The hormonal peptide Elabela guides angioblasts to the midline during vasculogenesis

A key step in the de novo formation of the embryonic vasculature is the migration of endothelial precursors, the angioblasts, to the position of the future vessels. To form the first axial vessels, angioblasts migrate towards the midline and coalesce underneath the notochord. Vascular endothelial growth factor has been proposed to serve as a chemoattractant for the angioblasts and to regulate this medial migration. Here we challenge this model and instead demonstrate that angioblasts rely on their intrinsic expression of Apelin receptors (Aplr, APJ) for their migration to the midline. We further show that during this angioblast migration Apelin receptor signaling is mainly triggered by the recently discovered ligand Elabela (Ela). As neither of the ligands Ela or Apelin (Apln) nor their receptors have previously been implicated in regulating angioblast migration, we hereby provide a novel mechanism for regulating vasculogenesis, with direct relevance to physiological and pathological angiogenesis. DOI: http://dx.doi.org/10.7554/eLife.06726.001

analyze whether Vegfa signaling, as previously proposed, is required for angioblast migration to the midline, we analyzed loss-of-function zebrafish embryos with deficiencies in the Vegfa signaling pathway resulting from genomic mutations as well as morpholino (MO) -mediated knockdown. To rule out developmental delays, we counted the somites of the embryos and fixed them for confocal analysis, when they had developed 16-17 somites (equivalent to 17 hpf). Surprisingly, neither Vegfa ligand depletion (vegfaa/vegfab MO) nor a null mutation in the gene encoding the VEGFR2 ortholog (kdrl) interfered with angioblast migration in zebrafish embryos ( Figure 1H,J), although Vegfa signaling dependent phenotypes, like failure to form the intersegmental vessels, could be observed after 24 hpf (Figure 1-figure supplement 1). Additionally, pharmacological inhibition of Vegfr2 signaling from 6 to 17 hpf did not impair angioblast migration to the midline (Figure 1-figure supplement 1). As activity of Sonic Hedgehog (Shh) in the midline has been shown to induce vegfaa expression , we repeated the published experiments and blocked this process by chemical inhibition of Shh signaling using cyclopamine. In line with our previous experiments, angioblast migration was not affected after Shh inhibition ( Figure 1-figure supplement 2). The sum of these data strongly indicates that Vegfa signaling is dispensable for angioblast specification or migration in zebrafish.
In order to identify the endogenous signal(s) guiding angioblast migration, we further analyzed the expression pattern of factors involved in cell motility and directed cell migration. As previously published (Zeng et al., 2007), we detected the expression of Apelin, a short secreted peptide encoded by the apln gene, by the NC (Figure 2-figure supplement 1C). At the same time, transcripts of its two paralogous receptors apelin receptor a (aplnra) and apelin receptor b (aplnrb) were present in angioblasts (Figure 2-figure supplement 1A,B; [Scott et al., 2007;Zeng et al., 2007]). Using MO-mediated gene knockdown, we observed that both aplnra and aplnrb are important for midline migration of angioblasts. While absence of each individual receptor reduced the efficiency of migration towards the midline, double depletion completely abolished this process ( Figure 2A, Video 2). We used TALEN or CRISPR/Cas9-mediated gene editing (Hwang et al., 2013;Jao et al., 2013) to introduce mutations in either the aplnra or the aplnrb gene (Figure 2-figure supplement 2). As our MO-based analysis already indicated an additive effect of both receptor genes, we analyzed the phenotypes of the offspring of aplnra +/− ;aplnrb +/− double heterozygous parents. The embryos were all individually scored for their phenotype regarding angioblast midline eLife digest The circulatory system enables blood to move around the body and deliver substances including nutrients and oxygen to the cells that need them. In the embryos of animals with a backbone, blood flows from the heart through the aorta into branching smaller vessels to the cells. The blood then gets collected by progressively bigger vessels and flows back to the heart via the cardinal vein. The cells that make up these blood vessels develop from cells called angioblasts-but first, during development these angioblasts must move to the place where the vessels will form.
