Fig. 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.