Vascular development is a complex but orderly process that is tightly regulated. A number of secreted factors produced by surrounding cells regulate endothelial cell (EC) differentiation, proliferation, migration and coalescence into cord-like structures1,2. Vascular cords then undergo tubulogenesis to form vessels with a central lumen3,4. But little is known about how tubulogenesis is regulated in vivo. Here we report the identification and characterization of a new EC-derived secreted factor, EGF-like domain 7 (Egfl7). Egfl7 is expressed at high levels in the vasculature associated with tissue proliferation, and is downregulated in most of the mature vessels in normal adult tissues. Loss of Egfl7 function in zebrafish embryos specifically blocks vascular tubulogenesis. We uncover a dynamic process during which gradual separation and proper spatial arrangement of the angioblasts allow subsequent assembly of vascular tubes. This process fails to take place in Egfl7 knockdown embryos, leading to the failure of vascular tube formation. Our study defines a regulator that controls a specific and important step in vasculogenesis.
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Carmeliet, P. & Jain, R. K. Angiogenesis in cancer and other diseases. Nature 407, 249–257 (2000)
Hanahan, D. Signaling vascular morphogenesis and maintenance. Science 277, 48–50 (1997)
Hogan, B. L. & Kolodziej, P. A. Organogenesis: molecular mechanisms of tubulogenesis. Nature Rev. Genet. 3, 513–523 (2002)
Lubarsky, B. & Krasnow, M. A. Tube morphogenesis: making and shaping biological tubes. Cell 112, 19–28 (2003)
Folkman, J. Angiogenesis and angiogenesis inhibition: an overview. EXS 79, 1–8 (1997)
Doliana, R., Bot, S., Bonaldo, P. & Colombatti, A. EMI, a novel cysteine-rich domain of EMILINs and other extracellular proteins, interacts with the gC1q domains and participates in multimerization. FEBS Lett. 484, 164–168 (2000)
Callebaut, I., Mignotte, V., Souchet, M. & Mornon, J. P. EMI domains are widespread and reveal the probable orthologs of the Caenorhabditis elegans CED-1 protein. Biochem. Biophys. Res. Commun. 300, 619–623 (2003)
Stainier, D. Y., Weinstein, B. M., Detrich, H. W. III, L. I. & Fishman, M. C. cloche, an early acting zebrafish gene, is required by both the endothelial and hematopoietic lineages. Dev. Suppl. 121, 3141–3150 (1995)
Soncin, F. et al. VE-statin, an endothelial repressor of smooth muscle cell migration. EMBO J. 22, 5700–5711 (2003)
Nasevicius, A. & Ekker, S. C. Effective targeted gene ‘knockdown’ in zebrafish. Nature Genet. 26, 216–220 (2000)
Brown, L. A. et al. Insights into early vasculogenesis revealed by expression of the ETS-domain transcription factor Fli-1 in wild-type and mutant zebrafish embryos. Mech. Dev. 90, 237–252 (2000)
Fouquet, B., Weinstein, B. M., Serluca, F. C. & Fishman, M. C. Vessel patterning in the embryo of the zebrafish: guidance by notochord. Dev. Biol. 183, 37–48 (1997)
Liao, W. et al. The zebrafish gene cloche acts upstream of a flk-1 homologue to regulate endothelial cell differentiation. Development 124, 381–389 (1997)
Lyons, M. S., Bell, B., Stainier, D. & Peters, K. G. Isolation of the zebrafish homologues for the tie-1 and tie-2 endothelium-specific receptor tyrosine kinases. Dev. Dyn. 212, 133–140 (1998)
Lawson, N. D. & Weinstein, B. M. Arteries and veins: making a difference with zebrafish. Nature Rev. Genet. 3, 674–682 (2002)
Zhong, T. P., Rosenberg, M., Mohideen, M.-A. P. n. K., Weinstein, B. & Fishman, M. C. gridlock, an HLH gene required for assembly of the aorta in zebrafish. Science 287, 1820–1824 (2000)
Childs, S., Chen, J.-N., Garrity, D. M. & Fishman, M. C. Patterning of angiogenesis in the zebrafish embryo. Development 129, 973–982 (2002)
Sehnert, A. J. H. A., Weinstein, B. M., Walker, C., Fishman, M. & Stainier, D. Y. Cardiac troponin T is essential in sarcomere assembly and cardiac contractility. Nature Genet. 31, 106–110 (2002)
Isogai, S., Lawson, N. D., Torrealday, S., Horiguchi, M. & Weinstein, B. M. Angiogenic network formation in the developing vertebrate trunk. Development 130, 5281–5290 (2003)
