Endothelial Ca2+ oscillations reflect VEGFR signaling-regulated angiogenic capacity in vivo

Sprouting angiogenesis is a well-coordinated process controlled by multiple extracellular inputs, including vascular endothelial growth factor (VEGF). However, little is known about when and how individual endothelial cell (EC) responds to angiogenic inputs in vivo. Here, we visualized endothelial Ca2+ dynamics in zebrafish and found that intracellular Ca2+ oscillations occurred in ECs exhibiting angiogenic behavior. Ca2+ oscillations depended upon VEGF receptor-2 (Vegfr2) and Vegfr3 in ECs budding from the dorsal aorta (DA) and posterior cardinal vein, respectively. Thus, visualizing Ca2+ oscillations allowed us to monitor EC responses to angiogenic cues. Vegfr-dependent Ca2+ oscillations occurred in migrating tip cells as well as stalk cells budding from the DA. We investigated how Dll4/Notch signaling regulates endothelial Ca2+ oscillations and found that it was required for the selection of single stalk cell as well as tip cell. Thus, we captured spatio-temporal Ca2+ dynamics during sprouting angiogenesis, as a result of cellular responses to angiogenic inputs. DOI: http://dx.doi.org/10.7554/eLife.08817.001


Introduction
An extensive branched blood vessel network is crucial to deliver oxygen and nutrients to tissues and organs. Such branched structures of blood vessels form mainly by sprouting angiogenesis, which involves the emergence of new vessels from the pre-existing vasculature (Eilken and Adams, 2010;Herbert and Stainier, 2011;Phng and Gerhardt, 2009). In the early steps of sprouting angiogenesis, specific motile endothelial cells (ECs) activated by angiogenic cues become leading tip cells and migrate outward from the parental vessels (Gerhardt et al., 2003). As the tip cell migrates from the parental vessel, neighboring ECs migrate by following the tip cell as stalk cells to keep connection between the tip cell and the parental vessel. While tip cells guide the sprouts, stalk cells constitute the base of the sprouts and are, therefore, considered to be less active than tip cells.
Sprouting angiogenesis is triggered by extracellular stimuli. Among them, vascular endothelial growth factor (VEGF)-A induces EC motility and loosen interendothelial cell junction by activating a tyrosine kinase receptor, VEGF receptor-2 (VEGFR2), to induce sprouting (Ferrara, 2009;Lohela et al., 2009). VEGF-A stimulates proliferation of ECs as well as survival (Koch and Claesson-Welsh, 2012;Lohela et al., 2009). VEGFR3 (also known as Flt4), a receptor of VEGF-C, is also required for developmental or tumor angiogenesis . Especially, Vegfr3 is essential for venous sprouting from the posterior cardinal vein (PCV) in zebrafish (Hogan et al., 2009). However, little is known about how VEGF-A/VEGFR2 or VEGF-C/VEGFR3 signaling in ECs induces initial sprouting in vivo.
While sprouting angiogenesis is promoted by VEGFR, it is negatively coordinated by delta-like 4 (Dll4)/Notch signaling (Eilken and Adams, 2010;Lohela et al., 2009;Phng and Gerhardt, 2009). During sprouting angiogenesis, Dll4/Notch signaling between ECs determines the selection of single tip cells among ECs of the pre-existing vessel by restricting the angiogenic behavior of neighboring ECs (Hellströ m, et al., 2007). In studies using mouse and zebrafish, loss of Dll4/Notch signaling results in an increased number of ECs showing tip cell behavior (Hellströ m, et al., 2007;Leslie et al., 2007;Siekmann and Lawson, 2007;Suchting et al., 2007). While VEGF-A/VEGFR2 signaling and Dll4/Notch signaling have opposing roles in regulating blood vessel sprouting, they are tightly linked in a negative feedback loop. VEGFR2 activation in tip cells leads to an up-regulation of the Notch ligand Dll4 and subsequent activation of Notch signaling in the following stalk cells (Lobov et al., 2007;Suchting et al., 2007;Zarkada et al., 2015). Increased Notch activity leads to the downregulation of VEGFR2 and/or VEGFR3, thereby restricting VEGFR2-or VEGFR3-dependent signaling in the stalk cells (Benedito et al., 2012;Siekmann and Lawson, 2007;Zarkada et al., 2015). Indeed, VEGFR3 expression is suppressed by Dll4/Notch signaling in zebrafish and mice (Benedito et al., 2012;Siekmann and Lawson, 2007;Zygmunt et al., 2011). On the other hand, it is unclear to what extent VEGFR2 expression is suppressed by Dll4/ Notch signaling (Benedito et al., 2012;Jakobsson et al., 2010;Suchting et al., 2007). VEGFR2 and VEGFR3 are responsible for the aberrant angiogenesis induced by loss of Dll4/Notch signaling (Hogan et al., 2009;Leslie et al., 2007;Siekmann and Lawson, 2007;Suchting et al., 2007;Zarkada et al., 2015); however, it is still unclear how ECs behave in response to VEGFR-dependent angiogenic cues and how their behavior is spatio-temporally regulated by Dll4/Notch signaling.
