Endothelial Ca2+ oscillations reflect VEGFR signaling-regulated angiogenic capacity in vivo
- National Cerebral and Cardiovascular Center Research Institute, Japan
- National Institute of Genetics, Japan
- Japan Agency for Medical Research and Development, Japan
Figures
Figure 1 with 2 supplements
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 …
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 …
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 …
Figure 2 with 3 supplements
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 …
Figure 2—figure supplement 1
Defects in blood vessels and lymphatic vessels found in Vegfr2- or Vegfr3-inhibited embryos.
(A) Confocal 3D images of Tg (fli1:GFP) embryos at 24 ss (first column), and the corresponding subsequent time-lapse images at the indicated time after treatment with DMSO or ki8751. Note that tip …
Figure 2—figure supplement 2
Vegfr3 is not involved in Ca2+ oscillations in tip cells budding from the dorsal aorta (DA).
(A) 3D-rendered time-sequential images of Tg(fli1:Gal4FF);(UAS:GCaMP7a);(fli1:H2B-mC) embryos injected with vegfr3 MO at 25 ss. A green arrowhead indicates a budding tip cell. (B) The fluorescence …
Figure 2—figure supplement 3
Ca2+ oscillations in the venous sprouts from the posterior cardinal vein (PCV).
(A) 3D-rendered time-sequential images of Tg(fli1:Gal4FF);(UAS:GCaMP7a);(fli1:H2B-mC) embryos during vessel sprouting from the PCV injected with control MO (39 hpf). A green arrowhead indicates a …
Figure 3 with 1 supplement
Ca2+-oscillating cells were not restricted to specific regions within the DA before vessel sprouting.
(A) 3D-rendered time-sequential images of Tg(fli1:Gal4FF);(UAS:GCaMP7a);(fli1:H2B-mC) embryos before ISV sprouting (18 ss). Yellow dashed lines indicate positions of somite boundaries. (B) The DA is …
Figure 3—figure supplement 1
Vegfr2 is responsible for Ca2+ responses before ISVs sprouting from the DA.
(A) 3D-rendered time-sequential images of Tg(fli1:Gal4FF);(UAS:GCaMP7a);(fli1:H2B-mC) embryos treated with ki8751 to inhibit Vegfr before ISV sprouting. The embryos were treated with ki8751 from 17 …
Figure 4 with 3 supplements
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 …
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 …
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+ …
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 …
Figure 5 with 1 supplement
Ca2+ oscillations in stalk cells during budding from the DA.
(A) 3D-rendered time-sequential images of Tg(fli1:Gal4FF);(UAS:GCaMP7a);(fli1:H2B-mC) embryos during stalk cell budding from the DA (29 ss). Green and orange arrowheads indicate tip and stalk cells, …
Figure 5—figure supplement 1
Ca2+ responses during stalk cell budding from the DA are dependent upon Vegfr.
(A) 3D-rendered time-sequential images of Tg(fli1:Gal4FF);(UAS:GCaMP7a);(fli1:H2B-mC) embryos treated with ki8751 during stalk cell budding from the DA as in Figure 5A. The embryos were treated from …
Figure 6
Vegfr2 activation in stalk cells is crucial for their migration from the DA.
(A) The cells dissociated from Tg(fli1:Gal4FF);(UAS:NLS-mC) or Tg(fli1:Gal4FF);(UAS:Vegfr2-ΔC,NLS-mC) embryos (34 hpf) cultured on laminin-coated dish were kept untreated (-) or treated for 5 min …
Figure 7 with 2 supplements
Ca2+ oscillations in tip and stalk cells that completely come out from the DA.
(A) 3D-rendered time-sequential images of Tg(fli1:Gal4FF);(UAS:GCaMP7a);(fli1:H2B-mC) embryos after stalk cells migrate out of the DA (29 ss). Green and orange arrowheads indicate tip and stalk …
Figure 7—figure supplement 1
Ca2+ responses after stalk cell budding from the DA are dependent upon Vegfr.
(A) 3D-rendered time-sequential images of Tg(fli1:Gal4FF);(UAS:GCaMP7a);(fli1:H2B-mC) embryos treated with ki8751 after stalk cells completely migrate out of the DA as in Figure 7A. The embryos were …
Figure 7—figure supplement 2
An EC following a stalk cell exhibits significant Ca2+ oscillations.
(A) 3D-rendered time-sequential images of Tg(fli1:Gal4FF);(UAS:GCaMP7a);(fli1:H2B-mC) embryos at 31 ss when a EC following tip and stalk cells is budding from the DA. Green and orange arrowheads …
Figure 8
Dll4 attenuates Ca2+ oscillations in the entire DA before ISV sprouting.
(A) 3D-rendered time-sequential images of Tg(fli1:Gal4FF);(UAS:GCaMP7a);(fli1:H2B-mC) embryos before ISV sprouting injected with dll4 MO (17 ss). Yellow dashed lines indicate positions of somite …
Figure 9 with 4 supplements
Dll4 is involved in suppressing Ca2+ responses in ECs adjacent to tip cells.
