A Complete Orf Clones Of The Zebrafish Vegfd Gene And Its Function In The Vascular System Development Process

VEGF family members, including VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, placental growth factor (P1GF) and VEGF-F, are the most important secreted angiogenic factors for blood vessel development in embryo and angiogenesis of tumors. VEGF-D, like VEGF-C, is different from VEGF-A in structure and functions. Much data have shown that human VEGF-D could induce the growth of intra-tumor lymphatic vessels, promote the lymph node metastasis and be used as a prognostic factor in tumor development. However, it is indispensable for development of the lymphatic system in mice. Mouse VEGF-D can also activate rabbit VEGFR-2 and induce angiogenesis in the rabbit corneal assay. It is also demonstrated that VEGF-D is the strongest angiogenic and lymphangiogenic effecter among VEGFs delivered into skeletal muscle via adenoviruses. However, mouse VEGF-D can bind VEGFR-3 but not VEGFR-2. The zebrafish has become a widely used vertebrate model organism for genetic, developmental research and drug discovery, because of its fecundity, optical clarity and morphological and physiological similarity to mammals. A functioning vascular system including major and sprouts vessels have presented in zebrafish embryos in three days. In addition, zebrafish embryos can survive and develop for at least one week without a circulatory system. Zebrafish has been a great good candidate for vascular system analysis in vivo.In this study, we cloned in silicon the open reading frame of zebrafish VEGFD by using BLAST and GenScan and TWIINSCAN programs with human VEGF-D protein sequence. GenScan and TWIINSCAN are programs that predict directly gene ORFs from genome sequence. Then putative ORF of zVEGF-D was 816 bp encoding 272 amino acid residues. The predicted zVEGF-D contains six exons and five introns. Conserved domain analysis with RPSBLAST program showed that the 105-188 amino acid residues of putative zVEGF-D protein sequence is belonged to PDGF/VEGF conserved domain consensus sequence, and contains eight cysteine residues highly related to structure and function. The phylogenetic tree showed that in mammals opossum VEGF-D shared the closest evolutionary relationship with zebrafish VEGF-D. Multiple sequences alignment by CLUSTAL W shows that zVEGF-D protein shares sequence identity of 40% to human and 39% to mouse respectively and 33% to zebrafish VEGF-C. To clone the zebrafish VEGFD coding region, primers were designed nearby the start codon and stop codon. BamH I and Xho I sites were added for recombinating into pCDNA3. 1 vector. One step RT-PCR was performed by using adult zebrafish total RNA as template. The product was cloned into T-easy to sequence. The result was consistant with the prediction. To detect the temporal expression of zebrafish VEGF-D mRNA, total RNA from zebrafish embryos at different stage were isolated with the Trizol reagent. Semi-quantitative RT-PCR was performed with One-Step RT-PCR kit. The amount of EF1 was used as the internal control for normalization. RT-PCR results showed that zVEGF-D transcripts began present from 13-somite and upregulated from 24 hpf stage. In vitro transcription was performed by using the linearized VEGFD—pCDNA3.1 plasmid above as template. Then The product was added poly(A) tailing and pured for microinjection. To analyze the function of zVEGF-D in zebrafish vascularization during embryogenesis, we overexpressed zVEGF-D by microinjecting mRNA into Flk-1:EGFP early embryo at 1-cell stage, and observed them at 36 hpf. In zVEGF-D overexpressed embryos heartbeat and blood flow were slower, the dorsal aorta and caudal vein connected abnormally and formed short or blocked circulation, and amounts of blood cells even accumulated at the tail region and formed a stagnant pool. Observed in fluorescence field, intersomitic vessels (ISV) were truncated, or sprouted aberrantly and formed an abnormal pattern. The caudal plexus developed to an enlarged cavum and no obvious tube formation was observed. Stagnant blood just accumulated in this cavum. But overexpression of zVEGF-D unexpectedly did not promote vasculogenesis and angiogenesis.To determine whether the aberrant ISV could support circulation, we performed microangiography with rhodamin-dextran.In zVEGF-D overexpressed fish the vascular pattern is abnormal and blood flow only existed within dorsal aorta and cardinal vein, no circulation in ISVs was observed. In conclusion, we cloned and characterize zebrafish VEGF-D gene, and find it is critical for the normal development of vascular system in zebrafish embryo, indicating that the optically clear developing zebrafish provides an alternative model, in addition to mouse, to further reveal the role of VEGF-D in angiogenesis.