Apelin signaling drives vascular endothelial cells toward a pro-angiogenic state

Essential revisions:

1) The claim that apelin is expressed in tip cells is not well supported by the data provided. It appears that apln:EGFP staining at 26 hpf is very prominent outside the vasculature (presumably, notochord and spinal cord), which makes it difficult to appreciate the fluorescent signal in tip cells (Figure 1A). The authors should clarify whether apln expression is found outside the vasculature and provide clearer images of tip cell expression. In addition, it is important to know whether the sprouts shown on the far left in A' are composed of a single (tip) cell or multiple cells. In case of the latter, it is not obvious that tips are positive whereas stalk cells are not.

apln expression is enriched in tip cells but is not exclusive to these cells. apln expression is also observed in the notochord around 17 hpf (Helker et al., 2015) as well as in the neural tube, and due to the stability of the GFP protein, these tissues are still GFP positive at 26 hpf. Therefore, the EC specific signal is much more visible at 54 hpf when GFP in the notochord and neural tube has mostly been degraded. In Figure 1A’’, the arrowheads point to strong apln expression in tip cells, while the arrows point to weak apln expression in stalk cells. We added these points in the Results section as well as in the figure legend. In addition, we replaced picture 1A’ with a new one at 30 hpf – a time point when apln expression is a bit stronger and easier to visualize.

2) The cellular mechanism underlying the observed ISV sprouting defect in apelin mutants is not well-defined. Specifically, does apelin primarily regulate filopodia or endothelial cell numbers to alter ISV sprouting? Is it known that tip cell filapodia number correlate with sprout length/distance?

Phng et al. reported that the inhibition of filopodia formation by Latrunculin B treatment reduces ISV sprout length (See Figure 5C from Phng et al., 2013), consistent with our hypothesis that the ISV sprouting defects in apln mutants are caused by the filopodia defects. However, one cannot exclude the possibility that defects in EC numbers are also contributing to the ISV sprouting defects. These points are now included in the Discussion.

If so, do Notch-deficient embryos that show excessive sprouting have increased numbers of tip cell filopodia?

Several studies have reported that inhibition of Notch signaling increases filopodia numbers (Hellstrom et al., 2007; Leslie et al., 2007; Siekmann et al., 2007; Suchting et al., 2007).

Does taking Apelin signaling away from dll morphant embryos decrease filapodia number?

Phng et al. reported that ectopic sprouting in dll4 morphants occurs in the absence of filopodia (Phng et al., 2013). Therefore, it is unlikely that the reduction of filopodia observed in apln/aplnrb mutants is the reason for the rescue of the hypersprouting phenotype in apln or aplnrb mutants injected with the dll4 morpholino.

It is argued that the overexpression of apln does not lead to ectopic sprouting. Nevertheless, the images shown in Figure 2—figure supplement 3 suggest that filopodia numbers, morphology, or extension might be altered. Please clarify.

We thank the reviewers for this valid point. Indeed, overexpression of apln leads to defects in lymphatic endothelial cell sprouting. We labeled these defects in Figure 2—figure supplement 3 using arrows and added this additional point to the Results section.

3) There is an internal inconsistency regarding heightened Apelin expression must be rectified. The authors claim that heightened Apelin expression in Notch-deficient embryos is responsible for the observed hyperspouting phenotype. However, overexpression of Apelin in their hsp70:apelin transgenic line fails to show a hypersprouting phenotype. One possibility is that expression of the Apelin receptor is limiting. Is there a constitutively active apelin receptor that could be used to test whether the Apelin signaling axis is sufficient to promote angiogenesis? Can the hyper-sprouting phenotype observed in Notch-deficient embryos also be suppressed by loss of the Apelin receptor? (Dll4 morpholino into alpnr-/- embryos)? Experimental clarification of the model is needed.

We are thankful for these comments and questions. The Apelin receptor is a GPCR and to the best of our knowledge there is no constitutively active form of it available to date. However, we injected the dll4 morpholino into aplnrb mutant embryos, and found that the hypersprouting phenotype observed in Notch-deficient embryos was also suppressed by the loss of the Apelin receptor. These new data can be found in supplemental Figure 4—figure supplement 1.

4) The link between Apelin and Notch is interesting, but it is unclear whether this effect is mediated by VEGF or increased hypoxia, which were previously linked to the regulation of apln expression. The authors should try to clarify the link between Notch and Apelin.

The transcriptional regulation of Apelin Signaling components by Notch signaling is very interesting. However, it is very unlikely that apln is a direct Notch target gene since activation of Notch signaling leads to a downregulation of APLN expression. Therefore, we speculate that apln expression is regulated by downstream Notch target genes such as those encoding members of the HES/HEY Transcription factor family, which are known to act as transcriptional repressors. This point has now been included in the Discussion. Thus, further work is required to obtain a detailed view of the regulation of Apelin signaling components by Notch signaling.

5) The claim that Apelin drives angiogenesis by increasing endothelial cell glycolysis/metabolism is not well supported. Specifically, the connection between apln and metabolic alterations has been only explored in cultured cells in vitro and its relevance for the observed phenotypes in zebrafish remains unclear. The authors should validate their key findings in vivo and explore how apelin regulates the metabolic properties of ECs. For example, could 2-DG be used to inhibit glycolysis in zebrafish embryos and ISV sprouting examined? Is glycolysis/metabolism increased in Notch-inhibited vessels that show hypersprouting? Can this phenotype be suppressed by 2-DG? Is there a method to artificially increase metabolism in order to learn whether this is sufficient to promote ISV sprouting? If so, does it rescue sprouting in apelin mutants?

It has been shown that stimulation of Apelin signaling induces the phosphorylation, and therefore nuclear exclusion, of FOXO1 (Figure 3B from Hwangbo et al., 2017). Furthermore, expression of a constitutively active FOXO1, which remains in the nucleus, has been shown to reduce MYC protein levels (Figure 3K from Wilhelm et al., 2016), and reduce metabolic activity (Figure 3E from Wilhelm et al., 2016). In agreement with these in vitro data, deleting Foxo1 in mouse ECs enhances metabolic activity and promotes EC sprouting in vivo (Figure 1B/C from Wilhelm et al., 2016).

Since 2-DG treatments not only affect ECs but the whole organism, we overexpressed pfkfb3 specifically in ECs in the aplnrb mutant background. These experiments show that enhancing the metabolic activity of ECs can rescue the sprouting phenotype of aplnrb mutant embryos. These new data can be found in Figure 5.