Fig. 3

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.