(A) The number of Ca2+-oscillating ECs at each somite boundary of the embryo injected with control MO or dll4 MO was quantified as in Figure 4A. (B) 3D-rendered time-sequential images of Tg(fli1:Gal4…
Figure 9—figure supplement 1
Ca2+ oscillations were maintained in two neighboring ECs in notch1b morphants during tip cell budding from the DA.
3D-rendered time-sequential images of Tg(fli1:Gal4FF);(UAS:GCaMP7a);(fli1:H2B-mC) embryos during tip cell budding injected with notch1b MO (26 ss). Green and blue arrowheads indicate the two …
Figure 9—figure supplement 2
Vegfr is responsible for ectopic Ca2+ oscillations observed in dll4 morphants.
(A) 3D-rendered time-sequential images of Tg(fli1:Gal4FF);(UAS:GCaMP7a);(fli1:H2B-mC) embryos injected with dll4 MO and treated with ki8751 during tip cell budding (26 ss). (B) The fluorescence …
Figure 9—figure supplement 3
Vegfr3 is partially involved in increases in oscillating cells in dll4 morphants.
(A) The number of Ca2+-oscillating ECs at each somite boundary of Tg(fli1:Gal4FF);(UAS:GCaMP7a);(fli1:H2B-mC) embryos injected with both dll4 and vegfr3 MOs was quantified at somite boundaries …
Figure 9—figure supplement 4
The expression of neither vegfr2 nor kdr is not altered in the trunk vessels of dll4 morphants.
Whole-mount in situ hybridization (WISH) analyses of the embryos (26–27 ss) uninjected or injected with dll4 MO using antisense probe for vegfr2 (also termed kdrl) and kdr (kdrb), a paralog of vegfr2…
Figure 10
Dll4 is involved in the selection of single stalk cells.
(A) The number of Ca2+-oscillating ECs following tip cells in each ISV of uninjected, or control MO- or dll4 MO-injected Tg(fli1:Gal4FF);(UAS:GCaMP7a);(fli1:H2B-mC) embryos during stalk cell budding …
Figure 11
Dll4 does not regulate Ca2+ oscillations in stalk cells after they completely migrate out of the DA.
(A) 3D-rendered time-sequential images of Tg(fli1:Gal4FF);(UAS:GCaMP7a);(fli1:H2B-mC) embryos after stalk cell budding from the DA injected with dll4 MO (27 ss). Green and orange arrowheads indicate …
Figure 12
A schematic representation of endothelial Ca2+ responses during sprouting angiogenesis from the DA.
Ca2+ oscillations occur widely within the DA before ISV sprouting. Dll4/Notch signaling attenuates Ca2+ responses in the entire DA at this stage. Ca2+ oscillations are restricted to single or two …
Videos
Video 1
Ca2+ oscillations in tip cell during budding from the dorsal aorta (DA).
Time-lapse recording of 3D-rendered light sheet images of the Tg(fli1:Gal4FF);(UAS:GCaMP7a);(fli1:H2B-mC) embryos at 24 somite stage (ss). Green, GCaMP7a fluorescence; red, H2B-mC fluorescence. …
Video 2
Ca2+ oscillations occur widely within the DA before vessel sprouting.
Time-lapse recording of 3D-rendered light sheet images of the Tg(fli1:Gal4FF);(UAS:GCaMP7a);(fli1:H2B-mC) embryos at 18 ss. Green, GCaMP7a fluorescence; red, H2B-mC fluorescence. Elapsed time is in …
Video 3
The process of tip cell selection.
Time-lapse recording of 3D-rendered light sheet images of the Tg(fli1:Gal4FF);(UAS:GCaMP7a);(fli1:H2B-mC) embryos just after vessel sprouting from the DA (22 ss). Elapsed time is in seconds (s). …
Video 4
Ca2+ oscillations occur in stalk cell during budding from the DA.
Time-lapse recording of 3D-rendered light sheet images of the Tg(fli1:Gal4FF);(UAS:GCaMP7a);(fli1:H2B-mC) embryos at 29 ss. Elapsed time is in seconds (s). Scale bar, 10 μm.
Video 5
Ca2+ oscillations occur both in tip and stalk cells after stalk cell migrates out of the DA.
Time-lapse recording of 3D-rendered light sheet images of the Tg(fli1:Gal4FF);(UAS:GCaMP7a);(fli1:H2B-mC) embryos at 29 ss. Elapsed time is in seconds (s). Scale bar, 10 μm.
Video 6
Ca2+ oscillations are maintained in two neighboring cells in the absence of dll4 during tip cell budding.
Time-lapse recording of 3D-rendered light sheet images of the Tg(fli1:Gal4FF);(UAS:GCaMP7a);(fli1:H2B-mC) embryos during tip cell budding from the DA injected with dll4 morpholino antisense oligo …
