Squid vascular endothelial growth factor receptor: a

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signature in the convergent evolution of closed circulatory systems. Masa-aki Yoshida,a,1,Г ... open circulatory system with only one peristaltic tubular heart.
EVOLUTION & DEVELOPMENT

12:1, 25 –33 (2010)

DOI: 10.1111/j.1525-142X.2009.00388.x

Squid vascular endothelial growth factor receptor: a shared molecular signature in the convergent evolution of closed circulatory systems Masa-aki Yoshida,a,1, Shuichi Shigeno,b Kazuhiko Tsuneki,a and Hidetaka Furuyaa a

Department of Biology, Graduate School of Science, Osaka University, Osaka 560-0043, Japan Department of Neurobiology, The University of Chicago, Chicago, IL 60637, USA

b

Author for correspondence (email: [email protected]) 1

Present address: Ochadai Academic Production, Ochanomizu University 2-1-1, Otsuka, Bunkyo, Tokyo 112-8610, Japan.

SUMMARY The highly specialized cephalopod cardiovascular system has long been considered a valuable model for understanding the evolution of circulatory systems. Despite the number of studies devoted to this topic, the developmental regulatory mechanisms remain largely unexplored. Here, we focus on the vascular endothelial growth factor receptor (VEGFR). This factor is known to mediate levels of endothelial growth factor that is involved in hematopoiesis and vasculogenesis including

multichambered heart development in vertebrates. We found a squid VEGFR ortholog that is expressed in the developing blood vessels, notably in the sheet-like endothelial cells of the systemic and branchial hearts. The highly restricted localization of VEGFR in the vascular endothelial cells and its shared expression pattern in the developing hearts of cephalopods and vertebrates suggest a shared molecular signature of closed circulatory systems that has been independently elaborated during evolution.

INTRODUCTION

Metazoan animals have evolved an incredible diversity of hearts and heart-like structures. The most elaborate case in invertebrates is observed in coleoid cephalopods: they exhibit an elaborate closed circulatory system (Schipp 1987; Budelmann et al. 1997). Their heart possesses a kind of advanced output structure similar to that of the human heart, which differs largely from molluscan typical nonendothelium primitive chambered hearts (see Kling and Schipp 1987; Schipp 1987). Neither morphological nor molecular data give strong support to a close phylogenetic relationship between vertebrates and cephalopods, suggesting that the closed circulatory systems and complicated hearts were formed independently in each lineage, and have converged during their evolution. However, recent findings have revealed that the cardiovascular systems of the Drosophila and vertebrates are comparable in terms of the similarity of their molecular regulatory pathways (Bodmer and Venkatesh 1998; Chen and Fishman 2000; Hartenstein and Mandal 2006). Indeed, the expression and function of homeobox transcriptional factor tinman/ Nkx2.5 during cardiogenesis are commonly shared in Drosophila and vertebrates (Bodmer and Venkatesh 1998). Notably, the vascular endothelial growth factor receptors (VEGFRs) are located on endothelial cells differentiating from mesodermal precursors in the vertebrates. The VEGFR is expressed throughout the endocardium of the developing heart

According to structural and functional criteria, cardiovascular systems can be divided into two groups; open and closed (reviewed in Brusca and Brusca 2003; Schmidt-Rhaesa 2007). Most invertebrates, such as the fruit fly Drosophila, possess an open circulatory system with only one peristaltic tubular heart (dorsal vessel) (Cripps and Olson 2002). The Drosophila heart contracts rhythmically and expels hemolymph into the hemal space of the body (body cavity). The open vascular systems of invertebrates are different from the closed ones of vertebrates in that they lack an endothelium (Opitz and Crark 2000; Schmidt-Rhaesa 2007). Large vessel-like structures of invertebrates are comprised of spaces located between the basement membranes of the endodermal and coelomic epithelia, or between two coelomic epithelia. Conversely, the closed circulatory system of vertebrates is composed of a highly efficient multichambered heart and a continuous network of blood vessels. The blood vessels are lined with an endothelium, an adluminal continuous layer of epithelial cells interconnected by special junctional complexes. The occurrence of endothelia is assumed to be related to the blood vascular system acting at high mechanical pressures with a small amount of blood (Prosser 1973; Wells 1978; Budelmann et al. 1997). & 2010 Wiley Periodicals, Inc.

