Fig. 5

Ccbe1 enhances Vegfc-driven sprouting and regulates levels of bioactive VEGFC in vitro. (A) Confocal projections at 32 hpf of Tg(shh:ccbe1), Tg(shh:vegfc) and Tg(shh:ccbe1;shh:vegfc) in a Tg(fli1a:EGFP) background. Co-overexpression of ccbe1 and vegfc in the floorplate leads to aberrant ectopic turning of the ISVs at 32 hpf (upper panels; n=32/36; P<0.0001). At 48 hpf, ccbe1 overexpression in the floorplate does not result in any phenotype, whereas vegfc-overexpressing animals display hyperbranching of the ISVs, and enhanced endothelial cell accumulation at the horizontal myoseptum (arrowhead). ccbe1 and vegfc co-overexpression in the floorplate also leads to hyperbranching ISVs, and to a marked accumulation of endothelial cells at dorsal aspects of the embryo (arrow). (B) Western blot of 293EBNA-1 cells (stably expressing VEGFC) indicate that CCBE1 is detected in the lysate of cells transfected with CCBE1 plasmid, but not in controls. (C) An increase in the levels of all forms of VEGFC is detected in the medium of CCBE1-transfected cells, compared with control cells. The mature form of VEGFC (detected at <23 kDa) is predominant. (C2) Relative intensity (split axis) of the different processed forms of VEGFC presented in C based on multiple exposures. Note the saturation of the mature form in C. (D) qPCR showing that CCBE1 transfection does not affect VEGFC mRNA levels in vitro in 293EBNA-1 cells stably expressing VEGF-C (D, left panel). Consistent with this, in zebrafish embryos the injection of vegfc or ccbe1 mRNA does not affect the endogenous levels of the other (D, right panel). Error bars represent s.e.m.