Binding of VEGF-A to VEGFR2 induces dimerization of VEGFR2 and autophosphorylation of tyrosine residues in VEGFR2, leading to the activation of various downstream signaling pathways. eLife digest Throughout life, new blood vessels grow out like branches from existing vessels in a process called "sprouting angiogenesis". This involves some of the endothelial cells that line the inner surface of the blood vessel migrating outwards, creating a vessel sprout made up of tip cells and stalk cells.
Sprouting is controlled by two opposing signaling systems. One pathway is triggered by a molecule called vascular endothelial growth factor (VEGF). This molecule binds to receptor proteins to activate a range of signaling processes that stimulate endothelial cells to become tip cells, and so encourage the formation of new sprouts. However, it was not known exactly when or how the endothelial cells respond to these signals.
By contrast, the Notch signaling pathway inhibits sprouting angiogenesis. The two signaling pathways interact with each other: VEGF signaling in tip cells activates Notch signaling in neighboring cells, which then prevents VEGF signaling in these cells. This feedback mechanism helps a new sprout to form by suppressing tip-like activity in the cells surrounding a new tip cell, forcing these cells to become stalk cells.
Activating VEGF receptors also causes brief increases, or oscillations, in the level of calcium ions inside the endothelial cells. Now, Yokota, Nakajima et al. have investigated VEGF activity by genetically engineering zebrafish embryos so that fluorescent proteins inside their endothelial cells emit more light when calcium ion levels inside the cell increase. As zebrafish embryos are transparent, this change in fluorescence can be seen in the living animal. Imaging the embryos revealed that calcium ion oscillations occur in both tip and stalk cells in response to VEGF signaling as they bud from vessels. Notch signaling can also regulate the calcium ion oscillations; this controls whether an individual cell becomes a tip or a stalk cell, and restricts the number of stalk cells in the sprout.
The flow of blood through the vessels is also thought to influence calcium ion oscillations in endothelial cells. Future studies could therefore use the imaging technique developed by Yokota, Nakajima et al. to investigate how blood flow influences the development of new blood vessels.
Autophosphorylated VEGFR2 associates with phospholipase C-g (PLCg) and increases intracellular Ca 2+ via accumulation of inositol 1,4,5-triphosphate (IP 3 ) (Koch and Claesson-Welsh, 2012;  (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 (DF/F 0 ) 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 Ca 2+ -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   Moccia et al., 2012) and store-operated Ca 2+ entry (Li et al., 2011). Besides VEGF-A, various chemical and mechanical stimuli induce an increase of intracellular Ca 2+ as a common second messenger in ECs (Ando and Yamamoto, 2013;Moccia et al., 2012). However, little is known about when and where the Ca 2+ responses in ECs occur during vascular development in vivo. We considered that we could precisely monitor Ca 2+ responses in vivo by using genetically encoded fluorescent Ca 2+ monitoring probes which can be used for detecting rapid Ca 2+ responses in living animals (Muto et al., 2013;Rose et al., 2014). We hypothesized that Ca 2+ dynamics might represent one of EC responses to angiogenic cues during sprouting angiogenesis.
In this study, we succeeded in visualizing the intracellular Ca 2+ dynamics in ECs at single-cell resolution in zebrafish by performing high-speed, three-dimensional (3D) time-lapse imaging, and uncovered how Ca 2+ dynamics are spatio-temporally regulated during sprouting angiogenesis. Intracellular Ca 2+ oscillations occurred in migrating tip and stalk cells, in a manner dependent upon VEGFR signaling. By investigating when and how Dll4/Notch signaling regulates endothelial Ca 2+ oscillations, we demonstrated how suppressive Dll4/Notch signaling was involved in the selection of tip cells from the dorsal aorta (DA) and that of stalk sells in intersomitic vessels (ISVs).

Ca 2+ oscillations in budding tip cells from the DA
To understand how individual EC responds to angiogenic stimuli, we examined the dynamics of intracellular Ca 2+ in ECs during sprouting angiogenesis in vivo. We conducted in vivo Ca 2+ imaging by expressing a genetically encoded Ca 2+ indicator, GCaMP7a (GFP-based Ca 2+ probe) (Muto et al., 2013) in ECs of zebrafish. GCaMP7a is an improved version of GCaMP (Nakai et al., 2001), an engineered GFP that increases fluorescence upon the Ca 2+ elevation (Figure 1-figure supplement 1A). Firstly, we established a transgenic fish line, Tg(fli1:Gal4FF);(UAS:GCaMP7a), in which GCaMP7a was driven by the endothelial specific promoter fli1 via the Gal4/UAS system (Asakawa et al., 2008). This Tg line showed an increase of fluorescence exclusively in ECs in response to Ca 2+ elevation ( Figure 1-figure supplement 1B). Secondly, to distinguish each EC, we developed a Tg fish line, Tg(fli1:H2B-mC), in which EC nuclei was labeled by H2B-mCherry, and crossed this line with the Tg(fli1:Gal4FF);(UAS:GCaMP7a) line. We confirmed that almost all ECs expressed GCaMP7a in developing trunk vessels of these triple Tg embryos (Figure 1-figure supplement 2A), although the expression of GCaMP7a varied among ECs. To monitor fast Ca 2+ dynamics in ECs (see Figure 1-figure supplement 2B,C), we used a light sheet microscopy, which allows rapid acquisitions in living embryos by illuminating the sample with a focused light sheet perpendicularly to the direction of observation (Huisken et al., 2004).
