Homologs of vascular endothelial growth factor and receptor, VEGF and VEGFR, in the jellyfish Podocoryne carnea

Abstract

Vascular endothelial growth factors (VEGF) are the major inducers of vasculogenesis and angiogenesis in vertebrates. Their effects are mediated by receptor tyrosine kinases of the VEGF receptor (VEGFR) family located on endothelial cells and include stimulation of cell survival, proliferation, migration, and tube formation as well as regulation of vascular permeability. Here, we report the presence of VEGF and VEGFR homologous genes in a basal invertebrate of the phylum Cnidaria. The marine jellyfish Podocoryne carnea features a gastrovascular system consisting of the feeding organ, or manubrium, the radial and ring canals, and the tentacle bulbs. Expression analysis indicates that both genes are involved in tentacle and gastrovascular canal formation, indicating an early recruitment of the VEGF signalling pathway for morphogenetic processes leading to tube formation in metazoans. The evolutionary origin of the VEGF signalling pathway resides in the common ancestor of the Cnidaria and Bilateria. Developmental Dynamics 231:303–312, 2004. © 2004 Wiley-Liss, Inc.

INTRODUCTION

Internal transport of nutrients and waste products as well as gas exchange in primitive multicellular organisms such as cnidarians and turbellarians is governed by gastrovascular systems with a single opening to the ambient water. In the highly developed vertebrates, internal transport of molecules is achieved by the closed cardiovascular and lymphatic systems, whereas the connection to the outside is made by specialized structures like the digestive tract, urinary tract, and gas exchanging organs like lungs or gills. The formation of the vertebrate vascular system is induced mainly by vascular endothelial growth factor (VEGF) and mediated by receptors located on endothelial cells differentiating from mesodermal precursors known as angioblasts. VEGF stimulates endothelial cell proliferation and migration during vasculogenesis and angiogenesis (Ferrara and Davis-Smyth, 1997). The importance of VEGF in vascular development was emphasized by the observation that mice lacking one of the VEGF alleles form abnormal blood vessels and die during embryogenesis (Carmeliet et al., 1996; Ferrara et al., 1996). Furthermore, loss of VEGF function by antisense oligonucleotide inhibition results in blood vessel deficiencies in zebrafish (Nasevicius et al., 2000). Members of the VEGF family and their tyrosine kinase receptors regulate vasculogenesis, angiogenesis, and endothelial cell differentiation (Risau and Flamme, 1995; Risau, 1997; Yancopoulos et al., 2000). The related growth factors of the PDGF family also have important functions during vertebrate embryogenesis, in particular for the development of the kidneys, blood vessels, lungs, and central nervous system (Heldin and Westermark, 1999). All members of the vascular endothelial/platelet-derived growth factor (VEGF/PDGF) family have a VEGF/PDGF homology domain (VHD) characterized by the precise spacing of eight cysteine residues (Neufeld et al., 1994). In addition to VEGF, the VEGF/platelet-derived growth factor (PDGF) family contains several related ligands, including VEGF-B, VEGF-C (Joukov et al., 1996), c-fos–induced growth factor FIGF/VEGF-D (Yamada et al., 1997), the placenta growth factor PlGF (Maglione et al., 1991), and viral homologs (VEGF-E). VEGF-B is expressed in the developing heart during embryogenesis and in adult cardiac and skeletal muscle. It has been proposed that VEGF-B may have a function in regulating the vascularization of adult and embryonic tissues, in particular of muscle (Olofsson et al., 1996). Mice lacking VEGF-B are normal and fertile, but their hearts are reduced in size, indicating that VEGF-B has a major role in coronary vascularization and growth (Bellomo et al., 2000). VEGF-C and VEGF-D constitute a distinct subgroup of VEGF-like growth factors, characterized by N- and C-terminal extensions flanking the VHD. The C-terminal extension contains extra cysteine-rich motifs characteristic of the protein component of silk produced by the larval salivary glands of the midge, Chironomus tentans. Overexpression of VEGF-C in mice results in increased formation of lymphatic vessels (Olofsson et al., 1999). VEGF-D is expressed in the lung during embryogenesis and in many adult tissues, most abundant in lung and heart. VEGF-D is a mitogen for lymphatic endothelial cells (Yamada et al., 1997; Achen and Stacker, 1998). PlGF is predominantly expressed in the placenta and has been proposed to play a role in placental angiogenesis (Maglione et al., 1991), but mice lacking PlGF show a normal phenotype (Persico et al., 1999; Carmeliet, 2000).

