Fig. 2

The Ca²⁺ oscillations during tip cell budding depend upon Vegfa/Vegfr2 signaling.

(A) 3D-rendered time-sequential images of Tg(fli1:Gal4FF);(UAS:GCaMP7a);(fli1:H2B-mC) embryos treated with a Vegfr inhibitor, ki8751, during tip cell budding. The embryos were treated from 22 ss with ki8751 and time-lapse imaged at 24 ss. A green arrowhead indicates a tip cell outlined by a dashed line. The elapsed time (s) after starting imaging of an embryo is indicated at the left upper corner. (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. (C) Quantification of Ca²⁺ oscillation frequency (left) and mean ΔF/F₀ (right) as in Figure 1D in ki8751-treated embryos. The embryos were treated from 22 ss with ki8751 and imaged at 24–27 ss (n ≥ 10). (D) 3D-rendered time-sequential images of Tg(fli1:Gal4FF);(UAS:GCaMP7a);(fli1:H2B-mC) embryos during tip cell budding (24 ss) injected with vegfr2 (kdrl) morpholino (MO). (E) The fluorescence changes in GCaMP7a (ΔF/F₀) of individual ECs from D indicated by arrowheads at the left panel are shown as a graph. (F) Quantification of Ca²⁺ oscillation frequency (left) and mean ΔF/F₀ (right) in tip cells and other ECs within the DA in control MO- or vegfr2 MO-injected embryos during tip cell budding at 24–27 ss (n ≥ 11). (G) 3D-rendered time-sequential images of Tg(fli1:Gal4FF);(UAS:GCaMP7a) embryos at 24–27 ss injected with control UAS:NLS-mC plasmid (upper) or UAS:sFlt1,NLS-mC plasmid (lower) which drives the expression of NLS-mC or both sFlt1 and NLS-mC simultaneously in ECs in a mosaic manner, respectively. Green and red arrowheads indicate NLS-mC-expressing ECs and both sFlt1- and NLS-mC-expressing ECs, respectively. Yellow dashed lines indicate positions of somite boundaries. (H) The fluorescence changes in GCaMP7a (ΔF/F₀) of individual ECs from G indicated by arrowheads at the left panel are shown as a graph. (I) Quantification of Ca²⁺ oscillatory activity in ECs expressing NLS-mC and both sFlt1 and NLS-mC at 24–27 ss. Graphs show Ca²⁺ oscillation frequency (left) and mean ΔF/F₀ (right) of NLS-mC-positive ECs within the DA close to somite boundaries (NLS-mC, n = 18; sFlt1+ NLS-mC, n = 20). Horizontal lines represent mean ± s.d.. Scale bars, 10 µm in A, D and G. **p < 0.01, ***p < 0.001; NS, not significant.