We examined intracellular Ca 2+ dynamics in budding ECs of the DA close to somite boundaries at 24-27 somite stages (ss). We defined these budding ECs as tip cells, because we confirmed that they eventually became tip cells. These tip cells showed sustained and non-periodic Ca 2+ oscillations  The time to reach the peak of individual oscillations was varied 5.6-18.7 s (average, 9.0 s) ( Figure 1C). Therefore, hereafter we performed 3D time-lapse imaging analyses at 5 s intervals to capture all Ca 2+ oscillations. Intracellular Ca 2+ levels of individual ECs were quantified at each time point by measuring fluorescence intensity of GCaMP7a, while tracking H2B-mC-labelled cell nuclei over time (Figure 1-figure supplement 2D; see Materials and methods). We analyzed Ca 2+ oscillations by the frequency and average increases in relative fluorescence intensity of GCaMP7a from the base line (mean DF/F 0 ). Frequency of Ca 2+ oscillations is elevated by increased levels of agonists in some cases in ECs (Carter et al., 1991;Jacob et al., 1988;Moccia et al., 2003;Mumtaz et al., 2011) and non-ECs (Woods et al., 1986). Meanwhile, the amplitude of Ca 2+ rise and total Ca 2+ increases may possibly reflect the dose of agonists (Brock et al., 1991;Fewtrell, 1993;Sage et al., 1989). Thus, in this study, we quantified the oscillations to describe the oscillatory activity in individual EC (see 'Materials and methods'). Our quantification analyses clearly revealed that budding tip cells exhibited oscillatory activity at 24-27 ss ( Figure 1D,E). Repetitive Ca 2+ transients were not detected in other ECs within the DA ( Figure 1A,B,D). These results indicate that the Ca 2+ imaging method we used precisely detects the endogenous intracellular increase or decrease of Ca 2+ in vivo.
Vegfa/Vegfr2 signaling, but not Vegfr3 signaling, is responsible for Ca 2+ oscillations in ECs sprouting from the DA Intracellular Ca 2+ oscillations are known to occur in response to physiological concentrations of agonists in vitro in many cell types (Fewtrell, 1993;Woods et al., 1986) including ECs (Jacob et al., 1988;Moccia et al., 2003;Sage et al., 1989), suggesting that Ca 2+ oscillations detected here may represent EC response to angiogenic stimuli. To examine which angiogenic stimuli are responsible for Ca 2+ oscillations during vessel sprouting from the DA, we first tested the involvement of Vegfr2 since VEGF-A/VEGFR2 signaling is essential for sprouting angiogenesis (Koch and Claesson-Welsh, 2012;Lohela et al., 2009) and can increase intracellular Ca 2+ in vitro (Figure 1-figure supplement 1C) (Brock et al., 1991). Firstly, we examined the effect of inhibiting Vegfr2 on Ca 2+ oscillations by using an inhibitor of VEGFR2, ki8751 (Kubo et al., 2005). When we treated 22 ss embryos with ki8751 and performed Ca 2+ imaging analyses at 24-27 ss, the Ca 2+ oscillations detected in budding tip cells of control embryos ( Figure 1 , confirming that ki8751 inhibits Vegfr2 of zebrafish. ki8751 might also inhibit Vegfr3 activity, because, in our previous results, ki8751 treatment blocked Vegfr3-dependent venous sprout from the PCV in zebrafish (Kwon et al., 2013). Therefore, we regarded ki8751 as a zebrafish Vegfr inhibitor in this study. To confirm the specific contribution of Vegfr2 in budding ECs from the DA to Ca 2+ oscillation there, we knocked-down vegfr2 (also termed kdrl) by injecting antisense oligonucleotides (MO) (Wiley et al., 2011). Although we observed partial sprouts from the DA in the morphants as reported in vegfr2 mutants (Covassin et al., 2006), Ca 2+ oscillations were markedly reduced in these budding cells ( Figure 2D-F), suggesting that Ca 2+ oscillations in ECs budding from the DA are dependent upon Vegfr2 activation.
We further tried to confirm whether Ca 2+ oscillations depend upon Vegfa/Vegfr2 signaling by examining the effect of forced expression of soluble Flt1 (sFlt1), a potent Vegfa trap, on Ca 2+ oscillations. sFlt1 was transiently and specifically expressed in ECs in a mosaic manner by injecting plasmids at one-cell stage. While expression of control NLS-tagged mCherry (NLS-mC) did not affect Ca 2+ oscillations in budding ECs (  . Collectively, our results indicate that Vegfa/Vegfr2 signaling is required for the Ca 2+ oscillations during tip cell budding from the DA. VEGFR3 signaling is another signaling pathway that facilitates sprouting angiogenesis (Covassin et al., 2006;. To investigate the role of Vegfr3 in Ca 2+ oscillations in the budding ECs from the DA, we examined Ca 2+ oscillation in vegfr3 morphants (Hogan et al., 2009). vegfr3 morphants exhibited loss of venous sprout from the PCV ( While ISV sprouts from the DA are mainly driven by Vegfa/Vegfr2 signaling, those from the PCV are driven by Vegfc/Vegfr3 signaling (Hogan et al., 2009). We observed that tip cells in the venous sprouts from the PCV exhibited Ca 2+ oscillations in a manner dependent upon Vegfr3 (Figure 2figure supplement 3A-D). Thus, Ca 2+ oscillations are not only specific for arterial sprouting, but also occur in venous sprouting that is regulated by Vegfc/Vegfr3 signaling.
