Figure 4. Iinitial tip cell selection in the DA.

(A) The number of Ca²⁺-oscillating ECs within the DA at each somite boundary of Tg(fli1:Gal4FF);(UAS:GCaMP7a);(fli1:H2B-mC) embryos at 19–22 and 24–27 ss. Graph shows percentage of the number of a Ca²⁺-oscillating cell (1), two cells (2), and three cells (3) at a somite boundary among the total number of somite boundaries (indicated at the top) observed. Two each representative 3D-rendered images of Tg(fli1:Gal4FF);(UAS:GCaMP7a);(fli1:H2B-mC) at 19–22 and 24–27 ss are shown in the left. Arrowheads indicate Ca²⁺-oscillating cells. (B) 3D-rendered time-sequential images of Tg(fli1:Gal4FF);(UAS:GCaMP7a);(fli1:H2B-mC) embryos from 22 ss. Green arrowheads indicate an EC which maintained Ca²⁺ oscillations, whereas red arrowheads indicate an EC which lost Ca²⁺ oscillations. Similar results were obtained in five independent experiments. (C) The fluorescence changes in GCaMP7a (ΔF/F₀) of the ECs indicated by arrowheads in B and indicated at the left panel are shown as a graph. (D) Schematic illustration of Ca²⁺ dynamics during tip cell budding. Ca²⁺ oscillations are detected mostly in a single or two-neighboring EC(s) at the onset of vessel sprouting. Finally, only single budding tip cell exhibits Ca²⁺-oscillation at later stages. Scale bars, 10 μm in A and B. SB, somite boundary; DA, dorsal aorta.

DOI: http://dx.doi.org/10.7554/eLife.08817.014

Figure 4—figure supplement 1. ECs close to somite boundaries have potential to sprout.

Light-sheet z-stack fluorescence images of Tg(fli1:Gal4FF);(UAS:GCaMP7a) (lower) and corresponding 2D-slice bright-field (BF) images at the level of somite boundary (upper) just after vessel sprouting (22 ss). Yellow arrowheads indicate somite boundaries. Yellow dashed lines indicate positions of somite boundaries. White arrowheads indicate Ca²⁺-oscillating cells, either of which extends protrusions dorsally. Note that double (left) or single (right) Ca²⁺-oscillating cells are located at somite boundary. Scale bar, 10 μm.

DOI: http://dx.doi.org/10.7554/eLife.08817.015

Figure 4—figure supplement 2. Tip cell selection between two neighboring ECs exhibiting Ca²⁺ oscillations.

3D-rendered time-sequential images of Tg(fli1:Gal4FF);(UAS:GCaMP7a);(fli1:H2B-mC) embryos during tip selection between two neighboring cells exhibiting Ca²⁺ oscillations. An EC maintaining Ca²⁺ oscillations became tip cell (green arrowheads), whereas an EC losing Ca²⁺ oscillations stayed in the DA (magenta arrowheads). A representative case among those we observed are shown (n = 18). Scale bar, 10 μm.

DOI: http://dx.doi.org/10.7554/eLife.08817.016

Figure 4—figure supplement 3. PlexinD1 is necessary to confine Ca²⁺-oscillating sprouts in the vicinity of somite boundaries.

(A) 3D-rendered time-sequential images of Tg(fli1:Gal4FF);(UAS:GCaMP7a);(fli1:H2B-mC) embryos during tip cell budding injected with plxnD1 MO (18 ss). Arrowheads indicate Ca²⁺-oscillating ECs budding from the DA. Yellow dashed lines indicate positions of somite boundaries. Note that plxnD1 morphants display ectopic EC sprouts that exhibit Ca²⁺ oscillations. (B) Fluorescence changes in GCaMP7a (ΔF/F₀) of individual ECs indicated by arrowheads in A are shown as a graph. Scale bar, 10 mm in A.

DOI: http://dx.doi.org/10.7554/eLife.08817.017