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and required for cardiac valve formation in zebrafish (Lee et al. 2006). In Drosophila embryos, a single VEGFR gene (Pvr) is expressed in developing and mature hemocytes (Heino et al. 2001; Cho et al. 2002). The three ligands, Pvf1–3, are expressed ubiquitously in the whole embryo and are involved in blood cell development (Cho et al. 2002). A recent study has revealed that the VEGF pathways are also required for cardiac valve development of the adult Drosophila heart (Zeitouni et al. 2007). These findings suggest the presence of a conserved molecular developmental program between the vertebrate and Drosophila cardiovascular systems. This raises a hypothesis that the closed circulatory systems and multiply divided hearts have been brought by the action of a group of genes highly conserved across bilaterian phyla. To explore this possibility, we chose to study a cephalopod circulatory system that provides a powerful lens for understanding cardiovascular evolution. Their cardiovascular system is considerably similar to vertebrates in several respects such as high oxygen binding capacity, high concentrations of proteins, and short circulation time (Schipp 1987). Each vessel in the cephalopod is constructed similarly to vertebrate vessels, with an endothelial lining on a basement membrane (Budelmann et al. 1997), although the cephalopod blood vessel lining does not have the cellular junction typical among vertebrate species. Most invertebrates have no endothelium in their vascular walls so the cephalopods are unusual in that they are invertebrates with vertebrate type blood vessels. As the other molluscs have open vascular system, the peculiar blood vessel configuration in the cephalopods is in all probability secondarily developed similarly to the vertebrate among chordates (Ruppert and Carle 1983). In this study, we discovered a VEGF receptor ortholog in squid and identified the expression in blood vessels of embryos. This is the first evidence to show commonality in signaling molecules between closed circulatory systems in the cephalopods and vertebrates. We also found that domains in developing cardiovascular systems of squid embryo express the VEGFR, together with the FGFR. In addition to the finding of conserved patterns of transcription factor Nkx2.5/ Csx gene in cardiac progenitor cells (Elliott et al. 2006), these conserved genes play roles as part of developmental pathway in heart development. Thus, we propose that the conserved molecular developmental program for cardiovascular systems were recruited independently to the closed circularity systems of cephalopods and vertebrates.

compact fish tank according to Yamamoto (1988). Spawned eggs were transferred to a Petri dish and kept at 181C. Embryonic stages were determined using a standardized scale (see Yamamoto 1988). The chorion was removed with forceps from stage-22 to stage-27 embryos. Before stage-21 the chorion was softened and removed according to Yamamoto et al. (2003), because the embryo had a narrow perivitelline space. Embryos from stage-27 onwards were anesthetized in seawater containing 1% ethanol and fixed overnight at 41C in PBS containing 4% paraformaldehyde and 0.1% Tween 20. Embryos for in situ hybridization were progressively dehydrated with gradual series of methanol (25/75, 50/50, 75/25, and 100/0) and stored at 201C until further use.

Molecular cloning Full-length cDNAs of squid VEGFR, FGFR, and Actin II were obtained using degenerated PCRs followed by the rapid amplification of cDNA ends methods. Details of primers and PCR conditions are provided in supporting information. The sequences are available under following accession numbers: VEGFR, AB500117; FGFR, AB500118; Actin II, AB500119.

Molecular phylogenetic analysis A sequence file was made for tyrosine kinase receptors using amino acid sequences of the kinase domain. Multiple sequence alignments and phylogenetic analyses based on the neighbor-joining method were performed using Clustal X (Thompson et al. 1997). Sequences were aligned using default parameters (see supporting information, for accession numbers for each protein). Positions with gaps were excluded from the phylogenetic analyses. The support values were given by bootstrap percentiles of 1000 replicates.