ECs close to somite boundaries have potential to sprout from the DA To know when and how single tip cells are selected, we looked at Ca 2+ dynamics in ECs of the DA before ECs sprouted from the DA. Prior to sprouting from the DA, Ca 2+ oscillations occurred widely within the DA ( Figure 3A,B and Video 2) and were completely dependent upon Vegfr2 (  , Ca 2+ -oscillating cells were not restricted to specific regions within the DA before vessel sprouting (17-19 ss, Figure 3C). These results suggest that single tip cells are not specified among the ECs of the DA before sprouting as far as Ca 2+ oscillation is used as an indicator ( Figure 3D).
Next, we examined endothelial Ca 2+ dynamics just after vessel sprouting (19-22 ss). Since ISV sprouts emerge bilaterally from the DA, we analyzed one side of sprouting ISVs. When ECs within the DA started to sprout dorsally (19-22 ss), Ca 2+ oscillations were detected in two neighboring ECs (45.2%) or single ECs (52.4%) at somite boundaries, either of which sprouted dorsally ( Figure 4A and Figure 4-figure supplement 1). We confirmed that two neighboring ECs were not the result of a recent division (86%, n = 7, data not shown). These results suggest that the initial selection of single tip cells is not completed just after the onset of vessel sprouting. Ca 2+ response became restricted to single tip cells at later stage (24-27 ss) ( Figure 4A), suggesting the completion of tip cell selection. Then, to visualize the process of tip cell selection between two neighboring cells exhibiting Ca 2+ oscillations, we performed Ca 2+ imaging for a longer period and found that only one cell maintained the oscillations over time ( Figure 4B,C and Video 3). We also noticed that the ECs losing Ca 2+ response retracted their sprout ( Figure 4B), implying that these cells lost their ability to respond to angiogenic cues. The ECs showing sustained Ca 2+ oscillations eventually became tip cells (18/18, Figure 4-figure supplement 2). Thus, our results provide evidence that the initial selection of tip cells is determined even after ECs start to migrate from the DA ( Figure 4D).
Sema-PlexinD1 (PlxnD1) signaling is required to determine the position of tip cell budding in the vicinity of somite boundaries . We observed ectopically budding ECs Stalk cells also exhibit Ca 2+ oscillations during budding from the DA We then examined Ca 2+ dynamics at later stages of ISV formation. Sustained repetitive Ca 2+ oscillations in tip cells were observed when ECs following tip cells were budding from the DA ( Figure 5A-C). We defined those following ECs as stalk cells, because they eventually became stalk cells. Interestingly, Ca 2+ oscillations also occurred in budding stalk cells ( Figure 5A,B and Video 4), although their oscillation frequency and mean DF/F 0 were lower than those in tip cells ( Figure 5C,D). We further found that the Ca 2+ oscillations both in tip cells and budding stalk cells were Vegfr-dependent

Vegfr2 in tip cells and stalk cells is required for their migration from the DA
To investigate the significance of the Vegfr2 activity in tip and stalk cells, we examined the behavior of ECs expressing dominant-negative Vegfr2. We generated a dominant-negative Vegfr2 by deleting the C-terminus of zebrafish Vegfr2 (Vegfr2-DC) (Millauer et al., 1994). Firstly, to validate whether Vegfr2-DC can block Vegfa/Vegfr2 signaling in zebrafish ECs, we established a Tg fish line, Tg(fli1: Gal4ff);(UAS:Vegfr2-DC,NLS-mC), that expresses Vegfr2-DC simultaneously with NLS-mC in ECs. In primary culture cells dissociated from the Tg embryos, Vegfaa-induced Erk phosphorylation was blocked in the Vegfr2-DC-expressing ECs ( Figure 6A,B; Vegfr2-DC+NLS-mC). In contrast, in the cells dissociated from the control Tg(fli1:Gal4ff);(UAS:NLS-mC) embryos that expressed NLS-mC alone in ECs, the Erk phosphorylation was not blocked ( Figure 6A,B; NLS-mC). These results confirmed that Vegfaa/Vegfr2 signaling is blocked by Vegfr2-DC expression. Consistently, ISV sprouting was The Ca 2+ -oscillatory activity in stalk cells coming out completely from the DA becomes comparable to that in tip cells We then investigated Ca 2+ dynamics after stalk cells completely came out from the DA. Ca 2+ oscillations in stalk cells out of the DA were comparable to those in tip cells ( Figure 7A-C and Video 5). The Ca 2+ oscillations were also dependent upon Vegfr2 ( Figure 7C and Figure 7-figure supplement 1).
Ca 2+ increases generally occur by two modes: one is intracellular Ca 2+ waves which only propagate within a single cell; the other is intercellular Ca 2+ waves (ICWs) which transmit from one cell to the next via gap junctions or extracellular messengers. To investigate whether Ca 2+ oscillations were due to either mechanism, we measured how often the oscillations in tip or stalk cells were synchronized with adjacent stalk or tip cells, respectively. We found that 78.9% of Ca 2+ oscillations in stalk cells occurred independently of tip cells, and 91.6% of oscillations in tip cells occurred independently of stalk cells ( Figure 7D). These results clearly indicate that most Ca 2+ oscillations observed in tip and stalk cells occurs independently of each other and not through ICWs. Therefore, our findings suggest that not only tip cells but also stalk cells have potential to respond to angiogenic cues ( Figure 7E). Similarly to the earlier stage, Ca 2+ oscillations were not detected in the ECs within the DA ( Figure 7A-C), except in ECs that were budding from the DA and following stalk cells (Figure 7-figure supplement 2).

