EGFL7 overexpression impairs formation of the CVV in the CVP in zebrafish. Zebrafish embryos expressing enhanced green fluorescent protein in their vasculature (green) were injected with (A) fli1:DsRed, (B) fli1:EGFL7, (C) fli1:EGFL7 RAD (having the EGFL7 RGD motif mutated into RAD), (D) fli1:EGFL7 ΔRGD (EGFL7 lacking the RGD motif), (E) integrin αv (itgαv) morpholino (MO), or (F) fli1:EGFL7 together with integrin αv (itgαv) MO and were subsequently stained with anti-DsRed (A red) or anti-EGFL7 antibody (B-D red). The overview of the tail circulation (left panel) has been captured with a ×10 lens; the white rectangles show the region highlighted in the right panels captured with a ×40 lens. Alternatively, regions of the ×40 magnification pictures were cropped and magnified (right panels in A-F). (A) The fli1 promoter-driven mosaic expression of DsRed in the zebrafish tail circulation did not affect the development of the CVV. (B) Mosaic expression of EGFL7 specifically disrupts the structure of the CVV. EC of the CVV with extracellular EGFL7 staining appeared more closely spaced (arrowhead, red lines) as compared with adjacent cells devoid of EGFL7 staining (arrow, white lines). (C-D) Mosaic expression of the mutant forms of EGFL7 impaired in integrin binding (EGFL7 RAD and EGFL7 ΔRGD) failed to disrupt the structure of the CVV-EC that are positive for EGFL7 (arrows). (E) Wild-type-like spacing of CVV-EC in itgαv morphant embryos and (F) in itgαv morphants overexpressing EGFL7 implicated a central role of integrin αv for the observed EGFL7 phenotype. (G) A quantitative summary of the frequencies of the phenotype in zebrafish embryos observed in panels A-F. (H) HEK293T cells were transfected with Flag-tagged wild-type EGFL7, EGFL7 ΔRGD, EGFL7 RAD, or EGFL7 lacking EMI and both EGF-like domains (EGFL7 ΔEEE). The cells were cotransfected with constructs encoding for αv and β3 integrin subunits. Immunoprecipitates were prepared using anti-Flag antibody and were analyzed for the presence of αv and β3 integrin.