The biological effects of VEGF and its homologs are mediated by three vertebrate receptors, VEGFR-1/Flt-1, VEGFR-2/Flk-1, VEGFR-3/Flt-4 (Neufeld et al., 1999; Kärkkäinen and Petrova, 2000). They belong to the VEGF receptor (VEGFR) subfamily of receptor tyrosine kinases characterized by the presence of seven extracellular immunoglobulin homology domains and a split tyrosine kinase intracellular domain. VEGFR-1 and VEGFR-2 are generally expressed in vascular endothelial cells, whereas VEGFR-3 is expressed in lymphatic endothelial cells. In Drosophila, an invertebrate organism without endothelial cells or blood vessels, the single VEGFR gene (Pvr) is expressed in developing and mature hemocytes (blood cells) and three VEGF genes (Pvf1, Pvf2, Pvf3) are expressed ubiquitously in the embryo. Mutants lacking VEGFR arrest blood cell migration. Inactivation of all three VEGF genes results in a similar phenotype. Hence, the Drosophila VEGF signaling pathway is required for hematopoietic instead of endothelial cells (Duchek et al., 2001; Heino et al., 2001, Cho et al., 2002).

The jellyfish Podocoryne carnea is a member of the basal phylum Cnidaria. Cnidarians are model systems for evolution and regeneration (Galliot and Schmid, 2002). Unlike other cnidarians, Podocoryne carnea features a complete life cycle such that all aspects of cnidarian development are accessible. Molecular studies have revealed that a substantial part of the genetic machinery regulating vertebrate development is also present in the jellyfish, including genes for homeobox transcription factors (Yanze et al., 2001) and several basic helix-loop-helix transcription factors (Spring et al., 2000, Müller et al., 2003; Seipel et al., 2004). Here, we analyzed the structure and expression of candidate genes for VEGF and VEGFR in the hydrozoan jellyfish Podocoryne carnea. The gene expression analysis throughout the cnidarian life cycle indicates that both genes are involved in tentacle and tube formation. Developmental and evolutionary aspects of the gene structures and expression patterns are discussed.

RESULTS

Identification of VEGF and VEGFR Homologous Genes in Podocoryne carnea

The VEGF cDNA is 1204 bp with a putative start codon at position 41 followed by an open reading frame ending at position 1082. The gene product of 346 amino acid residues is most related to the Drosophila VEGF-like protein Pvf1 and the human VEGF-D (Fig. 1A), with an overall sequence identity of 18% and 12.5%, respectively. A 23-residue hydrophobic sequence fulfilling the criteria for a secretory signal sequence is located at the N-terminus. The most conserved region is the VHD, with eight conserved cysteine residues. The Podocoryne VHD domain is most similar to the human VEGF-B protein with 35% identity and 54% similarity (Fig. 1B,C). Moreover, Podocoryne VEGF has several repeats of cysteine-rich motifs CX10CXCXC at the C-terminus similar to motifs at the C-terminus of the 185-kDa silk protein of the midge (Joukov et al., 1996). This additional pattern of cysteine residues has also been found in human VEGF-C and VEGF-D (Fig. 1D).

Figure 1. Sequence conservation in the vascular endothelial growth factor (VEGF) protein. A: Full-length sequence alignment of the Podocoryne carnea VEGF protein with the most related bilaterian proteins. The platelet-derived growth factor (PDGF)/VEGF homology domain (VHD) is underlined. B: VHD sequence alignment of the human (Hs), Drosophila (Dm), and Podocoryne carnea (Pc) VEGF and PDGF proteins. C: Phylogenetic tree of the VHD sequence alignment shown in B. Bootstrap values are indicated. D: Sequence alignment of the cysteine-rich motifs CX10CXCXC at the C-terminus of the human (Hs) and Podocoryne carnea (Pc) VEGF proteins and the Chironomus pallidivittatus (Cp) 185-kDa silk protein. Asterisks indicate the conserved cysteine residues.