In situ hybridization Whole mount in situ hybridization was carried out using established procedures (Grove et al. 1998) with some modification (see supporting information for details).

Beads injection To visualize the embryo’s vasculature, rhodamine-conjugated retrobeads (Lumafluor, New York, NY, USA) were diluted to 50% with PBS and injected into the embryo’s optic sinus. The injected embryos were let stand for 1 h and then were fixed in 4% paraformaldehyde in PBS. After progressive dehydration in glycerol/ Tris-buffered saline11% Tween 20 series, the embryos were observed with a BX61 microscope (Olympus, Tokyo, Japan).

RESULTS

Squid VEGFR gene and molecular phylogenetic analysis MATERIAL AND METHODS Samples Adult individuals of a model cephalopod, Idiosepius paradoxus, were collected in Zostrea beds around the Ushimado Marine Laboratory, Okayama University, Japan. The squids were kept in a

The squid VEGFR cDNA is 5093 bp with a start codon at position 660 followed by an open reading frame of 3834 bp, ending at position 4492. The gene product has 1278 amino acids (see supporting information). The signal sequence is followed by six immunoglobulin (Ig)-like domains, a transmembrane region, and a split-type tyrosine kinase domain.

Yoshida et al. The catalytic domain contains tyrosine protein kinase-specific active site and ATP-binding signatures with aspartic and lysine residues that are important for its catalytic activity and ATP binding. The signature sequence GxHxivNLLGACT, typical for split-type kinase domain of receptor tyrosine kinase Classes III–V (Grassot et al. 2003), was slightly modified to GqHlnivNLLGAVT in the squid VEGFR, as well as in the Drosophila Pvr gene. The protein blast suggests the intracellular tyrosine kinase domain of the squid VEGFR has a homology to amphioxus VEGFR (E 5 1e 62) and chick VEGFR3/FLT4 (E 5 5e 59). The squid VEGFR was most closely related to the Drosophila Pvr (Fig. 1). The Class V receptors of tyrosine kinase, such as the vertebrate VEGFR and Drosophila Pvr genes, are characterized by seven Ig-like domains. However, the extracellular region of the squid VEGFR contains only six Ig domains. Class IV receptors, such as the vertebrate PDGFR, include five Ig domains. FGFRs are Class III receptors characterized by three Ig domains. Drosophila has no Class IV receptor, so the Pvr gene is regarded as a homolog of both VEGFR and PDGFR genes. The phylogenetic tree suggests the squid VEGFR is also a homolog of the VEGFR/PDGFR of vertebrates.

VEGFR is expressed in developing circulatory systems The embryonic development of cephalopods is quite different from the other molluscs and characterized by an egg with a large yolk, meroblastic blastoderm, possible epibolic gastrulation, and direct development without typical molluscan larval stages. At the beginning of embryonic development, a blastodisc is formed on the animal pole of an ellipsoidal egg.

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The blastodisc progressively grows and expands over the surface of yolk. When the 2/3 of the egg surface is cellulated, various organ primordia, such as the eyes, statocysts, shell gland, and mouth, appear in the form of ectodermal placodes. In this stage embryo, open blood space thoroughly covers whole embryo body including outer yolk sac. The blood space changes its form from narrow space to tubules as the embryos become elaborated, the first of which are the primitive paired venae cavae (Boletzky 1975). Primary vena cava were observed in the visceral mass using the fluorescent beads in stage-21 embryo (Fig. 2A). The vena cavae located in the ventral midline branches into a pair of future branchial hearts. Microangiography showed vena cava, brachial sinus, ophthalmic (optic) sinus, and a pair of branchial hearts in stage24 embryos (Fig. 2B). The initially broad spaces differentiated into restricted area and lined the organ rudiments. However, no peripheral vessels were observed in the brain or arms (Fig. 2B). In stage-27 embryos, small diameter vessels were evident in the mantle via microangiography (Fig. 2C). The blood vessel network appeared to surround the arm and flow into the brachial sinus (Fig. 2D). The peripheral blood vessels appear to be formed between stage-24 and stage-27. In situ hybridization was performed with the VEGFR cDNA (antisense) probe, Actin II probe (cytoplasmic Actin, for positive controls), and VEGFR sense probe (for negative controls). Transcripts of Actin II were detected in the whole embryo. Using a VEGFR sense probe nonspecific signals were often seen in a shell sac (data not shown). No transcript of the squid VEGFR was detected during early epibolic stages (data not shown). Then, the embryonic body begins to rise up from the egg surface, with the transcripts first appearing in stage-21 embryos (Fig. 2, E and F). The VEGFR transcripts appeared in two spots at both lateral