Sumoy, L., Keasey, J. B., Dittman, T. D. & Kimelman, D. A role for notochord in axial vascular development revealed by analysis of phenotype and the expression of VEGR-2 in zebrafish flh and ntl mutant embryos. Mech. Dev. 63, 15–27 (1997)
Vokes, S. A. & Krieg, P. A. Endoderm is required for vascular endothelial tube formation, but not for angioblast specification. Dev. Suppl. 129, 775–785 (2002)
Odenthal, J. & Nusslein-Volhard, C. fork head domain genes in zebrafish. Dev. Genes Evol. 208, 245–258 (1998)
Schulte-Merker, S., Ho, R., Herrmann, B. & Nusslein-Volhard, C. The protein product of the zebrafish homologue of the mouse T gene is expressed in nuclei of the germ ring and the notochord of the early embryo. Development 116, 1021–1032 (1992)
Strahle, U., Blader, P., Henrique, D. & Ingham, P. Axial, a zebrafish gene expressed along the developing body axis, shows altered expression in cyclops mutant embryos. Genes Dev. 7, 1436–1446 (1993)
Parker, L. H., Zon, L. I. & Stainier, D. Y. Vascular and blood gene expression. Methods Cell Biol. 59, 313–336 (1999)
Fong, G. H., Zhang, L., Bryce, D. M. & Peng, J. Increased hemangioblast commitment, not vascular disorganization, is the primary defect in flt-1 knock-out mice. Development 126, 3015–3025 (1999)
Ye, W., Shimamura, K., Rubenstein, J. L., Hynes, M. A. & Rosenthal, A. FGF and Shh signals control dopaminergic and serotonergic cell fate in the anterior neural plate. Cell 93, 755–766 (1998)
We thank M. C. Fishman, B. Weinstein and N. Lawson for fish strains, plasmids and helpful discussions; N. Ferrara and H. Gerber for advice and for reviewing the manuscript; J. Lee for the zebrafish cDNA library; L. Rangell for electron microscopy; S. Greenwood for general lab assistance; R. Vandlen, D. Yansura, R. Corpuz and H. Kim for recombinant EGFL7 proteins; A. Chuntharapai and C. Reed for monoclonal antibodies; and W. Wood, H. Clark and J. Tang for bioinformatics assistance. S.J. is supported by the American Heart Association. D.B. is a Human Frontier Science Program Organization fellow.
The authors declare that they have no competing financial interests.
Supplementary figure legends and references. (DOC 62 kb)
EGFL7 is conserved during vertebrate evolution. (JPG 307 kb)
Human and murine Egfl7 is expressed in vasculatures associated with active cell proliferation, and is down regulated in mature vasculatures. (JPG 138 kb)
Egfl7 knockdown causes specific vascular tube formation defect. (JPG 111 kb)
Failure of vascular tubulogenesis is a specific and primary defect in the Egfl7 knockdown embryos. (JPG 153 kb)
Egfl7 regulates de novo vascular tubulogenesis. (JPG 89 kb)
A model depicting the key steps of vasculogenesis, using the trunk region of the zebrafish embryo as an example. (JPG 105 kb)
48 hpf embryos injected with control (left panel) or Egfl7 antisense (right panel) oligos were recorded with a video camera. The head and anterior trunk are shown. Rigorous heartbeats are evident in both fish. Circulation in the major trunk and head vessels is visible in the control fish, but is absent in the Egfl7 knockdown fish. (MOV 4862 kb)
48 hpf embryos injected with control (left panel) or Egfl7 antisense (middle and right panel) oligos were recorded with a video camera. Posterior trunks are shown here. Completeknockdown was achieved in some embryos resulting in the absence of circulation (the embryo shown in the middle panel is an example, and is the same embryo shown in the right panel of movie 1). Partial loss of function was achieved in a subset of embryos injected with lower dose of the antisense oligo. These embryos showed a weaker phenotype (embryo in the right panel is an example). In the embryo shown in the right panel, a segment of the two major trunk vessels failed to form lumen, thus resulting in premature returning of circulation anterior to the affected vessels. (MOV 6882 kb)
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Parker, L., Schmidt, M., Jin, S. et al. The endothelial-cell-derived secreted factor Egfl7 regulates vascular tube formation. Nature 428, 754–758 (2004) doi:10.1038/nature02416
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