A protein called Vascular endothelial growth factor (VEGF) had been suggested to help guide and align the angioblasts as the embryo develops. Now, Helker, Schuermann et al. have examined developing zebrafish embryos using new technologies. This revealed that VEGF is in fact not essential for the dorsal aorta and cardinal vein to develop. Instead, the angioblasts only move to the correct part of the embryo if they can produce the Apelin receptor protein, which forms part of a signaling pathway.
There are two hormones-called Apelin and Elabela-that can bind to and activate the Apelin receptor. Helker, Schuermann et al. show that Elabela alone is needed to guide the angioblasts to the right part of the embryo during blood vessel development. However, in embryos where there is not enough Elabela, the Apelin hormone can compensate for this deficiency and the first blood vessels will later develop correctly. Future research will address whether this signaling pathway not only guides angioblasts to establish a circulatory system, but also guides blood vessel growth. As blood vessel growth is very relevant to human disease, identifying the mechanisms that regulate it will have an impact on biomedical research. migration into four categories (normal, mild, strong, stronger; Figure 2B) and then subjected to genotyping. Homozygous mutant embryos phenocopied the MO-induced phenotypes, additional loss of one or two copies of the second receptor increased the severity of the phenotype ( Figure 2C). Additionally, we detected aplnrb transcripts together with the angioblast specification marker etv2 indicating that both genes are expressed very early during angioblast specification. However, when we analyzed aplnra/aplnrb double deficient embryos, no difference in etv2 expression was detectable by in situ hybridization indicating that Apln receptors only regulate angioblast migration, but not specification (Figure 2-figure supplement 1E).
Next, we analyzed the requirement for the ligand Apln by MO-mediated gene knockdown and, unexpectedly, observed no difference to control MO injected embryos (Figure 2A). To obtain undisputable genetic confirmation of this result, we again used gene editing to introduce mutations in the apln gene ( Figure 2-figure supplement 2, most likely resulting in functional null mutants). Consistent with the MO-mediated depletion, homozygous apln mu267 mutant embryos ( Figure 2A) showed no vasculogenesis defects, which indicated that endogenous Apln is not sufficient to regulate angioblast migration to the midline.
Previous studies have identified a requirement for Apln receptors in myocardial development in zebrafish (Scott et al., 2007;Zeng et al., 2007) and mice (Charo et al., 2009), which could not completely be phenocopied by Apln deficiency (Scott et al., 2007;Zeng et al., 2007;Charo et al., 2009). These results led to the proposal that Apln may not be the only ligand for Aplnrs. Recently, a novel peptide hormone named Elabela (Ela, also known as Apela or Toddler) was identified in zebrafish and shown to bind and activate Aplnrs (Chng et al., 2013;Pauli et al., 2014). By in situ hybridization we detected ela expression by the NC (Figure 2-figure supplement 1D, [Pauli et al., 2014]), consistent with a possible role in attracting angioblasts to the midline. We next analyzed angioblast migration in zebrafish embryos carrying a homozygous null mutation in the ela gene (ela br13 ). Ela deficiency led to a strong impairment of angioblast migration (Figure 2A), indicating that indeed Ela-Aplnr represent a novel bona fide ligand-receptor pair and therefore a novel signaling pathway regulating angioblast migration.
