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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2006 Apr 14;103(17):6554–6559. doi: 10.1073/pnas.0506886103

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

L D Covassin *, J A Villefranc *, M C Kacergis *, B M Weinstein †, N D Lawson *,‡
PMCID: PMC1458922  PMID: 16617120

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.

Keywords: differentiation, endothelial

The members of the Vegf family of proteins are soluble molecules required for blood vessel formation during vertebrate development. Vegfa is crucial for embryonic blood vessel development, and loss of only a single allele in mice causes severe defects in endothelial cell differentiation and blood vessel formation (1). Vegfa also plays a major role in blood vessel homeostasis and vascular permeability in mature blood vessels (2). Vegfa binds to two related endothelial cell-specific receptors, Vegfr-1 (Flt1) and Vegfr-2 (Kdr/Flk1; ref. 3). Vegfr-2 functions as a typical receptor tyrosine kinase in response to Vegfa binding, i.e., Vegf binding causes dimerization of Vegfr-2, autophosphorylation of tyrosine residues within the Vegfr-2 cytoplasmic domain, and association with a number of proteins containing Src-homology domains such as phosphoinositol 3′-kinase (4) and phospholipase C γ-1 (5). The activation of these and other molecules by Vegfa elicits a wide range of effects on endothelial cells, including survival, proliferation, migration, and increased permeability (6). Consistent with the importance of Vegfr-2 in Vegf signal transduction, mouse embryos that lack vegfr2 exhibit defects similar to Vegfa-deficient embryos (7).

In contrast to Vegfr-2, Vegfr-1 inhibits Vegf signaling during embryonic blood vessel formation through the action of an alternatively spliced form that encodes the soluble Vegfr-1 extracellular domain (8). Vegfr-1 displays a higher affinity for Vegfa (9) and lower kinase activity upon binding to Vegfa than Vegfr-2 (10), and sVegfr-1 is thought to act as a Vegf sink that negatively regulates Vegfr-2 signaling (11). Accordingly, mouse embryos lacking vegfr1 display abnormal blood vessel formation due to an overproliferation of endothelial cells (12, 13), whereas deletion of only the Vegfr-1 kinase domain does not affect blood vessel formation (14). A third Vegf receptor, Vegfr-3 (Flt4), is essential for lymphatic development and is activated by binding to Vegfc. Flt4-deficient embryos also show defects in the formation of the circulatory system (15), and there is growing evidence that Flt4 can modulate Vegfr-2 signaling (16, 17). For example, Vegfa and Vegfc can synergize to drive blood vessel formation of endothelial cells in culture, and Vegfr-2 and -3 can heterodimerize (18). However, there are few details concerning the defect in vascular endothelial development in Flt4 knockout embryos, and little is known about the interaction between Vegfa and Vegfc signaling pathways at the organismal level.

Recent work demonstrates an important role for Vegfa in determination of arterial endothelial identity and the formation of arteries. Reduction of Vegfa levels in zebrafish embryos or loss of specific Vegfa isoforms in mice prevents the expression of artery-specific markers, such as ephrin-B2, and blocks formation of arteries, whereas veins are largely unaffected (19–21). Conversely, exogenous Vegfa can induce ephrin-B2 in endothelial cells (19, 20) and can lead to the formation of ectopic arteries (22). Zebrafish embryos bearing mutations in phospholipase c γ 1 (plcg1) fail to respond to Vegfa and display specific defects in the differentiation and morphogenesis of arteries, whereas veins form normally (23, 24). Much less is known about the signaling mechanisms that may contribute to vein morphogenesis and differentiation. Recent work demonstrates a specific role for the orphan nuclear receptor COUP-TFII in defining venous endothelial identity (25). However, it is not known whether Vegf signaling components contribute to vein development in a specific manner similar to their role in artery development.

In this study, we find that different blood vessel types in the developing vertebrate embryo are formed through distinct genetic interactions among multiple Vegf receptors. We show that loss of zebrafish vegfr-2 (kdra) kinase activity causes artery-specific morphogenesis defects that are milder than those previously described in plcg1 mutant embryos (23), whereas treatment with SU5416, which blocks the catalytic activity of all Vegf receptors, completely blocks segmental artery formation, suggesting that other Vegf receptors cooperate with kdra. Accordingly, loss of the vegfr-3 ortholog flt4 or of kdrb, a newly identified vegfr-2 ortholog, in kdra mutant embryos can mimic the artery defects associated with loss of plcg1. Interestingly, genetic interaction between these receptors is apparent during artery morphogenesis but not during differentiation. Additionally, we find that distinct combinations of these receptors are required for formation of veins during development. These results demonstrate heterogeneity in the requirement for Vegf signaling during artery and vein formation and suggest a mechanism by which these pathways may contribute to blood vessel diversity during development.

