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...