Fig. 6

Quantification of lymphatic capillary development after xenotransplantation.

Green fluorescent images (fli1:EGFP) reveal blood vessels, red fluorescent images (Texas Red-LMD) reveal lymphatic capillaries, and blue fluorescent images (CellTracker) reveal transplanted cells in 3-dpf Tg(fli1:EGFP)^y1 zebrafish. Results of quantitative morphometric analyses are displayed in bar graphs. A–D, At 3 dpf (n = 32), the inhibitory effect of the vegfc morpholino MO (n = 32) was rescued by transplantation of HUVECs (n = 18) and B16 cells (n = 20), but not 293 cells (n = 25). *P = 0.0005, **P<0.0001. Scale bars, 50 μM. All cell types (blue) were detected in major blood vessels, including the dorsal aorta (white arrowheads). HUVECs and 293 cells were also seen extravascularly, bordering lymphatic capillaries (white arrows). Note: fewer ISVs (green) in images of B16- or 293-injected morphants relates to the particular focal plane imaged, since alternative focal planes revealed approximately normal numbers of ISVs in these and other similarly-treated zebrafish (data not shown). E, Immunoblot to detect human VEGF-C in cultured HUVEC, B16, and 293 cells. The active, proteolytically-processed form of VEGF-C (21 kilodaltons, kDa) was only detected at appreciable levels in HUVEC lysate. B16 and 293 cell lysates contained VEGF-C in a dimerized precursor form (80 kDa). No VEGF-C was detected in conditioned media from any of the cells (data not shown). An antibody against α-tubulin was used as a loading control.