Results

We identified the y17 mutation in a screen for zebrafish embryos that lack segmental arteries (N.D.L., unpublished observation). Bulk segregant analysis of wild-type and mutant sibling embryos demonstrates that y17 is ≈10 cM from marker Z6847 on linkage group 14 near the previously described vegf receptor-2 (vegfr-2) and the vegf receptor-3 (vegfr-3) orthologs (Fig. 1A). According to zebrafish nomenclature, we refer to the original vegfr-2 (26) as kdra because of the identification of a second vegfr-2 ortholog (see below) and vegfr-3 as flt4 (27). Analysis of the kdra coding sequence reveals a T to G mutation that changes L846 to R in y17 mutant embryos (Fig. 1A). Furthermore, linkage analysis of this mutation using dCAPS (28) finds no recombinant mutants in 284 meioses, indicating tight linkage between y17 and kdra. Because the y17 mutation was located in the Kdra ATP-binding motif that is highly conserved in protein kinases (29), we determined the effect of this mutation on Kdra catalytic activity. Although the recombinant Kdra cytoplasmic domain can autophosphorylate in vitro, Kdra containing the y17 mutation fails to do so (Fig. 1A Lower). These results indicate that y17 is a mutation in zebrafish kdra that eliminates its kinase activity.

Fig. 1.

Fig. 1.

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The kdray17 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 Kdray17 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 kdray17. 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) kdray17 mutant embryo. (G) Wild-type TG(fli1:egfp)y1 embryo treated with 1 μM SU5416.

We determined the effect of kdray17 on vascular morphology in TG(fli1:egfp)y1 embryos that express EGFP in all endothelial cells (30). By 30 h postfertilization (hpf), a single dorsal aorta carries blood caudally into the tail, whereas the posterior cardinal vein returns blood rostrally (Fig. 1B; see ref. 24). Segmental arteries emanate from the dorsal aorta along somite boundaries and branch to form dorsal longitudinal anastomotic vessels (DLAV; see Fig. 1B). Wild-type sibling embryos display fully formed segmental arteries and branched DLAVs at 30 hpf (Fig. 1C), and the boundaries of the dorsal aorta and posterior cardinal vein are clearly evident (red arrowheads in Fig. 1C). By contrast, partial segmental arteries are apparent in all kdray17/y17 mutant embryos, but DLAVs fail to form at 30 hpf (Fig. 1D). Embryos mutant for kdray17/y17 have a poorly formed dorsal aorta (red arrowheads, Fig. 1D) and display circulatory defects that include arteriovenous shunts (aberrant circulatory connections between the dorsal aorta and posterior cardinal vein; see Movies 1 and 2, which are published as supporting information on the PNAS web site) or absence of circulation (data not shown). The kdray17 mutation also leads to defects in arterial endothelial differentiation. In wild-type siblings, ephrin-B2a (efnb2a) is expressed in the dorsal aorta at 24 hpf (Fig. 1E), whereas kdray17/y17 mutant embryos show reduced efnb2a expression (Fig. 1F).

The kdray17/y17 artery defects are milder than those associated with loss of other Vegf signaling components in zebrafish (5, 23, 31). Interestingly, we find that embryos exposed to the pan-Vegf receptor inhibitor SU5416, which blocks the catalytic activity of the three mammalian Vegf receptors (32) and prevents angiogenesis in zebrafish embryos (33), completely eliminates segmental artery formation at 30 hpf in wild-type TG(fli1:egfp)y1 embryos (Fig. 1G). If Kdra is the only receptor required for segmental artery development, SU5416-treated embryos should phenocopy the kdray17 defects, because its mechanism of action mimics the y17 molecular defect. However, the more severe morphogenesis defects suggest that other Vegf receptors play a role in artery formation.