Fig. 1. The phylogenetic relationship of receptor tyrosine kinase domains. The split kinase domain receptors have three (Class III), five (Class IV), and seven (Class V) immunoglobulin (Ig)-like domains. PTK7 is a regular tyrosine kinase receptor with seven Ig-like domains and is used as an outgroup. The Idiosepius vascular endothelial growth factor receptor (VEGFR) is most closely related to Drosophila Pvr gene (a homolog of VEGFR/PDGFR), although the Idiosepius VEGFR gene has only six Ig domains. The Idiosepius FGFR has three Ig domains and is part of a cluster with the other FGFRs. Numbers at the nodes indicate the bootstrap support values (calculated by 1000 repetition). Bar length indicates the number of substitution per site.

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Fig. 2. Embryonic circulatory system and expression of vascular endothelial growth factor receptor (VEGFR) in Idiosepius embryos. (A–D) Visualization of the vascular system via injection of fluorescent beads. (E–H) Whole-mount in situ hybridization with probes for vascular endothelial growth factor receptor (VEGFR). I, schematic views of the embryonic circulation and the VEGFR expression in the embryos. (A) Large blood spaces were visualized by fluorescent beads injection at stage-21. Open blood space thoroughly covers whole embryonic body including outer yolk sac (y) and posterior sinus (ps), and surrounds the eyes (e). Two tubes of primary vena cava (vc) appeared in the center of the body. (B) Optic sinuses (os) surrounding the optic lobe (ol), brachial sinus (bs), pair of posterior sinuses (ps), mesenteric vein (mv), cephalic vein (cv), and vena cava (vc) appeared in stage-24 embryo, but no vessel structures were visible in the arms or mantle. (C) Small diameter vessels are present in the mantle of the stage-27 embryo. (D) High magnification of the arm III of the embryo in (C). Brachial arteries (ba) are observed in the embryo with beads injection. (E) Merged images of expression of the VEGFR and circulation visualized by microinjection at stage-21. Expression of a pair of VEGFR spots is located below the posterior sinus (ps) on the same plane as the gill buds (gb). The VEGFR transcripts were also observed in optic sinus (os), lateral side of eyes (e), and lower yolk vessel (lyv). See also the left diagram in Fig. 2I. (F) A horizontal optical section at the dotted line in (E). The specific staining is localized to putative primordial branchial heart (bh) and systemic heart (sh) laterally to vena cava. The anterior margin of vena cava shows the putative area to form the single systemic heart (arrowheads). (G) The VEGFR gene is strongly expressed by developing vessels (arrowheads) invading optic lobe (ol) of the brain, posterior sinus (ps), and brachial arteries (ba) at stage-24. The expression was also observed in the eye (e). Nonspecific signal was observed in shell sec (ss) at dorsal side of mantle (m). (H) The VEGFR remains to be expressed in the eye part (e) of the stage-27 embryo. No transcript was observed in systemic heart (sh) and branchial hearts (bh). (A), (E), and (F), stage-21. (B), (G), stage-24. (C), (D), and (H), stage-27. Yolk sacs (y) are removed in (B), (G), and (H). All embryos are viewed from ventral with posterior to the upper side, except (G), which is seen from dorsal. Scale bars 5 250 mm in (A), 5 200 mm in (B), 5 100 mm in (C), (E), and 5 50 mm in (F).

