Vascular Endothelial Growth Factor Receptor Family in Ascidians, Halocynthia roretzi (Sea Squirt). Its High Expression in Circulatory System-Containing Tissues

Abstract

The vascular endothelial growth factor (VEGF)-VEGF Receptor (VEGFR) system is an important pathway for regulation of angiogenesis. However, its evolutionary development, particularly the step from invertebrates to vertebrates, is still largely unknown. Here, we molecularly cloned the VEGFR-like gene from Halocynthia roretzi, a species belonging to the Tunicata, the chordate subphylum recently considered the sister group of vertebrates. The cDNA encoded a homolog of human VEGFR, including the transmembrane domain, and the tyrosine kinase domain with a kinase-insert region, which was designated S. sq VEGFR (GenBank AB374180). Similar to Tunicates including ascidians in the phylogenetic tree, the Amphioxus, another chordate, is located close to vertebrates. However, S. sq VEGFR has a higher homology than the Amphioxus VEGFR-like molecule (GenBank AB025557) to human VEGFR in the kinase domain-2 region. The S. sq VEGFR mRNA was expressed at highest levels in circulatory system-containing tissues, suggesting that S. sq VEGFR plays an important role in the formation or maintenance of circulatory system in Tunicates, Halocynthia roretzi.

Figure 1

Phylogenic tree of the chordate and the number of genes for Vascular Endothelial Growth Factor receptor (VEGFR) and Platelet-derived growth factor receptor (PDGFR) family.

Figure 2

Amino acid sequence of S. sq VEGFR, and its strong homology with human VEGFR. (a) Isolation of cDNA related to VEGFR gene from ascidian, H. roretzi; (b) Schematic diagram of VEGFR of H. roretzi in comparison to human Flt-1 (VEGFR1). A high degree of conservation of critical tyrosine-containing residues in S. sq VEGFR and human VEGFR; (c) Sequence homology of S. sq VEGFR with human VEGFR1 in the KD2 region (157 amino acid-long sequence; ATVD—RLAE in S. sq VEGFR). [*]: identical amino acids with the VEGFR-related sequence in the middle; (d) Sequence homology among ascidian VEGFRs (S. sq VEGFR, B. schlosseri VEGFR, and C. intestinalis VEGFR) in the KD2 region. [*]: identical amino acids with the VEGFR-related sequence in the middle.

Figure 3

Human VEGFR is more homologous to S. sq VEGFR than to Amphioxus VEGFR-like protein. (a) Comparison of the KD2 domain among S. sq VEGFR, Amphioxus VEGFR-like molecule and human VEGFRs; (b) Comparison of KD2 domain in S. sq VEGFR with invertebrate VEGFRs and human VEGFRs as well as PDGFRs; (c) Diagram of Phylogenic tree (the order of ascidians such as H. roretzi and amphioxus is not completely fixed).

Figure 4

The expression of the S. sq VEGFR gene in the H. roretzi tissues is correlated with the existence of circulatory system. (a) RT-PCR analysis of S. sq VEGFR gene expression in the different H. roretzi tissues. Upper panel: PCR was performed using a S. sq VEGFR-specific primer set and reverse-transcribed cDNA from the total RNA of each tissue as the template. Lower panel: PCR was preformed using the H. roretzi β-actin-specific primer set and reverse-transcribed cDNA from total RNA of each tissue as the template (positive control); (b) Anatomical scheme of H. roretzi.

Figure 5

Strong expression of S. sq VEGFR protein in H. roretzi stomach (heart-localized tissue) and intestine. Upper part, Histology of the different tissues of H. roretzi. Lower part, Western blot analysis of S. sq VEGFR in different H. roretzi tissues. The tissues examined are: Intestine (I), Stomach (S), Pharynx (P) and Gill (G). Scale bar indicates 120 m.