A likely candidate that may cooperate with Kdra during segmental artery formation is Flt4, a receptor for Vegfc. At the 18-somite stage, vegfc is expressed in the hypochord (Fig. 2A), a structure that lies ventral to the notochord and in contact with the dorsal aorta. By 24 hpf, vegfc expression is seen in the dorsal aorta (Fig. 2B) from which the segmental arteries are sprouting, whereas expression of flt4 becomes restricted to the posterior cardinal vein and segmental artery sprouts (Fig. 2C). To determine whether flt4 and vegfc play a role in segmental artery formation, we designed antisense Morpholino oligonucleotides (MO) to block splicing or translation of each transcript. The Flt4 SD1 MO targets the exon1/intron1 boundary of flt4 and eliminates subsequent exons (Fig. 2D) while Flt4B MO targets the flt4 5′ UTR (see Fig. 5, which is published as supporting information on the PNAS web site). For vegfc, we coinjected previously described MOs that target the 5′ UTR and ATG (34) or against the exon 3 splice donor site (see Fig. 5).

Fig. 2.

Fig. 2.

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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) kdray17/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) kdray17/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).

To determine the genetic interaction between flt4 or vegfc and kdra during artery development, we injected Flt4 MO or Vegfc MO into embryos derived from kdray17/+ carriers. Wild-type embryos injected with scrambled MO display normal segmental artery formation at 30 hpf (Fig. 2E, Table 1, which is published as supporting information on the PNAS web site), whereas kdray17/y17 mutant embryos have partial segmental arteries similar to uninjected siblings (for example, see Fig. 1D; Table 1). Embryos injected with 2 ng of Flt4 MO display variable defects in segmental artery formation, and the severity of these is inversely proportional to kdra dosage: approximately one-half of homozygous wild-type sibling embryos display partial segmental artery formation, whereas more than two-thirds of kdray17/+ heterozygous sibling embryos display similar defects after injection with Flt4 MO (for example, see Fig. 2F; Table 1). Accordingly, kdray17/y17 mutant embryos injected with Flt4 MO displayed no segmental arteries at all (Fig. 2G, Table 1). Reduction of Vegfc causes similar defects, including partial segmental artery formation in wild-type and kdray17/+ embryos (Fig. 2H) and loss of segmental arteries in kdray17/y17 mutant embryos (Fig. 2I; Table 1). Injection of 10 ng of Vegfa MO can eliminate segmental artery formation (5, 23, 31), whereas a 5-ng dose leads to partially formed segmental arteries (Table 1). Consistent with the genetic interaction between kdra and flt4, we find that injection of 5 ng of Vegfa MO and 10 ng of Vegfc MO together in wild-type TG(fli1:egfp)y1 embryos leads to complete loss of segmental artery formation (Fig. 2J).

Observation of circulatory defects revealed a similar genetic interaction between kdra and flt4. Homozygous wild-type sibling embryos injected with Flt4 MO occasionally displayed loss of caudal circulation caused by an arteriovenous shunt, whereas nearly one-half of injected sibling kdray17/+ heterozygous embryos displayed this phenotype (Table 1). Reduction of Flt4 in kdray17/y17 mutant embryos further enhanced the severity of circulatory defects. By contrast, reduction of flt4 does not affect artery-specific efnb2a expression in kdray17/y17 embryos (Table 1), although loss of Vegfc in kdray17/+ heterozygous embryos causes reduction in efnb2a, consistent with the ability of Vegfc to activate Kdr (35).

Given the cooperative role of flt4 and kdra during artery formation, we determined whether they act in a similar manner during vein development. In this case, we focused on the development of the midcerebral vein (MCeV) and primordial hindbrain channel (PHBC), which return cranial venous blood into the Ducts of Cuvier and back to the heart (Fig. 3A). These veins express kdra (26) and flt4 (Fig. 3B). In addition, vegfc expression is observed along the midbrain hindbrain boundary (along which the MCeV is located; Fig. 3C, white arrowhead) and in the anterior lateral branches of the dorsal aorta (along which the PHBC forms; Fig. 3C, black arrows), whereas vegfa is expressed in cells located ventral to each end of the PHBC (Fig. 3D), as well as at the dorsal aspect of the midbrain hindbrain boundary (Fig. 3D). Despite the expression pattern of kdra and vegfa, both the MCeV and the PHBC form normally in embryos mutant for kdray17 (data not shown) or injected with 10 ng of Vegfa MO (Fig. 3E). By contrast, the PHBC failed to form in embryos injected with Vegfc MO (Fig. 3F; Table 2, which is published as supporting information on the PNAS web site) or Flt4 MO (data not shown; Table 2). Furthermore, combined loss of kdra and flt4 exacerbated this phenotype (Fig. 3G; Table 2). Interestingly, we find that formation of the MCeV is normal even in kdray17/y17 mutant embryos injected with Flt4 MO (Fig. 3G, white arrow), although it fails to form in embryos treated with SU5416 (Fig. 3H).