Dll4/Notch signaling is crucial for suppressing Ca 2+ responses in ECs adjacent to tip cells during budding
To understand how tip cell behavior is spatio-temporally regulated by Dll4/Notch signaling, we examined the effect of the inhibition of Dll4/Notch signaling on Ca 2+ dynamics. Previous reports have shown that MO-mediated knockdown of Dll4 leads to an increased number of ECs exhibiting tip cell behavior in zebrafish ISVs, which phenocopies loss of Notch signaling (Siekmann and Lawson, 2007). Therefore, we analyzed Ca 2+ oscillations before and during vessel sprouting in dll4 morphants (Siekmann and Lawson, 2007). Before sprouting of ISVs, the number of Ca 2+ -oscillating ECs and their oscillatory activity were significantly increased in the entire DA in dll4 morphants ( Figure 8A-C), suggesting that Dll4 suppresses the Vegfr2-dependent angiogenic responses in entire ECs of the DA. When ECs start budding at 19-22 ss, the Ca 2+ responses were restricted to single ECs in 44.5% at somite boundaries in the control morphants, whereas those were, in 20.8% in dll4 morphants ( Figure 9A; 19-22 ss). Most dll4 morphants had two or more budding ECs exhibiting Ca 2+ oscillations (79.2%; Figure 9A; 19-22 ss). Thus, our findings support the hypothesis that Dll4 restricts the number of ECs showing tip cell behavior when they sprout from the pre-existing vessels. In the later stages (24-27 ss), while the Ca 2+ responses became restricted to single tip cells in the control embryos, two or more neighboring ECs exhibited Ca 2+ oscillations in dll4 morphants (74.1%; Figure 9A; 24-27 ss). Consistently in the dll4 morphants, repetitive Ca 2+ oscillations were maintained in the two neighboring ECs, both of which were budding from the DA ( Figure 9B,C and Video 6). Similar results were observed in notch1b morphants (Figure 9-figure supplement 1). These results suggest that Dll4/Notch signaling regulates the selection of single tip cell from two or more ECs showing Ca 2+ oscillation. In dll4 morphants, Ca 2+ responses were detected exclusively in budding ECs, but not in other ECs within the DA ( Figure 9D), suggesting that Dll4 suppresses the tip cell behavior especially in ECs adjacent to budding tip cells, but not in the entire DA after tip cell budding.
Next, we investigated the effect of inhibition of Vegfr in the dll4 morphants on Ca 2+ dynamics. Treatment with ki8751 completely abolished the Ca 2+ oscillations observed in the dll4 morphants ( Figure 9-figure supplement 2), suggesting that an increase in the number of oscillating cells in the dll4 morphants reflects an increase in the number of ECs showing either Vegfr2 or Vegfr3 activation. In zebrafish, arterial overproliferation and hypersprouting in the absence of Dll4/Notch signaling is, at least partially, attributed to ectopically enhanced Vegfr3 activation (Hogan et al., 2009;Siekmann and Lawson, 2007). Accordingly, Vegfr3 might be involved in the ectopic Ca 2+ oscillations during vessel sprouting in dll4 morphant. To assess the contribution of Vegfr3 to the ectopic Ca 2+ oscillations, we examined Ca 2+ oscillations in vegfr3/dll4 double morphants at 24-27 ss when the Ca 2+ responses became restricted to single tip cells in wild-type embryos ( Figure 4A). In vegfr3/ dll4 double morphants, Ca 2+ oscillations were detected in two or more neighboring cells in 65.8% at somite boundaries (Figure 9-figure supplement 3), although the phenotype in the double morphants was slightly milder than that in the dll4 morphants (74.1%; Figure 9A; 24-27 ss). Thus, Vegfr3 is partially involved in increases in oscillating cells in the absence of Dll4/Notch signaling. On the other hand, these results imply that ectopic Ca 2+ oscillations occur even in the absence of vegfr3 after inhibiting Dll4/Notch signaling. Considering that the Ca 2+ responses in dll4 morphants were completely abolished by ki8751 treatment (Figure 9-figure supplement 2), our findings suggest that Dll4/Notch signaling might restrict the tip cell number mainly by suppressing Vegfr2 signaling. Since Vegfr3 is responsible for hypersprouting of ISVs in the dorsal part of the dll4 morphants To investigate the mechanism how Dll4/Notch signaling suppresses Vegfr2 signaling in ECs of zebrafish, we tested the effect of inhibiting Dll4/Notch signaling on the expression of vegfr2 during sprouting of the ISVs. However, we could detect any increase in the expression of neither vegfr2 nor its paralog kdr in the trunk vessels of dll4 morphants (Figure 9-figure supplement 4), suggesting that Dll4/Notch signaling inhibits the Vegfr2 signaling cascade rather than vegfr2 expression.

