Fig. 4

The dll4 morphant hyperbranching phenotype is rescued in flt4^hu4602 mutants, vegfc morphants and sFLT4 mRNA-injected embryos. (A-D) Lateral views of uninjected control (A) and flt4^hu4602 mutant (B) as compared with wild-type (C) and flt4^hu4602 mutant (D) zebrafish embryos injected with dll4 MO. Images were taken in the TG(flt1:YFP)^hu4624 background, labelling trunk arteries to reveal the failure to form intersegmental venous sprouts in expando mutants (B, yellow arrows). The dll4 morphant arterial hyperbranching phenotype (C) is rescued in flt4^hu4602 mutants (D). Venous sprouts were identified by the absence of flt1:YFP expression in ISVs (brackets). Embryos at 72 hpf. (E) Arterial hyperbranching phenotypes were scored post-injection of dll4 MO and subsequently genotyped. The mutant genotype was almost completely absent from the severe category (e.g. C) (1/29 embryos, *P<0.01 by χ² test) and was enriched in the category not displaying hyperbranching (e.g. D) (21/55 embryos, ^#P<0.01 by χ² test). (F) In transgenic TG(flt1:YFP)^hu4624 embryos, co-injection with dll4 MO together with vegfc MO (green bars, n=57 embryos), or of dll4 MO together with sFLT4 mRNA (200 pg/embryo) (red bars, n=67 embryos), led to rescue of the arterial hyperbranching phenotype observed upon injection of dll4 MO alone (blue bars, n=81 embryos). Arterial hyperbranching phenotypes were categorised as no hyperbranching (none), mild or severe (see Fig. S1 in the supplementary material).