The VEGFR cDNA is 4,200 bp with a start codon at position 80 followed by an open reading frame of 3,765 bp, ending at position 3,844. The gene product of 1,255 amino acids is characterized by an N-terminal signal peptide followed by seven immunoglobulin (Ig)-like domains, a single transmembrane region, and a split tyrosine kinase domain (Fig. 2A). The catalytic domain contains tyrosine kinase–specific active sites with aspartic and lysine residues important for catalytic activity and ATP binding, respectively. The kinase domain also contains the signature sequence GxHxivNLLGACT typical for split tyrosine kinase domains of receptor tyrosine kinase classes III to V (Grassot et al., 2003). Class III proteins have five Ig-like domains and include PDGFR, macrophage colony stimulating factor (Fms), stem cell factor (Kit), and F1 cytokine (Flt3). Class IV proteins have three Ig-like domains and are receptors for fibroblast growth factors (FGFR). Class V proteins have seven Ig-like domains and are receptors for vascular endothelial growth factors (VEGFR). The Podocoryne VEGFR kinase domain is most closely related to the Hydra receptor tyrosine kinase U24116 (Fig. 2B). The size of the kinase insert is conserved to class IV proteins. The extracellular region of the Podocoryne receptor contains seven Ig-like domains characteristic of class V proteins. By conserved domain search, this region is most similar to the neural cell adhesion molecule (N-CAM) L1 (data not shown). The Ig-like domain 3 is a typical immunoglobulin domain, domain 6 is recognized as Ig-CAM, and domain 7 as immunoglobulin C-2 type domain. The Ig-like domains 1, 2, 4, and 5 are less conserved. In conclusion, the Podocoryne receptor combines a split tyrosine kinase domain with an overall class V structure characteristic for a VEGFR protein.

Figure 2. Conservation of the vascular endothelial growth factor receptor (VEGFR) domain structure. A: Domain structures and size of representative human (Hs), Podocoryne carnea (Pc), and Hydra vulgaris (Hv) receptor tyrosine kinases. The extracellular immunoglobulin (Ig) -like domains are followed by the transmembrane domain (TM) and the cytoplasmic tyrosine kinase domain. PTK7 is a regular tyrosine kinase receptor with seven Ig-like domains. The VEGF and fibroblast growth factor (FGF) receptors are members of the split kinase domain receptors. The Podocoryne receptor has an overall VEGFR structure. Its tyrosine kinase domain is most closely related to the Hydra receptor tyrosine kinase U24116. The asterisk indicates an incomplete C-terminus. B: Phylogenetic tree of the receptor tyrosine kinase domains. The split kinase domain receptors have three (class IV), five (class III), or seven (class V) Ig-like domains. The Podocoryne VEGFR has seven Ig-like domains and a split tyrosine kinase domain characteristic of class V receptors. The Hydra receptor tyrosine kinase U24116 has only three Ig-like domains characteristic of class IV receptors. PTK7 has been used as outgroup. Bootstrap values are indicated. aa, amino acids.

Expression Analysis of the VEGF and VEGFR Genes in the Hydrozoan Life Cycle

Podocoryne carnea is a hydrozoan with a full life cycle (Fig. 3A). The fertilized egg develops into a ciliated planula larva. After 30 to 35 hr, the larva is ready to attach to a suitable substrate at its anterior pole and to transform into the primary polyp. In the first phase of metamorphosis, the larval shape is resolved and the disc stage is formed; in the second phase, the primary polyp rises out of the disc. After stolon outgrowth at the base of the primary polyp, a colony of feeding polyps (gastrozoids) and medusa budding polyps (gonozoids) develop asexually. In the mid-body region of the gonozoid, medusa buds of different developmental stages form a whorl. Medusa development takes 7 to 8 days and has been described previously (Seipel et al., 2004). Expression analysis by reverse transcriptase-polymerase chain reaction (RT-PCR) shows that both VEGF and VEGFR are expressed in all life cycle stages, including the planula larva, both types of polyps, and the medusa (Fig. 3B,C). RNA of the VEGFR but not the VEGF gene is present in the egg as maternal message.

Figure 3. Expression of VEGF and VEGFR genes in the life cycle of Podocoryne carnea. A: Life cycle. B–E: Quantitative reverse transcriptase-polymerase chain reaction expression analysis for VEGF (B,D) and VEGFR (C,E) in all stages of the life cycle from egg to adult (B,C) and in different parts of the adult medusa (D,E). The ordinate values are relative expression levels normalized to the expression level of EF1α and displayed in arbitrary units. VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor.

