < 0.001, n.s. not significant.

The vegfc c.325+5G>A splice-site variant results in a truncated protein, which does not have dominant negative activity. (A) Diagram of the construct used for overexpression of vegfc-intron 2–3 depicting primers used for RT-PCR (arrows). (B) RT-PCR of zebrafish embryos expressing ShhVegfc-intron 2–3 wt or ShhVegfc-intron 2–3 c.325+5G>A. Embryos expressing the wt form of ShhVegfc-intron 2–3 and the ShhVegfc-intron 2–3 c.325+5G>A variant express a band of 660 bp corresponding to non-spliced RNA or integrated plasmid DNA as well as a second smaller band corresponding to wt ShhVegfc (440 bp) or mutant ShhVegfc (387 bp). Right lane: 100 bp ladder. Non-injected control, nic. (C) Sequencing of cDNA of embryos expressing ShhVegfc-intron 2–3 and the ShhVegfc-intron 2–3 c.325+5G>A variant showing a 53 bp deletion in the ShhVegfc-intron 2–3 c.325+5G>A variant (lower panel). (D) Schematic representation of predicted wild type (top) and mutant (bottom) proteins. Mutant protein consists of the first 91 amino acids of Vegfc containing only a part of the N-terminus of Vegfc but not the VHD or the C-terminus (see Figure 2 for legend). (E) Analysis of co-overexpression of ShhVegfc-intron 2–3 wt and ShhVegfc-intron2–3 c.325+5G>A in the floorplate using the transgenic line Tg(flt4^BAC:mCitrine)^hu7135 and Tg(flt1^enh:tdTomato), which marks venous and lymphatic cells in green and arterial vessels in red at 48 hpf. Expression of ShhVegfc-intron 2–3 wt and ShhVegfc-intron 2–3 c.325+5G>A within the same cell are monitored by the simultaneous expression of mturquoise and tagRFP, respectively. Forced expression of ShhVegfc-intron 2–3 wt in the floorplate led to excessive vessel sprouting comparable with co-overexpression of ShhVegfc-intron 2–3 wt and ShhVegfc-intron 2–3 c.325+5G>A in the floorplate. Arrow heads mark expression of tagRFP and mturquoise in the floorplate. (F) Quantification of lymphovenous sprouting by measuring the YFP positive area surrounding the site of mturquoise/tagRFP expression. Values are presented as means ± standard error of mean values (SEM). n.s. not significant.