Fig. 3.

Fig. 3.

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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) kdray17/y17 mutant embryo injected with 2 ng of Flt4 MO. (H) Wild-type Tg (Fli1:egfp)y1 embryo treated with 2.5 μM SU5416.

The persistence of MCeV and, to a lesser extent, PHBC formation in the absence of both Flt4 and Kdra (Table 2) suggested that other Vegf receptors contribute to vein development. By searching the available zebrafish genome for genes with homology to kdra, we identified an additional vegfr-2 ortholog (referred to hereafter as kdrb) that is expressed in all developing blood vessels in the head at 24 hpf (Fig. 4A). kdrb is expressed in segmental arteries and the dorsal aorta but not in the posterior cardinal vein (Fig. 4B), similar to kdra (26). The predicted Kdrb amino acid sequence is 51% identical to human Vegfr-2/Kdr and 38.5% identical to human Vegfr-1. Phylogenetic alignment clusters zebrafish Kdrb with mammalian Vegfr-2 orthologs, whereas zebrafish Kdra clusters with Vegf receptor-like molecules predicted from the genomes of Xenopus tropicalis (Ensembl no. ENSXETP00000009987; v3.0), Tetraodon nigoviridis (Ensembl no. GSTENT00031225001; v7), and Fugu rubripes (Ensembl no. SINFRUP00000139301; v2.0; see Fig. 4C).

Fig. 4.

Fig. 4.

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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 kdray17/y17 mutant embryo injected with 5 ng of Kdrb MO. (F) kdray17/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 kdray17/+ heterozygous embryo injected with 5 ng of Kdrb MO. (H) Absence of segmental arteries in a kdray17/y17 mutant embryo injected with 5 ng of Kdrb MO.

To determine the role of kdrb, we designed a splice donor MO targeting this gene. The Kdrb SD1 MO targets the splice donor site at exon 14 and blocks splicing into 3′ exons (Fig. 4D). Loss of Kdrb did not affect MCeV or PHBC formation even in kdray17/17 mutant embryos (Fig. 4E, Table 2). However, combined loss of all three receptors completely blocked the formation of both veins (Fig. 4F, Table 2). We also find that injection of kdrb MO into kdry17/+ leads to partial segmental artery formation, whereas injected sibling kdray17/y17 mutants display a complete loss of segmental arteries (Fig. 4H, Table 1). However, segmental artery formation is largely unaffected in homozygous wild-type embryos lacking Kdrb (Table 1). Similarly, loss of Kdrb alone does not affect circulation in wild-type embryos and only mildly affects kdray17/+ heterozygous embryos (Table 1). We did observe enhanced circulatory defects in kdray17/y17 mutant embryos after injection with Kdrb MO. Loss of kdrb did not have any effect on efnb2a expression in the dorsal aorta (data not shown; Table 1).

Discussion

In this study, we have taken advantage of the zebrafish to genetically dissect Vegf receptor function during vertebrate vascular development. Our findings reveal that multiple Vegf receptors cooperate through differential genetic interactions to drive formation of different blood vessel types during embryonic development in zebrafish.

We find that a kinase-dead allele of zebrafish kdra causes defects in artery development, whereas loss of a newly identified Vegfr-2 ortholog, kdrb, alone has no effect on wild-type embryos. Surprisingly, phylogenetic analysis based on amino acid sequence homology suggests that kdrb represents an ancestral form of Vegfr-2, whereas kdra is more closely related to mammalian Vegfr-1 and appears to have resulted from duplication of Vegfr-1 during teleost evolution. However, several observations suggest that kdra has evolved as the functional zebrafish homolog of Vegfr-2 in mammals. Most notably, loss of the kdra cytoplasmic domain (36) or kinase activity (this study) leads to defects in vascular development in zebrafish embryos. By contrast, mice lacking the cytoplasmic domain of Vegfr-1 display normal blood vessel formation (14). Additionally, the phenotypes associated with kdra mutations in zebrafish resemble Vegfa or plcg1 loss of function (19, 20), whereas loss of Vegfr-1 in mouse leads to hyperproliferation of endothelial cell progenitors, consistent with its role as a negative regulator of Vegf signaling (12, 13). Together, these observations indicate that zebrafish kdra is the functional receptor for Vegfa, whereas kdrb appears to play a compensatory or accessory role during embryonic blood vessel development.