Dll4 is required for the selection of single stalk cells
We further investigated the role of Dll4/Notch signaling in stalk cell selection. Of note, when stalk cells began to migrate from the DA, Ca 2+ oscillations were mainly detected only in single stalk cells in the DA, but not in adjacent cells apart from tip cells ( Figure 10A). Because Dll4/Notch signaling regulates the selection of single tip cells (Figure 9) (Eilken and Adams, 2010;Lohela et al., 2009;Phng and Gerhardt, 2009), we hypothesized that it might also regulate the selection of single stalk cell. To test this hypothesis, we examined the effect of knockdown of Dll4 on Ca 2+ dynamics during stalk cell budding. While Ca 2+ oscillations were mostly detected in single stalk cell in the control embryos, they were often detected in two or more neighboring ECs following tip cells in dll4 morphants ( Figure 10A-D). Longer time-lapse imaging confirmed that both Ca 2+ -oscillating stalk cell in the dll4 morphants eventually came out from the DA to form the ISVs. These results suggest that Dll4/Notch signaling regulates the selection of single stalk cell by restricting angiogenic behavior in the ECs adjacent to the stalk cells.
We then investigated the role of Dll4/Notch signaling after stalk cell budding from the DA. Even in the absence of dll4, we could not detect significant increases in oscillatory activity in both tip and stalk cells that came out of the DA ( Figure 11A-C). Thus, Dll4/Notch signaling does not suppress the Ca 2+ -responses in the initial sprouting from the DA to form ISVs.

Discussion
In the present study, we visualized Ca 2+ dynamics, for the first time, in ECs during sprouting angiogenesis in vivo. By quantitatively analyzing Ca 2+ dynamics in individual ECs, we have uncovered how Ca 2+ responses are spatio-temporally regulated at the single-cell level (Figure 12). Intracellular Ca 2+ oscillations occurred in ECs exhibiting angiogenic behavior, such as sprouting and migration. Therefore, visualizing Ca 2+ oscillations allowed us to monitor EC responses to angiogenic cues. While Ca 2+ oscillations depended upon Vegfa/Vegfr2 signaling in ECs sprouting from the DA, they were also detected in venous sprouts in response to Vegfc/Vegfr3. Thus, Ca 2+ oscillations in ECs occur during both arterial and venous sprouting and subsequent migration.
Oscillatory increases of intracellular Ca 2+ were dependent upon Vegfa/Vegfr2 signaling in ECs budding from the DA. VEGF-A treatment induces Ca 2+ rise in cultured human ECs, although their Ca 2+ response is not oscillatory (Brock et al., 1991;Favia et al., 2014;Li et al., 2011). In vitro, VEGF-A induces Ca 2+ rise by the PLCg-IP 3 -IP 3 R pathway (Koch and Claesson-Welsh, 2012;Moccia et al., 2012), by store-operated Ca 2+ entry involving Orai1 and CRAC channel (Li et al., 2011) and by lysosomal Ca 2+ release involving two-pore channel TPC2 (Favia et al., 2014). Since intracellular Ca 2+ oscillations generally occur through the concerted action between several Ca 2+ transporters (Smedler and Uhlen, 2014), further studies are needed to delineate whether Vegfr2 signaling directly regulates Ca 2+ store or Ca 2+ channels in ECs in vivo. Furthermore, it is also required to detect where intracellular Ca 2+ increases during sprouting angiogenesis. Although we observed an increase in Ca 2+ throughout the cytoplasm, if ECs respond to Vegfa, the initial increase of Ca 2+ might be found in a manner dependent upon the distribution of Vegfr2. In cultured ECs, they exhibit Ca 2+ oscillations only in the front of the cells but not in the rear during migration (Tsai et al., 2014). These polarized Ca 2+ oscillations might be detected if we can improve the time/ spatial resolution of imaging of zebrafish in vivo.
Ca 2+ imaging at single-cell resolution pointed to the new regulatory mechanism underlying the formation of stalk cells following tip cells in the ISV. Lateral inhibitory action of Dll4/Notch signaling is essential for selection of tip cells (Eilken and Adams, 2010;Lohela et al., 2009;Phng and Gerhardt, 2009); however, it is unknown how stalk cells are specified and migrate from the parental vessels. We found that stalk cells have Vegfr-dependent Ca 2+ oscillations when they start budding following tip cells and that Dll4/Notch signaling is required for the selection of single stalk cell similarly to tip cell selection ( Figure 12). VEGF-A/VEGFR2 signaling is active in stalk cells and is important for cell proliferation in mouse retina angiogenesis (Gerhardt et al., 2003). Here, we propose that Vegfr2 activation in stalk cells is also necessary for the selection of stalk cells and their exit from the parental vessels during ISV formation. Thus, our Ca 2+ imaging highlighted a novel role of Vegfr2 in stalk cells.
Tip cells and stalk cells are selected during sprouting angiogenesis. In the present study, we focused on the stage of ECs budding from the DA and observed that even two cells showing Ca 2+ oscillations budded from the DA and that one of them finally became single tip cell. Jacobsson et al. report that tip cells and stalk cells change their positions in elongating vessels sprouts (Jakobsson et al., 2010), whereas in the earlier stage, we did not observe interexchange of tip cells and stalk cells, suggesting that their plasticity of tip and stalk depends upon the stage and type of angiogenesis.
