Fig. 2

Delineation of V1^LP, V2^LP and V4^LP vascular connections by quantum dots.

QD655 injected Tg(lfabf:DsRed;elaA:egfp);Tg(fli1:efgp) larva is shown. The 3D rendered confocal microscopy images (the top panel: 3D) and their serial optical sections (bottom panels: Z-slice) of Tg(lfabf:DsRed;elaA:egfp);Tg(fli1:egfp) at 6 dpf are shown. The bottom five panels are Z-slices of the 3D rendered images shown at the top panel. The depth of each slice is indicated as µm at the top left in each panel of Z-slice. V1^LP and V2^LP vessels are sandwiched between white arrows and outlined by white arrows, respectively. V4^LP vessel is outlined by yellow arrows and sandwiched between yellow arrowheads. The islet of Langerhans is indicated (dotted circle in each panel). The islet of Langerhans, consisting of endocrine cells, was identified as an area devoid of EGFP reporter signals driven by the exocrine pancreas-specific elaA promoter. The left, middle and right panels in A and B show EGFP (green: fli1⁺ vessels, elaA⁺ exocrine-pancreas)/DsRed (orange: lfabf⁺ liver), QD655 (magenta) and merged images, respectively. A. The QD655 perfused V1^LP vessel. The fli1⁺ V1^LP vessel (green) is perfused with Q655 (magenta) albeit only faintly. B. The QD655 perfused V2^LP and V4^LP vessels. V2^LP and V4^LP vessels are outlined by white and yellow arrows, respectively. By following each serial Z-slices, the direct connection of V2^LP vessel (white arrows) to vascular plexus at the islet of Langerhans (dotted circle) is discernable. V4^LP vessel (yellow arrows and sandwiched between yellow arrowheads) is clearly connected to intrahepatic vasculature. There are three branches (two indicated by *, one indicated by**) stemming from V4^LP that appear to bridge between V2^LP and V4^LP vessels. By following the serial Z-slices, it is clear that two of these QD655 perfused vessels indicated by * become more discernable as V2^LP becomes fainter (compare the 4^th and 5^th Z-slices in the QD655 column), suggesting that those two indicated by * are not directly linked to either V2^LP or V4^LP vessels. Instead, they cross behind V2^LP vessel. In contrast, the one indicated by ** appears to be on the same Z-slice plane as V2^LP vessel (compare the 4^th and 5^th Z-slices in the QD655 column), suggesting that this vessel (**) is a branch that directly bridge between V2^LP and V4^LP vessels. C. Higher magnification of the V2^LP vessel showing co-localization of fli1⁺ vascular endothelial cells (green) and QD655 signal (magenta) (of the 4^th panel of the merge column as indicated by dotted rectangle). D. Higher magnification of the V4^LP vessel showing co-localization of fli1⁺ vascular endothelial cells (green) and QD655 signal (magenta) (of the 3^rd panel of the merge column as indicated by dotted rectangle). E. IsoSurface object image of QD655 perfused vessel connections of V2^LP and V4^LP and their branches. The QD655 perfused vessel image was treated by surface rendering method and IsoSurface object was built (Threshold = 12). In this surface rendered image, all three branches (? and **) stemming from V4^LP vessel appear to be fused to V2^LP vessel. However, as demonstrated by Z-slices shown in C., two (?) are crossing behind V2^LP vessel, and the one (**) fuses with V2^LP vessel. Scale bars: A, B: 50 μm; C–E: 25 μm.