Given that the ela -/phenotype was not as severe as the depletion of both receptors and that apln expression increases later in the NC (while ela expression becomes progressively reduced, Figure 2-figure supplement 1C,D), we analyzed the phenotypes of the offspring of apln +/− ;ela +/− double heterozygous parents. The embryos were all individually scored for their phenotype regarding angioblast midline migration into four categories (normal, mild, strong, severe) and then subjected to genotyping. Interestingly a mild phenotype was observed, when either one ela or both apln alleles were mutant, a strong phenotype when ela was homozygously mutant with increasing severity (severe phenotype) upon loss of the apln alleles ( Figure 2D,E). Homozygous ela −/− ; apln −/− double mutants phenocopied apln receptor deficiency (compare in Figure 2A,B). The observed dose dependency is indicative, that in this context Ela and Apln might act as novel chemoattractants for angioblast migration to the midline, with Ela acting as the major endogenous attractant and Apln having a minor additive role. Previous analysis of zebrafish embryos lacking the NC showed, that signals from the midline guide angioblast migration (Fouquet et al., 1997;Sumoy et al., 1997). We could show that indeed angioblast migration as well as ela expression is perturbed in the zebrafish mutants lacking a NC (notochord homeobox, noto; previous name: floating head) or exhibiting defective NC precursor differentiation (T, brachyury homolog a, ta; previous name: no tail) (Figure 3) (Halpern et al., 1993;Odenthal et al., 1996). While angioblasts in noto tk241 mutant embryos failed to migrate to the midline, ta b160 angioblasts migrated towards the midline but failed to reach this structure ( Figure 3A). In line with these results, ela expression was strongly diminished in the ta b160 midline and almost absent in noto tk241 mutant embryos ( Figure 3B). We next wanted to test whether midline ela expression would be sufficient to restore angioblast migration in ta b160 mutants. For this purpose, plasmid DNA with a shh promoter fragment enabling ela expression in the floorplate was injected together with Tol2 transposase mRNA to obtain mosaic Ela overexpression in the midline. Indeed, Ela overexpression did restore angioblast migration to the midline in ta b160 embryos ( Figure 3C), demonstrating that midline Ela expression is not only necessary, but sufficient to regulate angioblast migration during vasculogenesis.
Next, we wanted to analyze the function of Ela-Aplnr signaling more mechanistically. Recently, it was published that Ela does not act as a chemoattractant, but instead acts as a motogen in regulating mesoderm migration during gastrulation (Pauli et al., 2014). In contrast, the aplnr deficient (aplra/b MO injected) angioblasts were still partially motile and did send out filopodia, but failed to migrate to the midline (Video 2). To test whether Ela does act as a chemoattractant or just enables motility of angioblasts, we overexpressed Ela at ectopic locations in noto or ta mutant embryos, as both of these lack endogenous ela and apln expression in the NC. We used a heatshock promoter to control timing of expression and labeled Ela overexpressing cells by coexpression of RFP through an internal ribosome entry site (IRES) element ( Figure 4). We measured the distance of control (Cherry) or Ela/ RFP expressing cells from angioblasts in a 40 μm radius of the red labeled cell and considered an  angioblast as attracted, if it was less than 5 μm away from it. While only 20% of the angioblast were found in the vicinity of control cells, more than 52% of the angioblasts were close to Ela overexpressing cells ( Figure 4D,G). Together with the observed dose dependency of Ela-Apln, the motility of Aplnr deficient embryos and the rescue of NC mutants by midline Ela expression, our data support the interpretation, that Ela acts as a chemoattractant for angioblasts during midline migration.
To prove that Ela-Aplnr signaling is directly required in angioblasts and rule out that the observed migration defects were caused by changes in other cell types, we performed transplantation experiments. Mini-ruby injected (red fluorescence) transgenic fli1a:EGFP positive (angioblasts, green) cells from control MO injected, aplnra/b MO injected or wild type (WT) donor embryos were transplanted into either WT or aplnr loss-of-function (apnlra/b MO injected) host embryos ( Figure 5A). Analysis of angioblast migration to the midline at 17 hpf revealed that in average 93.6% of control MO-injected transplanted angioblasts migrated to the midline in a WT host embryo ( Figure 5B,C; n = 39 GFP+ angioblasts, 7 embryos). In contrast, only 9.4% of aplnra/b MO injected angioblasts arrived at the midline, whereas the majority of these cells remained in more lateral positions ( Figure 5B,D; n = 62 GFP+ angioblasts, 5 embryos). Transplanted WT angioblasts were perfectly capable to migrate to the midline in an aplnr deficient environment (average of 81.9%, Figure 5B,E; n = 15 GFP+ angioblasts, 6 embryos), which shows that there is indeed a cell autonomous requirement for Aplnr signaling in angioblasts.