Dll4/Notch signaling is required for selection of single tip cell and single stalk cell during budding from the DA. Our findings support the notion that Dll4 in tip cells suppresses tip cell behavior in adjacent cells for the initial selection of tip cell (Eilken and Adams, 2010;Herbert and Stainier, 2011;Lohela et al., 2009;Phng and Gerhardt, 2009). Meanwhile, it has not been determined whether tip cells also suppress angiogenic responses in stalk cells via Dll4/Notch signaling during vessel elongation. Our results indicate that inhibitory activity of Dll4 toward Ca 2+ responses in stalk cells diminishes after stalk cells come out from the DA. Instead, Dll4/Notch signaling restricts the number of stalk cells. Thus, our results suggest that Dll4/Notch-mediated lateral inhibition leads to suppression of angiogenic responses mainly in ECs within the DA adjacent to budding tip or stalk cells to restrict excess budding from the DA (Figure 12). Our Ca 2+ imaging provide novel insights into how Dll4/Notch signaling spatio-temporally controls angiogenic behavior.
Endothelial Ca 2+ responses can be used as an indicator of cellular responses to extracellular stimuli. In this study, we show that endothelial Ca 2+ oscillations occur during sprouting angiogenesis in response to Vegfa/Vegfr2 and Vegfc/Vegfr3 signaling. We could investigate endothelial Ca 2+ responses induced by other chemical or mechanical stimuli. Among them, blood flow is a well-studied input that induces an intracellular Ca 2+ increase in ECs (Ando and Yamamoto, 2013). Blood flow-dependent Ca 2+ increases were recently reported in zebrafish embryos (Goetz et al., 2014). Our Ca 2+ imaging analyses have captured flow-dependent Ca 2+ oscillations in various types of vessels (Yokota et al., unpublished data): therefore, Ca 2+ imaging analysis will be a useful system for understanding quantitatively how individual ECs are regulated by blood flow in vivo. Thus, in vivo Ca 2+ imaging in ECs presented here will become a powerful tool to investigate the dynamic behavior of individual ECs in vascular development and homeostasis in the future studies.

Zebrafish husbandry
Zebrafish (Danio rerio) were maintained and bred under standard conditions. The experiments using zebrafish were approved by the animal committee of National Cerebral and Cardiovascular Center (No. 14005) and performed according to the guidance of the Institute.

Transgenic zebrafish lines
To generate the Tg(fli1:H2B-mC), Tg(UAS:NLS-mC), and Tg(UAS:Vegfr2-DC,NLS-mC) zebrafish lines, the corresponding pTol2-based plasmid DNAs (15 pg) were microinjected along with Tol2 transposase mRNA (30 pg) into one-cell stage embryos of AB or Tg(fli1:Gal4FF) zebrafish. Tol2 transposase mRNAs were in vitro transcribed with SP6 RNA polymerase from NotI-linearized pCS-TP vector using the mMESSAGE mMACHINE kit (Ambion, Thermo Fisher Scientific, Waltham, MA). The embryos Figure 12. A schematic representation of endothelial Ca 2+ responses during sprouting angiogenesis from the DA. Ca 2+ oscillations occur widely within the DA before ISV sprouting. Dll4/Notch signaling attenuates Ca 2+ responses in the entire DA at this stage. Ca 2+ oscillations are restricted to single or two neighboring ECs just after vessel sprouting and then further restricted to single ECs that eventually become tip cells. Dll4/Notch signaling is required for the selection of single tip cells. During stalk cell budding, tip cells and stalk cells exhibit Ca 2+ oscillations, although the Ca 2+ -oscillatory activity in stalk cells is weaker than that in tip cells. Dll4/Notch signaling regulates the selection of single stalk cells. After stalk cells completely come out from the DA, strong Ca 2+ oscillations occur both in tip cells and stalk cells. Intensity of green reflects the frequency of Ca 2+ oscillations. DA, dorsal aorta. DOI: 10.7554/eLife.08817.036 showing transient mCherry expression in the vasculature were selected, raised to adulthood, and crossed with wild-type AB to identify germline transmitting founder fishes.

Zebrafish image acquisition and processing
For light sheet imaging, zebrafish embryos were dechorionated and anesthetized in 0.4 mg/ml rocuronium bromide (Merk Sharp and Dohme, Kenilworth, NJ) in E3 embryo medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl 2 , 0.33 mM MgSO 4 ). The anesthetized embryos were pulled into the small glass capillaries containing 1% low-melting agarose in E3 medium, using a metal plunger. After the gel set, the fish were slightly extruded from the capillary and immersed in the chamber containing E3 medium. Light sheet imaging was performed with a Lightsheet Z.1 system (Carl Zeiss, Germany) equipped with a water immersion 20x detection objective lens (W Plan Apochromat, NA 1.0), dual sided 10x illumination objective lenses (LSMF, NA 0.2), a pco.edge scientific CMOS camera (PCO) and ZEN software. For all 3D time-lapse datasets, a z-interval of 4 mm, and a time interval of 5 s were used. z-stack images were 3D volume rendered and analyzed with IMARIS 7.7.1 software (Bitplane AG, Switzerland). For 2D time-lapse imaging, a time interval of 100 ms was used.

