The endothelial-cell-derived secreted factor Egfl7 regulates vascular tube formation

Abstract

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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Figure 1: Egfl7 expression profile.
Figure 2: Egfl7 gene knockdown causes a vascular tubulogenesis defect in zebrafish embryos.
Figure 3: Endothelial cell number is unaltered in the Egfl7 knockdowns.
Figure 4: EGFL7 promotes endothelial cell adhesion.

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Acknowledgements

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.

Author information

Correspondence to Weilan Ye.

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The authors declare that they have no competing financial interests.

Supplementary information

Supplementary Information

Supplementary figure legends and references. (DOC 62 kb)

Supplementary Figure 1

EGFL7 is conserved during vertebrate evolution. (JPG 307 kb)

Supplementary Figure 2

Human and murine Egfl7 is expressed in vasculatures associated with active cell proliferation, and is down regulated in mature vasculatures. (JPG 138 kb)

Supplementary Figure 3

Egfl7 knockdown causes specific vascular tube formation defect. (JPG 111 kb)

Supplementary Figure 4

Failure of vascular tubulogenesis is a specific and primary defect in the Egfl7 knockdown embryos. (JPG 153 kb)

Supplementary Figure 5

Egfl7 regulates de novo vascular tubulogenesis. (JPG 89 kb)

Supplementary Figure 6

A model depicting the key steps of vasculogenesis, using the trunk region of the zebrafish embryo as an example. (JPG 105 kb)

Supplementary Movie 1

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)

Supplementary Movie 2

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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