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Fig. 3. Expression of the vascular endothelial growth factor receptor (VEGFR) in Idiosepius embryos. (A) An optical section to show the hearts and visceral complex of the mantle-removed specimen. The cells with specific staining appeared in branchial hearts (bh), atrium (sha), and ventricle (shv) of central systemic heart. (B) Enlarged view of (A) to show the details of localization of VEGFR in lining cells of the hearts (arrowheads). (C) A horizontal cryosection of the eye. Layer of retinal cells (re), lens (l), and optic sinus (os) are evident at stage-24. The transcripts were observed in the lens-surrounding tissue (lc) and the inner cell layer beside iris (ir). (D) Horizontal cryosection of the brain. VEGFRpositive capillary is shown (arrowheads). (A–D), stage-24. Scale bars 5 50 mm in (A), (C), (D), and 5 25 mm in (B).

sides of the primitive vena cava (Fig. 2, E and F). The spots were located in the same plane as the gill buds (Fig. 2, E and F), and placed laterally to the primary vena cava tubes (Fig. 2F). Together with an observation that paired systemic heart anlagen are formed on inner yolk mass (Boletzky 1987), the expression domain probably corresponds to the heart anlagen. By the late organogenesis stage (from stage -24 to -25), a mantle completely covered the gills. In stage-24 embryos, significant VEGFR-specific signal was observed in developing structures in the arms and brain (Fig. 2G). The VEGFRpositive cells appeared in the optic lobe of the brain and were elongated in shape with filopodia (Fig. 3D). There is strong staining in their visceral region (Fig. 2G). When the mantle was removed manually, the VEGFR transcripts were observed in both the systemic and branchial hearts (Fig. 3, A and B). Strong staining was noted in both atria and the incipient aorta. VEGFR expression was also noted in the developing eye of stage-24 embryos (Fig. 3C). Strong staining was found in lens-surrounding tissue and a cell layer in cornea. At the stage-27 the VEGFR was still expressed throughout the eye part of the embryo. However, no transcript was detected in the other organs (Fig. 2H).

VEGFR and FGFR expression in heart formation To further explore the molecular conserved nature of the vascular systems in cephalopods and other animals, we examined the expression patterns of squid FGFR orthologs (Fig. 4). In vertebrates and Drosophila, each FGFR is expressed in developing vascular systems particularly in heart anlagen (Zaffran and Frasch 2002; Lunn et al. 2006). The cephalopod cardiovascular system arises from a pair of mesoderm anlagen in the ventral plane of the body (Boletzky 1968). This region lies below the mantle and expressed the FGFR (Fig. 4, A and C). The mesodermal region expressing VEGFR was slightly larger than that expressing FGFR at stage-21. The FGFR was also expressed in the eye primordia and stomodeum (Fig. 4A). At later stages, the FGFR transcripts appeared in the arm base between first arm (arm I) and second arm (arm II), and suckers (Fig. 4C). Stain was not seen in the eye after closure of the eye vesicle. The FGFR transcripts were also observed in the systemic heart in the stage-24 embryo (Fig. 4D). The colocalization of both the VEGFR and the FGFR in heart formation supports presence of a conserved developmental pathway among bilaterian phyla (Fig. 5).

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Fig. 4. Expression of the FGFR in Idiosepius embryos. (A) Posterior view of the embryo. The FGFR transcripts are localized in primordia of eye (e), primordial buccal mass (bu), and ventral margin of mantle (m). (B) The FGFR transcript appeared in suckers of arms and posterior sinus (ps). Strong staining remained in the buccal complex (bu) including a pair of arm bases (ab) between arms I and II. Nonspecific signal was observed in shell sec (ss). (C) Enlarged view of visceral mass in (A). The specific staining was observed within ‘‘mesoderm anlagen’’ including gill buds (g), branchial hearts (bh), and primordial endoderm (en). (D) Visceral complex of mantle-removed specimen. The FGFR transcripts were localized in vena cava (vc), ventricle (shv), and atrium (sha) of systemic heart. (A), (C), stage-21. (B), (D), stage-24. Scale bars 5 100 mm in (A), (B), and 5 50 mm in (C), (D).