VEGF expression increases during larval development, reaching maximal levels after attachment of the larva to the substratum but before tentacle formation in the primary polyp (Fig. 3B). The organism is shaped like a disc in this phase of metamorphosis. By in situ hybridization, VEGF-specific staining localizes to the larval endoderm (data not shown). In the polyp, there is weak staining in the hypostomal endoderm and strong staining in the tentacle endoderm along the entire length of the tentacle but also in the tentacle base where cells are recruited continuously for tentacular growth (Fig. 4A,B). In early medusa bud stages, VEGF-specific staining localizes to the bud endoderm (Fig. 5A), with highest levels in the developing radial canals (Fig. 5B–D), later to the striated muscle, the tentacle bulb, radial canal, and plate endoderm (Fig. 5E–G). In older stages, VEGF-specific staining localizes to the tentacles and tentacle bulbs and the three layers of the developing medusa bell (Fig. 5H,I). In the adult medusa, VEGF is expressed in all medusa parts (Fig. 3D), with highest levels in the striated muscle and the tentacles. VEGF-specific staining localizes to the endoderm of the tentacles and the radial and ring canals with highest intensity close to the tentacle bulbs (Fig. 7A,B).

Figure 4. Expression of VEGF and VEGFR in Podocoryne carnea polyps. A–D: In situ hybridization with probes for vascular endothelial growth factor (VEGF; A,B) and VEGF receptor (VEGFR; C,D). Sagittal section (A,C) and cross-section through the polyp hypostome at the level of the tentacle base (B,D) of gastrozoid polyps. hy, hypostome; pe, polyp ectoderm; pen, polyp endoderm; te, tentacle ectoderm; ten, tentacle endoderm. Scale bar in A = 500 μm in A,C, 200 μm in B,D.

Figure 5. VEGF expression in Podocoryne carnea medusa development. Schematic drawings of Podocoryne medusa development and corresponding VEGF expression patterns as revealed by in situ hybridization of tissue sections. A–I: Sagittal sections (A–C,E,G–I), cross-section (F) with bud stages 1 (A), 2 (B), 4 (C,D), 6 (E,F), 7 (G), 8 (H), and 9 (I). Medusa bud stages 1 to 9 according to Frey (1968). be, medusa bud ectoderm; ben, medusa bud endoderm; e, entocodon (orange); ec, entocodon cavity; ex, exumbrella; go, gonads; m, mesoglea (extracellular matrix) separating entocodon from ectoderm and endoderm; ma, manubrium; maa, manubrium anlage; o, oocyte precursor cell; p, plate cells of the subumbrella; pe, polyp ectoderm; pen, polyp endoderm; rc, radial canal; sm, smooth muscle of the manubrium (dark green); sr, smooth muscle of the radial canal (light green); st, striated muscle (red); t, tentacle; tb, tentacle bulbs; v, developing velum; VEGF, vascular endothelial growth factor. Scale bar in I = 100 μm in A, 200 μm in B–D, 300 μm in E–I.

Significant levels of VEGFR RNA are present in the egg and early larva and in the adult medusa, while VEGFR expression is rather low during metamorphosis, in the polyp stage and during medusa development (Fig. 3C). By in situ hybridization, there is weak staining in the larval endoderm (data not shown). In the polyp VEGFR-specific staining is present in the tentacles with highest intensity at the tentacle base diminishing toward the tip (Fig. 4C,D). At the beginning of medusa development, VEGFR-specific staining localizes exclusively to the bud endoderm (Fig. 6A,B). After formation of the entocodon, VEGFR appears to be expressed in the entocodon-derived tissues, including the outer layer of the manubrium and the innermost layer of the medusa bell, the striated muscle (Fig. 6C,D). In the middle stages of medusa development, VEGFR-specific staining is present in the striated muscle, endoderm plate, and the developing radial canals (Fig. E,F), and in the late bud stages in the exumbrella and plate (Fig. 6G). In the adult medusa, VEGFR is expressed at low levels in all medusa parts (Fig. 3E), with visible staining in the tentacle endoderm (Fig. 7C,D).