Although the kdray17 phenotypes are similar to those associated with loss of Vegfa or plcg1 (23, 24), they are somewhat milder, suggesting that the kdray17 allele may be a hypomorph. Indeed, we have recently identified an additional allele of kdra that more closely phenocopies the artery defects associated with loss of plcg1, whereas veins remain unaffected (unpublished observations). Although Kdray17 does not possess catalytic activity, its cytoplasmic domain could still be a substrate for phosphorylation by other kinases and therefore allow subsequent interaction with downstream signaling molecules. The requirement of flt4 for segmental artery formation and its genetic interaction with kdra suggests that flt4 may play such a role. Indeed, the genetic interactions between kdra and flt4 pathways are consistent with recent biochemical evidence that shows these receptors can heterodimerize in endothelial cells (18). Interestingly, wild-type Flt4 is capable of phosphorylating kinase-dead Flt4 in human cell lines after stimulation with Vegfc (37). A similar mechanism in which Flt4 phosphorylates kinase-dead Kdra may explain partial segmental artery formation seen in kdray17 mutant embryos.

An intriguing outcome of our results is that each blood vessel analyzed in this study appears to display a different sensitivity to Vegf receptor perturbation. For the most part, flt4, kdra, and kdrb are coexpressed in blood vessels, suggesting that differential receptor expression is not responsible for diverse Vegf receptor sensitivities. One exception is the dorsal aorta, where flt4 expression is down-regulated as development proceeds. Accordingly, flt4 is not required for arterial differentiation of endothelial cells in the dorsal aorta. The variable expression patterns of vegfa and vegfc suggest that differences in ligand accessibility may be in part responsible for different receptor sensitivity. For example, vegfc is the predominant ligand expressed in close proximity to the PHBC, which appears most sensitive to loss of flt4, whereas vegfa is similarly expressed near segmental arteries that are most sensitive to kdra perturbation. In addition to these differences, we have previously shown that plcg1, which is known to act downstream of Vegfa and Kdra (5), is required for artery, but not vein, development (23). Together with our current findings, these observations suggest that ligand accessibility may determine Vegf receptor usage and lead to the activation of distinct downstream signaling cascades.

An important question raised by our observations is how these different signals are ultimately translated by developing endothelial cells. Additionally, why would different signaling pathways need to be used in different blood vessel types? These differences may reflect different cellular mechanisms by which each of these blood vessels forms. It is also likely that Vegf receptor outputs may play an important role in defining the identity and functional differences of blood vessels. This possibility is consistent with the known role of Vegfa signaling components in driving artery identity (19, 20). Finally, do the diverse interactions we observe between Vegf receptors in zebrafish occur in mammalian blood vessels? Recent evidence demonstrating that Vegfr1/2 and Vegf2/3 heterodimers can form in mammalian cell lines and sometimes display qualitatively different signaling capacities (38) suggests that our observations reveal a conserved aspect of Vegf signaling. Future use of the zebrafish will allow further genetic dissection and characterization of these diverse pathways and will help shed insight onto how they may act to drive blood vessel development.

Materials and Methods

Zebrafish.

Zebrafish were maintained according to standard protocols (39).

Genotyping and Mapping.

Bulk segregant mapping used to map y17 is described elsewhere (40). The kdra coding sequence was amplified from cDNA derived from wild-type and y17 mutant embryos and directly sequenced. A dCAPS approach (28) was used to determine linkage of the y17 mutation to kdra as follows: genomic DNA was PCR-amplified by using primers 5′-GCTTCTGTCGTTCATTCTTAA and 5′-ACTAAAGATAACCTGTTACAGTTACCTCTC, digested with DdeI (New England Biolabs), and separated on agarose gels.

Phenotype Analysis.