DISCUSSION

Formation of cephalopod closed circulatory systems In the cephalopods, aortic formation occurs after the embryonic body stands up on the yolk (Boletzky 1968, 1987). Expression patterns of squid VEGFR were apparently associated with sprouting processes of the peripheral blood vessels (angiogenesis), such as the arm vessels and the brain arteries. However, the microangiography with beads injection showed no signal in the corresponding regions in the stage-24 embryos. Subsequently, the blood vessel network was observed around their arms in stage-27 embryos. Microangiography probably labeled only the lumens of fully formed and functional vessels, so that this method may not be useful for visualizing the active behavior of vascular cells or their progenitors, such as sprouting and migration of endothelial cells (Cha and Weinstein 2007). This temporal discrepancy suggests that the VEGF signaling is correlated with early tubular formation in the squid embryo. In vertebrates, sprouts possess a tip region where the endothelial cells are organized as a compact string without a lumen (Patan 2000). Subse-

quently, the sprouts mature into new vessels with a lumen, and a continuous basement membrane is formed (Davis and Senger 2005). The expression patterns of squid VEGFR indicate that the peripheral blood vessels in squid embryos first appear as an endothelial cord, then penetrate into the tissue, and finally lumenize. Vertebrate blood vessels involve extension of filopodia, which utilize the ECM to drive the directional movement of cells. The presence of filopodia in the developing squid vessels supports the common angiogenic process between the cephalopod and vertebrates. In the vertebrates, vasculogenesis, de novo formation of endothelial cells from the lateral mesoderm, gives rise to the heart and the primitive single large artery and vein (Risau and Flamme 1995; Patan 2000). At an early stage of vertebrate development, progenitors of endothelial cells begin to express VEGFR2/flk-1 and then form primary tubes at the same time somites are formed. The early expression of VEGFR is possibly involved in early vasculogenesis as well as the peripheral angiogenic process. Histological studies showed that the octopus vascular system appears initially as lacunar (schizocoelic) spaces, and subsequently is surrounded by a basement membrane and endothelium (Boletzky 1975; Boletzky 1987).

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Fig. 5. Generalized scheme for a conserved molecular signature of the developing hearts in selected animals. A traditional phylogenetic tree and emergence of three types of circulatory systems (bars) adopted from Brusca and Brusca (2003) are shown below. Situation of the common ancestor is hypothesized as circles. In each adult body plan depicted above, the black area shows the position of hearts or heart-like structure. The embryonic origins of hearts or heartlike tissues are restricted in the embryos as flat anlagen (anterior direction is above in figures immediately above the phylogenetic tree). These anlagen are centralized and finally develop into the diffused gastrovascular system or tube-form hearts in adults (the main vascular directions of blood flow are shown with arrows in figures immediately below the diagrams of adult body plan). bh, branchial heart; v, ventricle. Data are sourced for: hydra (Shimizu and Fujisawa 2003), jellyfish (Seipel et al. 2004), Drosophila (Shishido et al. 1997; Bodmer and Venkatesh 1998; Zeitouni et al. 2007), vertebrates (Fishman and Chien 1997; Elliott et al. 2006; Lunn et al. 2006). In cnidarians, involvement of FGFR in the circulatory formation is not reported. In jellyfish, VEGFR is expressed in endodermal cells of their gastrovascular system, but coexpression with Nk-2 is not determined. In squids, Nkx2.5 is also expressed in the developing hearts (Elliott et al. 2006).

