CD146 modulates VEGF-C induced lymphatic development in zebrafish. (a) CD146 expression in the zebrafish embryos at 5 dpf was detected by whole-mount in situ hybridization (WISH). lyve1b was used as positive control. Black arrows indicate the position of TD. DA, dorsal aorta; PCV, posterior cardinal vein; and TD, thoracic duct. Scale bars: 30 μm. (b) The effect of knockdown cd146 or vegfc on blood vessels. Bright-field lateral views of the trunk of embryos injected with control, cd146 or vegfc specific MOs. Embryos were stained by WISH for a panel of arterial markers of dll4, tbx20 and ephrinB2 and venous markers of msr and flt4. Black arrows, blood vessels; DA, dorsal aorta; and PCV, posterior cardinal vein. Scale bars: 125 μm. (c) The effect of knockdown cd146 or vegfc on vasculature of blood and lymph. The control, cd146 or vegfc specific MO were injected into the transgenic zebrafish Tg (fli1a: EGFP) embryos, respectively. The embryos harvested at 36 hpf, 48 hpf and 72 hpf were analyzed. The confocal images were flanked by redrawing of the vessel contours. Transient lymphangiogenic structures (lymphangiogenic sprouts; PL cells) were labeled with light green. The other vessels (PCV, DA and ISV) were labeled with dark grey. Scale bars: 20 μm. (d) The effects of cd146 knockdown on development of lymphatic capillary. The control or cd146 specific MO was co-injected with control or human vegfc mRNA into the transgenic zebrafish Tg (fli1a: EGFP) embryos. Late-phase microangiography was used to visualize lymphatic capillaries in embryos at 3 dpf. Images showed the representative phenotypes of each group and the ratio indicated the embryos with such representative phenotype over the tested embryos. Scale bars: 50 μm.