Duplicate VegfA genes and orthologues of the KDR receptor tyrosine kinase family mediate vascular development in the zebrafish

Abstract

Vascular endothelial growth factor A (VEGFA) and the type III receptor tyrosine kinase receptors (RTKs) are both required for the differentiation of endothelial cells (vasculogenesis) and for the sprouting of new capillaries (angiogenesis). We have isolated a duplicated zebrafish VegfA locus, termed VegfAb, and a duplicate RTK locus with homology to KDR/FLK1 (named Kdrb). Morpholinodisrupted VegfAb embryos develop a normal circulatory system until approximately 2 to 3 days after fertilization (dpf), when defects in angiogenesis permit blood to extravasate into many tissues. Unlike the VegfAa 121 and VegfAa 165 isoforms, the VegfAb isoforms VegfAb 171 IntroductionDifferentiation of endothelial cells to form the vascular network of arteries and veins requires a critical mitogenic signal, vascular endothelial growth factor A (VEGFA), which is transduced to the nucleus through a number of functionally redundant receptors. These receptors include the type III receptor tyrosine kinases VEGFR1/FLT1, VEGFR2/FLK1 (human KDR), and type I nonkinase transmembrane receptors neuropilin 1 (NRP1) and NRP2, 1 which are critical mediators of both vasculogenesis (de novo formation of blood vessels) and angiogenesis (sprouting of vessels from preexisting vessels). 2 Mice hemizygous at their single VegfA locus show defective vasculogenesis and die between 11 and 12 days after conception (dpc). [3][4][5] Embryos homozygous for a targeted disruption of Flk1 (Vegfr2) die between 8.5 and 9.5 dpc from defects in both hematopoiesis and vasculogenesis, but disruption of Flt1 (Vegfr1) only leads to perturbed vascular patterning in full-term embryos. 6,7 The tight regulation of VEGFA levels and the requirement for its receptors in hematopoiesis and vasculogenesis point to its critical role in the formation of blood vessels during development.Differential splicing of the single VEGFA locus in humans gives rise to multiple protein isoforms, ranging in size from 121 to 206 amino acids (aa's) (VEGFA 121 , VEGFA 145 , VEGFA 165 , VEGFA 189 , and VEGF 206 ). 8,9 The VEGF isoforms differ mainly by the presence or absence of 2 heparin-binding domains encoded within exon 6 and 7 sequences. [10][11][12] Expression of a single VEGFA isoform rescues VegfA mutant mice through birth, although the vascular networks differ with expression of different isoforms. [13][14][15][16] VEGFA splicing is regulated during development and in disease, resulting in tissue-and stage-specific isoform ratios, which allow for distinct, context-dependent VEGFA signaling. 17 Zebrafish have proven useful for dissecting vascular developmental pathways. 18 A single zebrafish vegfA gene has been previously described, encoding the 121-and 165-aa isoforms. 19,20 Although VegfA 121 is the predominant isoform in early embryos and VegfA 165 in adults, other alternate transcripts exist in specific tissues. 21 The initial establishment of axial vasculature patterning does not require VegfA, but the sprouting of intersegmental vessels does. 22 Together, these results show that VegfA...

Anti-angiogenic Activity of Erxian Decoction, a Traditional Chinese Herbal Formula, in Zebrafish

“…46) Furthermore, a recent study reported the cloning and characterization of duplicated zebrafish genes kdra and kdrb, and they found that morpholinos targeting both forms resulted in defects in vasculogenesis. 65) The gene tested in this study is kdra. Is the other form kdrb involved in EXD inhibition of vessel formation in zebrafish embryos?…”

Molecular cloning and embryonic expression of zebrafish PCSK5 co‐orthologues: Functional assessment during lateral line development

“…How these expression patterns were established and maintained is the subject of much debate since the benefit to the organism of having two biologically distinct but functionally related genes is not always apparent (Force et al, 1999). Many examples of this phenomenon have been described including the following zebrafish coorthologues, hoxB1a and b (McClintock et al, 2002), sonic hedgehog (shh) and tiggywinkle hedgehog (twhh) (Hammond et al, 2003), protocadherin15 (pcdh15a and pcdh15b) (Seiler et al, 2005), VEGFAa and VEGFAb (Bahary et al, 2007), and progranulin A and B (Cadieux et al, 2005). In the present study, we have cloned two zebrafish cDNA sequences designated PC5.1 and PC5.2.…”

von Hippel-Lindau tumor suppressor mutants faithfully model pathological hypoxia-driven angiogenesis and vascular retinopathies in zebrafish

“…Like all vertebrates, with the exception of placental mammals, zebrafish have four Vegf receptors (Flt1, Kdr, Kdr-like and Flt4); Kdr-like (also known as Kdrl) was lost during the evolution of placental mammals (Bussmann et al, 2007;Bussmann et al, 2008). Although Kdr and Kdr-like represent separate classes of Vegf receptor, the duplicated VEGFA orthologs Vegfaa and Vegfab can bind to, and activate, both receptors in vitro (Bahary et al, 2007 …”

“…1A) showing the conservation of VEGF ligands in zebrafish. The duplicated zebrafish VEGFA orthologs vegfaa and vegfab (Bahary et al, 2007), as well as zebrafish vegfc (Ober et al, 2004) and vegfd (Song et al, 2007), have all been described previously. From our analyses, we identified zebrafish vegfb, which is syntenic to human VEGFB (not shown) and with orthologs that are present in other fish species.…”