Sprouting intersegmental arteries express flt4 and are sensitised to exogenously introduced Vegfc in the absence of dll4. (A-E) At 24 hpf, the expression of flt4 is present in all arterial cells (A) and is unaltered upon loss of dll4 (B) (n=40 uninjected control and n=40 dll4 MO-injected embryos scored). By 26 hpf (C), flt4 is downregulated in the dorsal aorta (n=20 embryos). Analysis of a flt4 promoter-driven transgenic line identifies YFP expression in all arterial cells at 24 hpf (D). The expression of YFP is unaltered in dll4 MO-injected embryos (E). (F,G) At 24 hpf, the expression of vegfc is present in the dorsal aorta (F) and is unaltered in dll4 MO-injected embryos (G) (n=20 uninjected and n=20 MO-injected embryos). (H,I) The expression of vegfd in the tailbud is unaltered in dll4 MO-injected (I) as compared with uninjected control (H) embryos (n=20 uninjected and n=20 MO-injected embryos). (J,K) The expression of the arterial marker gene hey2 is unaltered in dll4 MO-injected (K) as compared with uninjected control (J) embryos (n=20 uninjected and n=20 MO-injected embryos). (L-R) dll4 morphants are sensitised to exogenously introduced vegfc mRNA during arterial angiogenesis. Forced expression by the injection of mRNA encoding full-length zebrafish Vegfc into dll4 MO-injected embryos (n=133) (P) leads to aberrant ectopic bilateral turning of ISAs by 28 hpf (yellow arrows), which was never observed in wild-type embryos (L) or in those injected with dll4 MO only (n=90) (M). ISAs rarely turn bilaterally upon injection of vegfc mRNA only (n=106) (N), whereas injection of full-length vegfd mRNA robustly showed this phenotype without (n=211) (O) or with (n=227) (Q) dll4 MO. For each of the conditions tested, the percentage of embryos showing wild-type, mild (aberrant ISAs spanning two somites) or severe (aberrant ISAs spanning more than two somites) ISA phenotypes is shown in R.
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