Identification of Four nrp Genes in Zebrafish
We have identified four cDNAs encoding zebrafish homologues of the neuropilin gene family. The sequences of the cDNAs predict two proteins each that show homology to Nrp1 and Nrp2, respectively (Takagi et al., 1991; Chen et al., 1997). Based on sequence similarities, we designated the cloned zebrafish genes nrp1a, nrp1b, nrp2a, and nrp2b. We could not identify any further genomic sequence read in the ZFv3 Sanger assembly that showed similarity to the nrp gene family. Therefore, we predict that the zebrafish genome encodes for a total of two nrp1 and two nrp2 genes, whereas in other vertebrates only one of each has been described.
Expression Domains and Knockdown Phenotypes of Zebrafish nrps Suggest a Role in Arteriovenous Differentiation
The four nrp genes show overlapping, yet distinct expression patterns predominantly in neuronal and vascular tissues. The transcript distribution is consistent with a role of the respective genes as receptors for both Semaphorins and VEGF (He and Tessier-Lavigne, 1997; Soker et al., 1998) and with the expression described in other species (Kawakami et al., 1996; Chen et al., 1997; Herzog et al., 2001). With respect to the vasculature, nrp1 transcripts are localized in the dorsal aorta in chick and mouse embryos, whereas nrp2 is expressed in the posterior cardinal vein (Herzog et al., 2001; Moyon et al., 2001; Yuan et al., 2002). We confirm that zebrafish nrp1 and nrp2 are complementarily expressed in or around the corresponding vessels.
Differential expression of the nrp1 and nrp2 genes supports the idea of Nrps playing a role in arterial and venous blood vessel differentiation. In addition, we observed arteriovenous malformations when nrp function was knocked down. For the interpretation of this result, the following explanation is conceivable: Nrps function in a VEGF-dependent way (Lee et al., 2002, this study). Lawson et al. (2002) showed that VEGF is involved in the determination of arterial cell fate by acting upstream of the Notch pathway. Zebrafish mindbomb (mib) mutant embryos, which are defective in Notch signaling, display molecular defects in arteriovenous differentiation and exhibit ectopic connections between the dorsal aorta and the posterior cardinal vein within the trunk (Lawson et al., 2001). Thus, the shunt phenotypes of nrp knockdown larvae is in accordance with the speculation that Nrps might be involved in arteriovenous differentiation as coreceptors for the VEGF ligands.
In addition, a recent study in zebrafish provides in vivo evidence that Semaphorin3a1 affects the migration of angioblasts and the formation of the dorsal aorta (Shoji et al., 2003). Putative migrating angioblasts express nrp1a. Semaphorin3a1 is a possible ligand for Nrp1a. Based on these results, a defect in the dorsal aorta when nrp1a is knocked down was expected (Shoji et al., 2003). Our results together with the short-circuit blood flow of nrp1a morpholino-injected embryos reported by Lee et al. (2002) support a possible role of Nrp1a in proper formation of the dorsal aorta.
Remarkably, shunts were uniformly located within the tail of nrp knockdown larvae, whereas in mib mutant embryos shunts occur within the trunk. This observation could indicate a more sensitive response of the caudal artery and vein to a loss of nrps than the axial vessels within the trunk.
While the distinct expression patterns of the zebrafish nrp genes and the different phenotypes of the knockout of mouse nrp1 and nrp2 (Kawasaki et al., 1999; Yuan et al., 2002) would have suggested diverged functions of Nrps in zebrafish, three of four nrp knockdown larvae displayed rather similar shunt phenotypes. These results indicate that Nrps may have different responsibilities within the same process but that at least in the case of Nrp2a and Nrp2b the functions have diverged. Therefore, if either one of the components of this process is deficient, endothelial cells differentiate in the same improper way, resulting in arteriovenous shunts of the affected larvae.
In the nrp1a, nrp1b, and nrp2a knockdown larvae, the shunts were accompanied by defects in the intersegmental vessels. One could speculate about a role for Nrps in proper pathfinding of these vessels, similar to its function as a receptor for Semaphorins in axon repulsion (He and Tessier-Lavigne, 1997; Kitsukawa et al., 1997). However, many larvae with deficient genes relevant for vessel development display an intersegmental vessel phenotype (Nasevicius et al., 2000; Lawson, 2001; Childs et al., 2002; Habeck et al., 2002), indicating that these vessels are rather sensitive to molecular changes in their environment.
nrp2b is the only nrp gene whose inactivation leads to vascular phenotypes other than a shunt when knocked down by injections of antisense morpholino oligo. The vascular malformation in these larvae specifically affected the caudal artery. It is striking that nrp2b is expressed in cells ventral to the aorta; however, the neighboring artery is disrupted in the knockdown situation. Possibly, Nrp2b receptors on the caudal vein are necessary to guide arterial cell fate in a repulsive manner, such as reported in neuronal tissues. Here, binding of Semaphorin ligand to Nrp receptor proteins led to repulsion of growth cones (He and Tessier-Lavigne, 1997; Kitsukawa et al., 1997). When high amounts of morpholino were injected, nrp2b knockdown larvae displayed a heart phenotype that resulted in a lack of blood circulation. This finding is consistent with its expression in the heart. In contrast to this observation, nrp2-deficient mice were viable to adulthood and the vessel phenotype was rather subtle, effecting primarily the small lymphatic vessels and capillaries (Yuan et al., 2002). Of interest, lymphatic vessels have not been described in zebrafish to date. Taken together, both results, the differential expression and the knockdown phenotypes, are consistent with a model of Nrps being involved in proper embryonic vascular development.
All Four Nrps Interact With VEGF In Vivo
Lack of a single vegf allele results in abnormal vascular development and lethality of mouse embryos (Carmeliet et al., 1996). When zebrafish vegf is knocked down, injected larvae display a variety of phenotypes, ranging from defects in the intersegmental vessels to nearly complete loss of axial and intersegmental vessels (Nasevicius et al., 2000). A dosage sensitivity could also be observed in the nrp/vegf double knockdowns. Coinjections of morpholinos directed against vegf and each of the nrps led to severe vascular defects, whereas the same amounts of morpholino had no significant effect when injected alone (Lee et al., 2002, this study). These results are consistent with in vitro experiments showing that Nrp1 is a coreceptor for VEGFR2, thereby enhancing the binding affinity of VEGF165 ligand (Soker et al., 1998; Lee et al., 2002), while Nrp2 forms complexes with VEGFR1 (Gluzman-Poltorak et al., 2001). In addition, studies in mouse and zebrafish show that VEGF is required for arterial blood vessel development (Lawson et al., 2002; Stalmans et al., 2002). Together with the arteriovenous malformations observed in the nrp knockdown larvae, our data fit the idea of VEGF being an in vivo ligand for Nrps.
In summary, we provide strong evidence that Nrps are required for the correct development of the major axial vessels, the caudal aorta and the caudal vein. The results also demonstrate physiological interdependence of Nrps and VEGF during embryonic vascular development.