Figure 6. VEGFR expression in Podocoryne carnea medusa development. Schematic drawings of Podocoryne medusa development and corresponding VEGFR expression patterns as revealed by in situ hybridization of tissue sections. A–G: Sagittal sections (A–D,F) and cross-sections (E,G) with bud stages 1 (A), 2 (B), 4 (C); 6 (D,E); 8 (F,G). Medusa bud stages 1 to 9 according to Frey (1968). Abbreviations are as in Figure 5. VEGFR, vascular endothelial growth factor receptor. Scale bar in F = 100 μm in A,G, 200 μm in B,C, 300 μm in D–F.

Figure 7. Expression of VEGF and VEGFR in the adult male medusa. A–D: Whole-mount in situ hybridization with probes for VEGF (A,B) and VEGFR (C,D). Abbreviations are as in Figure 5. VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor. Scale bar in A = 300 μm in A,C, 150 μm in B,D.

DISCUSSION

Ancestral Function of VEGF Signalling in Tube Formation

The VEGF signalling pathways were discovered and named in vertebrates because of their effects on vascular endothelial cells and their roles in vasculogenesis and angiogenesis. Although Drosophila has blood cells, it is lacking endothelial cells and blood vessels (Heino et al., 2001). Thus, the functions of the recently identified Drosophila VEGF and VEGFR homologs could not be related to endothelial cells and blood vessels. Instead of a cardiovascular system, Drosophila has an open circulatory system, the tracheal system, responsible for delivering oxygen to all body parts of the fly. Both the vertebrate vascular system and the fly tracheal system are similarly formed by branching morphogenesis, based on the VEGF and the FGF signalling pathways, respectively. In Drosophila, the VEGF signalling pathway is involved in blood cell migration and differentiation (Duchek et al., 2001; Heino et al., 2001; Cho et al., 2002).

Cnidarians are the most basal phylum of the animal kingdom with tissue level organization and a complex nerve net. They do not feature blood vessels or blood cells. Nevertheless VEGF and VEGFR genes are present in Podocoryne carnea. This hydrozoan species is a cnidarian with a full life cycle, including a planktonic medusa stage with an elaborate gastrovascular system consisting of the feeding organ, or manubrium, the radial and ring canals, and the tentacle bulbs. In analogy to the vertebrate vascular system, the gastrovascular system functions to supply nutrients throughout the organism. In situ hybridization analysis indicates that the VEGF and VEGFR genes are expressed in the tentacles and in the developing radial and ring canals. The common feature of the canals and tentacles is the tubular structure. Both genes are also expressed in the endoderm of the developing medusa when undifferentiated cells start to migrate and differentiate into plate cells, which in later bud stages form the subumbrellar plate and the four radial canals. The subumbrellar plate consists of a thin epithelium sandwiched between the outer and inner extracellular matrix (ECM) connecting the four radial canals. The plate cells interact with the ECM and with the smooth muscle tissue. This process is reminiscent of blood vessel formation where vascular endothelial cells interact with the surrounding matrix and smooth muscle tissue. In conclusion, the VEGF signalling pathway appears to be involved in tube formation in cnidarians as well as in vertebrates. In Drosophila, however, the VEGF signalling pathway regulates blood cell migration (Duchek et al., 2001; Heino et al., 2001; Cho et al., 2002). This function appears to have evolved secondarily and in relation to the absence of endothelial cells. We propose tube formation as the ancestral function of the VEGF signalling pathway.

On the Origin of the VEGF Signalling Pathway

The Podocoryne VEGFR encodes a protein with a split tyrosine kinase and seven immunoglobulin-like domains. To our knowledge, this is the first report of a cnidarian receptor tyrosine kinase with seven Ig-like domains. The overall structure defines it as a member of the class V split tyrosine kinase receptors, which in Drosophila and vertebrates are VEGF receptors. The split kinase domain of the Podocoryne VEGFR is most closely related to a Hydra receptor tyrosine kinase containing only three Ig-like domains, indicating that the VEGFR may have evolved from a FGFR ancestor by addition of four Ig-like domains. Members of various cnidarian families will have to be studied to reveal the prevalence of genuine VEGF receptors within the Cnidaria. As for the vascular endothelial growth factors, homologs have been found in two hydrozoans, Hydra vulgaris (Diane Bridge, manuscript in preparation) and Podocoryne carnea. To our knowledge, there are no VEGF and VEGFR homologs in metazoans without tissue level organization, as in the sponges. In evolutionary terms, the VEGF signalling pathway appears to have originated in the common ancestor of the Cnidaria and Bilateria, based on an ancestral cell type giving rise to the cnidarian endoderm plate cells and the vertebrate vascular endothelial cells.

