Figure 1. Ca²⁺ oscillations in tip cells during budding from the dorsal aorta (DA).

(A) 3D-rendered time-sequential images of the trunk regions of Tg(fli1:Gal4FF);(UAS:GCaMP7a);(fli1:H2B-mC) embryos during vessel sprouting from the DA (24 somite stage (ss)). 3D images were acquired using a light sheet microscope. The merged images of GCaMP7a (green) and H2B-mC (red) images are shown in the following images, unless otherwise described. All the zebrafish images are lateral views and displayed as anterior to the left. A green arrowhead indicates a tip cell outlined by a dashed line. (i)-(v) and other images are those indicated by the arrowheads indicated at a graph in B. (B) The fluorescence changes in GCaMP7a (ΔF/F₀) of individual ECs from A indicated by arrowheads (green, light gray, dark gray, and black) at the left panel are shown as a graph. To measure the fluorescence intensity of GCaMP7a (green) in individual EC, the cell nucleus (red) was tracked over time (see ‘Materials and methods’). (C) Dot-plot graphs depicting the time to reach the peak of each Ca²⁺-oscillation in tip cells. Time-lapse 2D slice images of Tg(fli1:Gal4FF);(UAS:GCaMP7a);(fli1:H2B-mC) embryos taken every 100 ms as in Figure 1—figure supplement 2B were analyzed for quantification. Horizontal lines represent mean ± s.d. (n = 18). (D) Quantification of Ca²⁺ oscillation frequency (left) and mean ΔF/F₀ (right) in tip cells and other ECs within the DA during tip cell budding (24–27 ss) (see ‘Materials and methods’). Each dot represents the value for a single cell. Horizontal lines represent mean ± s.d. (n ≥ 8). (E) Schematic model of tip cells showing Ca²⁺ oscillations when they sprout from the DA. Intensity of green reflects the frequency of Ca²⁺ oscillations. Scale bar, 10 μm in A. ***p < 0.001. DA, dorsal aorta.

DOI: http://dx.doi.org/10.7554/eLife.08817.003

Figure 1—figure supplement 1. GCaMP7a works as a Ca²⁺ indicator in endothelial cells (ECs).

(A) HUVECs transfected with GCaMP7a expression plasmids were treated with DMSO (upper) or ionomycin (lower). GCaMP7a images before (-) and after the treatment (40 s) are shown. (B) Confocal stack fluorescence images of Tg(fli1:Gal4FF);(UAS:GCaMP7a) embryos at 26 ss treated with DMSO (upper) or 25 μM ionomycin (lower). GCaMP7a images before (-) and after the treatment (30 min) are shown. (C) Fluorescence images of HUVECs transfected with GCaMP7a expression plasmids pretreated with DMSO or 25 μM BAPTA-AM for 30 min and treated with 50 ng/ml VEGF-A before (-) and after the treatment (100 s). Note that enhancement of GCaMP7a fluorescence by VEGF-A is blocked by pretreatment with BAPTA-AM. Scale bars, 10 μm in A-C.

DOI: http://dx.doi.org/10.7554/eLife.08817.004

Figure 1—figure supplement 2. Quantitative analyses of intracellular Ca²⁺ dynamics in ECs.

(A) Confocal stack fluorescence images of Tg(fli1:Gal4FF);(UAS:GCaMP7a);(fli1:H2B-mC) embryos at 28 ss. The merged images of GCaMP7a (green) and H2B-mC (red) images are shown with enhanced brightness. Note that almost all ECs express GCaMP7a in developing trunk vessels. (B) Time-lapse 2D slice images of Tg(fli1:Gal4FF);(UAS:GCaMP7a);(fli1:H2B-mC) embryos taken every 100 ms at 24 ss using a light sheet microscope. A green arrowhead indicates a budding tip cell outlined by a dashed line. The elapsed time (s) after starting imaging of an embryo is indicated at the left upper corner. (C) The fluorescence changes in GCaMP7a (ΔF/F₀) of individual ECs from B indicated by arrowheads (green, light gray, dark gray, and black) at the left panel are shown as a graph. (D) Quantification analyses for intracellular Ca²⁺ levels in individual EC. Time-lapse 3D images of Tg(fli1:Gal4FF);(UAS:GCaMP7a);(fli1:H2B-mC) embryos were analyzed for quantification using IMARIS software. The merged images of GCaMP7a (green) and H2B-mC (red) images are shown in the left panel. To quantify intracellular Ca²⁺ of individual EC at each time point, the cell nucleus was tracked over time. Trajectories of individual nuclei were shown in the right panel. To mark the individual EC which we analyze, we set a spherical region of interest (ROI) as shown in the right panel (see ‘Materials and methods’). We then defined the highest voxel intensity of the GCaMP7a fluorescence (green) within the ROI as the fluorescence intensity (F) in the EC. Scale bars, 10 μm in A, B, and D. ISV, intersomitic vessel; DA, dorsal aorta.

DOI: http://dx.doi.org/10.7554/eLife.08817.005