Distinct genetic interactions between multiple Vegf receptors are required for development of different blood vessel types in zebrafish

Abstract

Recent evidence indicates a specific role for vascular endothelial growth factor a (Vegfa) during artery development in both zebrafish and mouse embryos, whereas less is known about signals that govern vein formation. In zebrafish, loss of vegfa blocks segmental artery formation and reduces artery-specific gene expression, whereas veins are largely unaffected. Here, we describe a mutation in the zebrafish vegf receptor-2 homolog, kdra, which eliminates its kinase activity and leads to specific defects in artery development. We further find that Flt4, a receptor for Vegfc, cooperates with Kdr during artery morphogenesis, but not differentiation. We also identify an additional zebrafish vegfr-2 ortholog, referred to as kdrb, which can partially compensate for loss of kdra but is dispensable for vascular development in wild-type embryos. Interestingly, we find that these Vegf receptors are also required for formation of veins but in distinct genetic interactions that differ from those required for artery development. Taken together, our results indicate that formation of arteries and veins in the embryo is governed in part by different Vegf receptor combinations and suggest a genetic mechanism for generating blood vessel diversity during vertebrate development.

Fig. 1.

The kdra^y17 mutation affects artery development. (A Top) Line drawing of LG14 region linked to y17. Ratios are number of recombination events in mutant embryos over total meioses assayed. (Middle) Drawing of Kdra indicating location of ATP-binding domain and the L846→R mutation caused by y17. (Bottom) In vitro kinase assay with wild-type and Kdra^y17 cytoplasmic domains. Western blot was sequentially probed with antibodies against phosphotyrosine and the myc epitope tag. (B) Camera lucida drawing indicating position of DLAV, segmental arteries (SeA), dorsal aorta (DA), and posterior cardinal vein (PCV) in a 30-hpf zebrafish embryo. (C, D, and G) Confocal images of TG(fli1:egfp)^y1 embryos at 30 hpf; anterior is to the left, and dorsal is up. (C) Wild-type TG(fli1:egfp)^y1 sibling; white arrow indicates a DLAV branch. Red arrowheads denote dorsal wall of the dorsal aorta. (D) TG(fli1:egfp)^y1 embryo mutant for kdra^y17. White arrows show partial sprout. Red arrowheads indicate dorsal wall of the dorsal aorta. (E and F) efnb2a expression in nontransgenic embryos; lateral views, anterior to the left, and dorsal is up. (E) Wild-type embryo. Arrow indicates expression in the dorsal aorta. (F) kdra^y17 mutant embryo. (G) Wild-type TG(fli1:egfp)^y1 embryo treated with 1 μM SU5416.

Fig. 2.

vegfc and flt4 contribute to segmental artery development. (A) vegfc expression in dorsal aorta (DA, black arrow) at 18-somite stage. (Inset) Higher-magnification image of hypochord expression. (B) vegfc expression in lateral dorsal aorta (black arrow) at 24 hpf. (C) flt4 expression in posterior cardinal vein (PCV) and segmental arteries (arrowhead) at 25 hpf; arrow indicates reduced expression in dorsal aorta (DA). (D Left) flt4 exons 1–3 and location of PCR primers and SD1 MO. (D Right) RT-PCR amplification of fragments from Flt4 SD1 MO injected embryos. (E–J) Confocal images of TG(fli1:egfp)^y1 embryos. Lateral views, anterior to the left, dorsal is up. (E) Wild-type embryo injected with 5 ng of scrambled MO. (F) Partial segmental artery formation in embryo injected with 2 ng of Flt4 MO. (G) kdra^y17/y17 mutant embryo injected with 2 ng of Flt4 MO. (H) Partial segmental artery formation in embryo injected with Vegfc MOs (5 ng each). (I) kdra^y17/y17 mutant embryo injected with Vegfc MOs. (G) Wild-type TG(fli1:egfp)^y1 embryo coinjected with 5 ng of Vegfa MO and Vegfc MOs (5 ng each).

Fig. 3.

vegfc and flt4 are required for PHBC formation. (A) Camera lucida drawing of 30-hpf zebrafish head indicating positions of the MCeV and PHBC; DC, Duct of Cuvier. Box indicates region imaged in B–H. (B) flt4 expression in MCeV and PHBC at 24 hpf. (C) vegfc expression in lateral dorsal aorta (black arrows) and along midbrain hindbrain boundary (white arrowhead) at 24 hpf. (D) vegfa expression at 24 hpf. (E–H) Confocal images of TG(fli1:egfp)^y1 embryos; lateral views, anterior to the left, dorsal is up; arrow indicates MCeV, asterisk indicates PHBC; in cases where vessel is absent, arrow or asterisk indicates where vessel would have formed. (E) Wild-type embryo injected with 10 ng of Vegfa MO. (F) Wild-type embryo injected with 5 ng each of Vegfc MOs. (G) kdra^y17/y17 mutant embryo injected with 2 ng of Flt4 MO. (H) Wild-type Tg (Fli1:egfp)^y1 embryo treated with 2.5 μM SU5416.

Fig. 4.

Identification of a second Vegfr-2 ortholog in zebrafish. (A and B) kdrb expression by whole-mount in situ hybridization; lateral views, anterior to the left, dorsal is up. (A) kdrb is expressed in all blood vessels in the head; LDA, lateral dorsal aorta (only left branch is shown). (B) kdrb expression in segmental arteries (SeA) and dorsal aorta (DA); PCV, posterior cardinal vein. (C) Phylogenetic tree of selected vertebrate Vegf receptor amino acid sequences using clustalv. Dr, Danio rerio; Fr, Fugu rubripes; Hs, Homo sapiens; Mm, Mus musculus; Tn, Tetraodon nigoviridis; Xt, Xenopus tropicalis. (D Left) kdrb exons 14–18 and location of PCR primers and Kdrb MO. (D Right) RT-PCR amplification of fragments from Kdrb SD1 MO-injected embryos. (E–H) Confocal images of Tg(fli1:egfp)y1 embryos; lateral view, anterior to the left, dorsal is up. (E) Head veins in a kdra^y17/y17 mutant embryo injected with 5 ng of Kdrb MO. (F) kdra^y17/y17 mutant embryo injected with 1 ng of Flt4 MO and 2.5 ng of Kdrb MO; arrows and asterisk indicate where MCeV and PHBC, respectively, would normally form. Arrowheads indicate EGFP expression in nonendothelial arch mesenchyme. (G) Partial segmental arteries in a kdra^y17/+ heterozygous embryo injected with 5 ng of Kdrb MO. (H) Absence of segmental arteries in a kdra^y17/y17 mutant embryo injected with 5 ng of Kdrb MO.