EXPERIMENTAL PROCEDURES

Animals

Podocoryne carnea M. Sars (Cnidaria, Hydrozoa, Anthomedusae) colonies are reared in the laboratory in aerated artificial seawater at 16°C. Animals were cultured on glass jars and fed every second day with 2-day-old artemia. Medusa buds were staged according to Frey (1968). Eggs and larval stages were obtained as described (Aerne et al., 1991). Metamorphosis in larva was induced by CsCl treatment as described previously (Müller and Buchal, 1973).

Molecular Cloning and Sequence Comparison

A partial VEGF clone was isolated incidentally from a Podocoryne carnea HybriZAP II cDNA library in a yeast two-hybrid screen (Müller et al., 2003). The sequence was extended by 5′ rapid amplification of cDNA ends (RACE) on SMART cDNA using nested SMART primers NUP1 (5′-AAG CAG TGG TAA CAA CGC AGA G-3′) and NUP2 (5′-TAA CAA CGC AGA GTA CGC GGG-3′) together with the gene-specific primer VEGF-R1 (5′-ACT GTA GAT GTG TAC AAC ACC-3′). The full-length cDNA of 1,204 bp was gel-purified (Qiagen), subcloned into the pCRII-TOPO vector, and sequenced. The VEGF coding sequence has been submitted to the DNA databases with accession no. AY508721.

A short fragment with homology to RTK class III tyrosine kinase domains was isolated from medusa cDNA by homology PCR using the nested degenerate primers VEGFR-F3 5′-CA(CT) (AC)G(AGCT) GA(CT) AT(AGCT) GC(AGCT) GC(AGCT) (AC)G(AGCT) AA-3′ and VEGFR-F1 5′-GT(AGCT) AA(AG) AT(ACT) TG(CT) GA(CT) TT(CT) GG-3′ together with VEGFR-R1 5′-AT(CT) TCC CA(AGCT) A(AG)(AGCT) A(AG)(AGCT) AC(AGCT) CC-3′. To obtain the full-length cDNA, a Podocoryne carnea cDNA library (lambda gt11) was screened in three consecutive rounds. Phages were picked and amplified by 5′ RACE and 3′RACE. The probes for screening were prepared by PCR with gene specific primers VEGFR-F1 (5′-TTA GCT CGT GAT ATT TAT AAA GAT-3′) and VEGFR-R1 (5′-TTT GAC AGG AAG CAA ACC AGT TGT-3′) for the first round, FGFR-F5 (5′-TTC ATG GAG GAT GGG AGC TGA C-3′) and FGFR-R3 (5′-CCA ATG ACG TCA CTT CTT CTA C-3′) for the second, and FGFR-F7 (5′-CAT TGG GCC AAA GAT GGA CAG-3′) and FGFR-R8 (5′-AAT GGA GCT GAA GTT TGT TGA CG-3′) for the third round. The 5′ end of the gene was obtained by 5′RACE on larval SMART cDNA using the nested SMART primers NUP1 and NUP2 together with the gene-specific primers FGFR-R11 (5′-GAC AAC AGT GCA CCT GCT TGA GCC-3′) and FGFR-R12 (5′-GAG CCA TGC GAA ATG GAA GTT CG-3′). The 3′ end of the gene was obtained by 3′ RACE on larval first-strand cDNA synthesized with the modified oligo(dT) primer XT20V (5′-GGC AGG TCC TCG TTG ACT CGA GAC GT(20)A GC-3′) by AMV reverse transcriptase (Roche). The 3′ RACE was done with the XT20-specific primers X1 (5′-GGC AGG TCC TCG TTG ACT CG-3′) and X2 (5′-GCA GGT CCT CGT TGA CTC GAG ACG-3′) together with the gene-specific primers FGFR-F3 (5′-TTT AGA CTT CAC TGA TGC GAA-3′) and FGFR-F4 (5′-GAA AAT GAT TAC CTC ACC CCA G-3′). The resulting 4,200-bp VEGFR cDNA has been submitted to the GenBank with accession no. AY508722. Nucleotide and deduced amino acid sequences were analyzed using the GCG software package. BLAST searches (Altschul et al., 1997) were performed using the BLAST network service at the EBI (http://www.ebi.ac.uk). Multiple sequence alignments and phylogenetic trees based on the neighbor-joining (N-J) method were generated with Clustal X (Jeanmougin et al., 1998). Sequence alignments were generated with gap opening 10, gap extension 0.2 using the protein weight matrix of the Blosum series. Positions with gaps were excluded for the N-J tree construction. Accession numbers for protein sequences used in Figures 1 and 2 are as follows: PDGFA-Hs, 1205299A; PDGFB-Hs, 1109245A; PDGFC-Hs, AAF80597; PDGFD-Hs, AAK56136; PLGF-Hs, P49763; Pvf1-Dm, AJ401391; Pvf2-Dm, AY079182; Pvf3-Dm, AY079183; VEGFA-Hs, CAC60149; VEGFB-Hs, CAC60150; VEGFC-Hs, CAC60151; VEGFD-Hs, CAC60153; and in Figure 2 as follows: FGFR1-Hs, P11362; FGFR2-Hs, P21802; FGFR3-Hs, P22607; FGFR4-Hs, P22455; FGFRL-Hv, AY193769; Flt3-Hs, P36888; Fms-Hs, P07333; Kit-Hs, P10721; PDGFRα-Hs, P16234; PDGFRβ-Hs, P09619; PTK7-Hs, Q13308; Pvr-Dm, AY079187; Silkprot-Cp, AAA99803; U24116-Hv, U24116; VEGFR1-Hs, P17948; VEGFR2-Hs, P35968; VEGFR3-Hs, P35916. RTK classification is according to the RTK database (Grassot et al., 2003).