Embryos were observed at 30 hpf with a MZFLIII dissection microscope (Zeiss) equipped with epifluorescence. Segmental artery phenotypes were classified as followed: presence of a DLAV, indicating normal segmental artery development (DLAV+); presence of segmental arteries but absence of DLAV (DLAV−); or complete absence of segmental artery formation (SeA−). All embryos were genotyped as above for the y17 mutation. Circulation was scored at 48 hpf by using an MZ12 microscope. Whole-mount in situ hybridization was performed as described (41). Blood vessels were imaged by whole-mount immunostaining of TG(fli1:egfp)y1 embryos with an antibody against GFP (Molecular Probes) and an AlexaFluor 488-conjugated secondary antibody. Stacks of images were obtained at ×200 by using a Leica (Deerfield, IL) TCS SPII confocal microscope. Single vertical projections were generated as TIF files by using the included software. Transmitted light images were captured on an MZ12 dissection scope or a Axiophot2 by using an AxioCam MRc digital camera (Zeiss).

Riboprobes.

To identify kdra orthologs, we performed blastn and blastx searches of the available zebrafish genomic and predicted peptide sequences, respectively (www.ensembl.org/Danio_rerio/blastview) by using the full length kdra coding sequence. A kdrb fragment was PCR-amplified, cloned into pCR2.1, and sequenced (GenBank accession no. AY833405). The resulting plasmid was digested with NotI, and a digoxigenin-labeled antisense riboprobe was synthesized with SP6 polymerase. The efnb2a and flt4 riboprobes were prepared as described (41). The vegfc riboprobe was synthesized from the pCS2vegfc plasmid (N.D.L., unpublished work).

Flt4 5′ RACE and Morpholinos.

The coding sequence 5′ flt4 (GenBank accession no. AY833404) was obtained by 5′ RACE (SMART RACE kit, BD Biosciences, Palo Alto, CA) and compared to genomic sequence to deduce exon-intron boundaries. Flt4 SD1 MO is 5′-TTAGGAAAATGCGTTCTCACCTGAG and overlaps the exon 1 splice donor site. The kdrb intron-exon boundaries were determined by comparing the sequence described above (see Riboprobes) to available genomic sequence. The sequence of Kdrb SD1 MO is 5′-GTTTTCTTGATCTCACCTGAACCCT. MOs were obtained from Gene Tools (Philomath, OR). To confirm efficacy of splice MOs, they were injected into wild-type embryos, RNA was isolated by using TRIzol reagent at 24 hpf, reverse-transcribed, and subjected to PCR spanning targeted exon junctions. MOs against Vegfa and Vegfc have been described (31, 34). For Vegfc MO injections, 5 ng of each published MO was coinjected to observe the described phenotype. Additional MO and PCR primer sequence are available in Supporting Text, which is published as supporting information on the PNAS web site.

SU5416 Treatment.

SU5416 (EMD Biosciences, San Diego) was dissolved in DMSO and stored at −20°C. TG(fli1:egfp)y1 embryos were treated beginning at shield stage with indicated concentration of SU5416 or 0.1% DMSO alone diluted in standard egg water.

Kinase Assay.

Kdra cytoplasmic domain was cloned into pTrcHis2A (Invitrogen). The y17 mutation was introduced by PCR. Plasmids were used to transform BL21 bacteria, and recombinant protein was isolated by nickel affinity purification with His-Trap columns (Amersham Pharmacia Biosciences). For the in vitro kinase assay, 100 ng of protein was incubated at room temperature for 30 min in a reaction containing 50 mM Hepes (pH 7.5), 150 mM NaCl, 0.1% Triton X-100, 10 mM MgCl2, 10 mM MnCl2, and 5 mM ATP. Reactions were stopped with sample buffer, separated by SDS/PAGE, and subjected to Western blotting. Blots were incubated with 4G10 antibody against phosphotyrosine (Upstate Biotechnology, Lake Placid, NY). To detect total recombinant protein, blots were stripped and incubated with anti-myc 9e10 monoclonal antibody (Sigma). A horseradish peroxidase-conjugated secondary antibody and chemiluminescent detection were used in both cases for detection (Bio-Rad).

Acknowledgments

We thank Charles Sagerstrom and Arndt Siekmann for critical reading of this manuscript. This work was supported by National Cancer Institute Grant R01CA107454 (to N.D.L.) and the Richard and Susan Smith Family New Investigator Award (to N.D.L.).

Abbreviations

hpf

hours postfertilization

DLAV

dorsal longitudinal anastomotic vessels

MO

Morpholino oligonucleotides

MCeV

midcerebral vein

PHBC

primordial hindbrain channel.

Footnotes

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

Data deposition: The sequences reported in this paper have been deposited in the GenBank database [accession nos. AY833405 (partial kdrb) and AY833404 (flt4)].

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