The squid VEGFR appeared as spots at both lateral sides of the primitive vena cava. Fioroni (1978) reported that ‘‘coelom–mesoblast complex’’ is located beside the primitive vena cava in Octopus embryos. These regions correspond to mesodernal anlagen including systemic and branchial hearts. Because the VEGFR is localized in developing heart at the stage-24, the regions with VEGFR expression are possibly associated with early cardiac formation.

Conserved VEGFR expression patterns and evolution of closed circulatory system The angiogenic process is observed in both vertebrates and cephalopods, but not in insects (Heino et al. 2001). The presence of VEGFR in the cephalopod vascular formation is likely associated with the secondary derivation of cephalopod vasculature from a molluscan ancestor, although the genes in VEGF pathway have not been reported in the other molluscs. Occurrences of vessels regulated by the VEGF pathway

across vast phylogenetic distances suggest the pathway was commonly found in bilaterians and was recruited independently to the vessel formations in the vertebrates and cephalopods. In that case, what was the first function of the VEGF pathway in the bilaterian ancestor? It is generally accepted that similarities are found in the initial morphogenetic stages and in the developmental regulatory networks between vertebrates and insects, but that adult morphologies are quite different (Zaffran and Frasch 2002; Hartenstein and Mandal 2006). The similarity between them is that the linear hearts are initially formed from fusing paired anlagen at the midline; this process has also been observed in cephalopods (Boletzky 1987; Salvini-Plawen and Bartolomaeus 1995). In this study we showed that the squid FGFR is expressed within mesoderm anlagen including heart primordia. The FGF pathway is known to be related to development of the insect and vertebrate hearts (Cripps and Olson 2002). In addition, the VEGFR gene is expressed in heart tubes of vertebrates and insects (Fishman and Chien

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1997; Zeitouni et al. 2007). Pvr, homolog of VEGFR in Drosophila, is expressed specifically in precursor cells of adult valves and is required for cardiac valve formation (Zeitouni et al. 2007), although the heart has only one cell layer which behaves as both the myocardium and endocardium. The expression of squid VEGFR was observed in the whole ventricle and was especially strong in the atria and base of the posterior aorta, which form heart valves. It is therefore suggested that, although inducing very different cellular processes, the VEGF pathway plays an evolutionary conserved function in cardiac development (Fig. 5). Presence of homologs of VEGFR/Pvr and tinman/Nkx in cnidarians, which is expressed in the peristaltic endodermal wall (Shimizu and Fujisawa 2003; Seipel et al. 2004), suggests that the pathway has originated in a prebilaterian metazoan ancestor. The expression of VEGFRs is commonly observed in the endothelial and endocardial cells of vertebrates, as well as endothelial cells of cephalopods. The vertebrate endothelium is similar to their endocardial cells with respect to having junctional complexes including VE-cadherin (Larson et al. 2004). Both types of cells are known to be derived from a multipotent progenitor by the presence of a common transcriptional network (Ferdous et al. 2009). Taken together, we propose (a) that the VEGF pathway may have been involved in cardiac formation in the common bilaterian ancestor and (b) that both vertebrates and cephalopods recruited the evolutionarily conserved pathway and expanded their tubular networks using this genetic pathway. Acknowledgments We thank Prof. T. Sakamoto and Mr. W. Godo of Ushimado Marine laboratory, Okayama University for their kind help of collecting specimens. We also would like to express our gratitude to Prof. H. Nishida and his colleagues of the Department of Biology, Osaka University, for kind help in microinjection and microscopic methods. This study was supported by the grants from the Research Institute of Marine Invertebrate Foundation and from the Japan Society for the Promotion of Science (research grant 18570087).

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Squid VEGFR and the closed circulatory system

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SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Appendix S1. Detailed methods Appendix S2. The co-option of VEGF pathway in retinal development Fig. S1. Amino acid sequence of the squid VEGFR. The signal sequence is followed by six immunoglobulin (Ig)-like domains, a transmembrane region, and a split-type tyrosine kinase domain. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.