RT-PCR Expression Studies

The mRNA for expression analysis was extracted from unfertilized eggs, planula larvae, gastrozoid and gonozoid polyps, medusae, and medusa parts by using the Dynabeads mRNA direct kit (Dynal) and reverse transcribed into single-strand cDNA with AMV reverse transcriptase and random hexamer primers (first-strand cDNA synthesis kit for RT-PCR, Roche). RT-PCR expression analysis was performed with the FastStart DNA Master SYBR Green I kit (Roche) as described (Spring et al., 2000). The ubiquitously expressed elongation factor 1 alpha (EF1α) was included in each set of experiments as a reference to compensate for variations in the quantity and quality of preparations. PCR analysis was done in duplicates and in independent experiments. VEGF expression was analyzed with primers VEGF-F1 (5′-ACC ACG AGA AGC AAT GCC AA-3′) and VEGF-R2 (5′-GAT GCT CTA CAC CGA AGA CAG TC-3′). VEGFR expression was analyzed with primers FGFR-F1 (5′-GGT GTA TAC TTC CAT GAG TG -3′) and FGFR-R1 (5′-CAG CAC GTT TGT CAA TCG ATC-3′). Primers for EF1α have been described (Yanze et al., 1999).

In Situ Hybridization

A 481-bp fragment, including the VEGF/PDGF homology domain, was amplified with VEGF-F1 (5′-AAA TGC CAA CCA CGA GAA GC-3′) and VEGF-R2 (5′-GTA TTT GGA TCT CGA ATG TGT GC-3′). A 390-bp fragment at the 3′end of the VEGFR was amplified with the primer pair FGFR-F4 (5′-GAA AAT GAT TAC CTC ACC CCA G-3′) and FGFR-R5 (5′-GAT CAT CCT TTC GTA AAT CAT GTT GG-3′). The PCR products were subcloned into the pCRII-TOPO vector (TOPO TA cloning Dual Promotor kit, Invitrogen) and used as template to generate digoxigenin (DIG) -labeled antisense probes by in vitro run-off transcription with the DIG-RNA Labeling Kit (Roche) using Sp6 RNA-polymerase (Roche). In situ hybridization was carried out as described previously (Seipel et al., 2004). Detection was performed by immunochemical staining with anti–DIG-Fab-AP (Roche) using nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) as a substrate. Specimens were fixed for 2 hr at room temperature with freshly prepared 4% paraformaldehyde or Lavdowsky solution. For sectioning, stained specimens were post-fixed for 2 hr at room temperature in 4% paraformaldehyde, dehydrated in ethanol, embedded in Paraplast (Sigma) and sectioned to 10 μm.

Acknowledgements

We thank Dr. Jürg Spring for critical reading and helpful comments. We also thank Roky Bamert and Brigitte Winniger for technical support.