Quantitative analysis of intracellular Ca 2+ oscillations
In our Ca 2+ imaging using light sheet microscopy, we analyzed the ISVs and DA located in middle trunk region of embryos at somite levels 8 to 14. Time-lapse images were collected every 5 s for 1000-2000 s with a z-interval of 4 mm. Z-stack images were 3D volume-rendered and analyzed with IMARIS 7.7.1 software (Bitplane AG). ISV sprouts emerge bilaterally from the DA. Thus, to focus on sprouting angiogenesis occurring on one side, our analyses were performed at either side (left or right side) of the embryos by cropping. To quantify intracellular Ca 2+ levels of individual ECs at each time point, the cell nuclei labeled with H2B-mC were automatically (or manually in some cases) tracked over time using IMARIS software. Then, for each individually tracked EC, we set a spherical region of interest (ROI) of 4-11 mm in diameters that is slightly larger than the nucleus (see Figure 1-figure supplement 2D). The diameters of every spherical ROI were carefully set to cover the nucleus and part of the cytoplasm but do not overlap with adjacent ECs. We then defined the highest voxel intensity of the GCaMP7a fluorescence within the ROI as the florescent intensity (F) of GCaMP7a in the EC. Because intracellular Ca 2+ waves spread rapidly and uniformly throughout the cytoplasm of each EC (see Figure 1-figure supplement 2B), the spherical ROIs including part of the cytoplasmic region are enough to detect intracellular Ca 2+ waves. DF/F 0 was calculated as (F À F 0 )/ F 0 , where F 0 is the baseline florescent intensity of GCaMP7a averaged over a 50-s period. Fluorescence changes in GCaMP7a of individual ECs are represented as DF/F 0 traces in the graphs, and mean DF/F 0 was calculated by taking the average of every DF/F 0 . The frequency of intracellular Ca 2+ oscillations was counted as the number of Ca 2+ oscillations per min. DF/F 0 increase of 20% from the baseline was defined as an oscillation, while 100% is the average of the three highest DF/F 0 peaks in oscillating ECs of wild-type embryos. The ECs, which underwent mitosis through the time-lapse images, were removed from our quantification analyses because we found that ECs never exhibit any Ca 2+ rise during M phase.
HUVECs expressing GCaMP7a were time-lapse imaged with an inverted fluorescence microscope (IX-81; Olympus) equipped with a Plan-Apochromat 40x/1.00 NA oil immersion objective lens (Olympus) and with a pE-1 LED excitation system (CoolLED) with a cooled charge-coupled device camera (Neo5.5 sCMOS; Andor Technology, UK). The cells were imaged at 37 o C with 5% CO 2 using a heating chamber (Tokai Hit, Japan).

Preparation of zebrafish embryo primary cells, immunocytochemistry, and quantification
Tg(fli1:Gal4FF);(UAS:NLS-mC) or Tg(fli1:Gal4FF);(UAS:Vegfr2-DC, NLS-mC) embryos at 34 hpf were dechorionated and collected into a 1.5-ml tube after removing the yolk, washed in phosphate buffered saline (PBS), and digested with 500 ml of protease solution (PBS with 1 mg/ml trypsin, 2.7 mg/ ml Collagenase P and 1mM EDTA, pH 8.0) for 15 min at 28 o C under occasional pipetting. Digestion of the embryos was terminated with 50 ml of stop solution (PBS with 30% fetal bovine serum [FBS] and 6 mM calcium chloride). The dissociated cells were filtered with 35-mm cell strainers (BD Falcon, Thermo Fisher Scientific) and subsequently cultured on glass-base dish coated with laminin in L15 medium (Life Technologies, Carlsbad, CA) with 50 U/ml penicillin and 0.05 mg/ml streptomycin at 28 o C. The cells spread on the culture dishes for 3.5 hr were used for experiments.
The supernatants of HEK293T cells transfected with or without the plasmid DNA encoding Vegfaa-myc were collected 36 hr after the transfection and added to zebrafish primary cells. After stimulation, the cells were fixed with 4% paraformaldehyde (PFA) in PBS for 15 min at RT, permiabilized with 0.2% Triton X-100 for 30 min at RT and blocked with 3% BSA in PBS for 30 min at RT. The cells were immunostained with anti-phospho-ERK (pERK) antibody (#4370, Cell Signaling Technology, Danvers, MA) in 3% BSA in Can Get Signal Immunostain solution (TOYOBO, Japan) at 4˚C overnight. Protein reacting with the antibody was visualized with species-matched Alexa-Fluor 488-labeled secondary antibody (Invitrogen). Fluorescence images were taken with an inverted fluorescence microscope (IX-81) equipped with a Plan-Apochromat 60x/1.40 NA oil immersion objective lens, an X-cite 110LED excitation system (Excelitas Technologies, Waltham, MA) and a cooled charge-coupled device camera (CoolSNAP HQ; Roper Scientific, Photometrics, Tucson, AZ). The microscope and image acquisition were controlled by MetaMorph software (Molecular Devices, Sunnyvale, CA).
Quantification of the fluorescence intensity of pErk staining was performed with MetaMorph software. We defined a region of interest (ROI) kept at constant size (4 mm in diameter) inside the nucleus of NLS-mC-expressing ECs, and measured the mean fluorescence intensity of pErk staining within the ROI after subtracting the background.

Whole-mount in situ hybridization
Whole-mount in situ hybridization (WISH) of zebrafish embryos was performed as described previously (Fukuhara et al., 2014).

Statistical analyses
. Data were analyzed using GraphPad Prism software. Statistical significance for paired samples was determined using Student's t test. Data were considered statistically significant at p < 0.05.