Based on the sum of the results presented above, we propose that Ela expression at the embryonic midline provides a chemoattractive signal for Aplnr-positive angioblasts. While Ela and Apln both signal to the Apln receptors, the initial ela expression at the midline appears stronger than apln, which peaked at later developmental time points (Figure 2-figure supplement 1C,D). Thus, ela and apln may have partially redundant functions, but are required at different stages of vascular morphogenesis. Surprisingly, while Ela-Aplnr signaling was strictly required for the guided migration of angioblasts, this process was not controlled by Vegfa-mediated signaling.
Our studies identified Ela-Aplnr signaling as a novel signaling pathway for angioblasts regulating vascular patterning in the developing vertebrate embryo. As therapeutic intervention with vascular growth is clinically highly relevant and, so far, predominantly focuses on the VEGF-A signaling cascade, the Ela-Aplnr signaling pathway may also represent a novel therapeutic target in human disease.

Zebrafish husbandry and strains
Zebrafish (Danio rerio) were maintained in a recirculating aquaculture system under standard laboratory conditions (Westerfield, 1993). Embryos were staged by hours post fertilization (hpf) at 28.5˚C (Kimmel et al., 1995), for 17 hpf embryos were incubated after gastrulation at 21˚C and staged on the following morning by counting somites (16 somites equal 17 hpf [Kimmel et al., 1995]).
The target sequences of apln exon 1 (5′-GAATGTGAAGATCTTGACGC-3′) and aplnrb exon 1 (5′-CTACATGCTCATCTTCATCC-3′) were cloned into the gRNA expression vector pDR274 as previously described (Hwang et al., 2013). The apln sgRNA or aplnrb sgRNA were transcribed using DraI-digested gRNA expression vector as template and the T7 mMessage mMachine kit (Ambion, Life Technologies, Germany). apln sgRNA or apnrb sgRNA and nls-zCas9-nls-encoding mRNA were co-injected into one-cell stage zebrafish embryos. Each embryo was injected with 2 nl of solution containing 12.5 ng/μl sgRNA and 300 ng/μl nls-zCas9-nls mRNA. A HgaI (New England BioLabs, Frankfurt, Germany) restriction site was used for genotyping of apln mu267 and a FokI (New England BioLabs) restriction site was used for genotyping of aplnrb mu270 . 6% of ctr MO injected (n = 39 GFP + angioblasts, 7 embryos), but only 9.4% of aplnra/b deficient (aplnra/b MO injected; n = 62 GFP+ angioblasts, 5 embryos) donor angioblasts migrated to the midline in WT host embryos. In contrast, 81.9% of WT donor angioblasts (n = 15 GFP+ angioblasts, 6 embryos) migrated to the midline in aplnra/b deficient host embryos. Error bars represent SEM, calculated using the standard deviation of percent of angioblasts in the midline per embryo. Statistical analysis showed significance using 2 way ANOVA and t-test, with p *** = 3.44577E-08 for aplnra/b MO in WT, and p = 0.232995135 (not significant, n.s.) for WT in aplnra/b MO. see also  Figure 5 continued on next page aplnra mutants were generated by TALEN mutagenesis. TALENs were assembled using the Golden Gate method (Cermak et al., 2011). For targeting the aplnra locus, a 5′ RVD (NI HD NI HD HD NH NI NH NI HD NI NG NI HD NH NI NG) and a 3′ RVD (HD NI HD NI HD HD HD NI NH NI NH NG HD NI NG NG NI NG NI) were generated. A BsrI (New England BioLabs) restriction site was used for genotyping of aplnra mu296 . mRNA was generated using the T3 mMessage mMachine Kit (Ambion) and injected using 100 pg of the TALEN mix.

Microinjections
Microinjections of mRNA or MOs were performed as previously described (Nasevicius and Ekker, 2000).

In vivo time-lapse analysis and confocal microscopy
Embryos were manually dechorionated and mounted in 0.3% agarose (with subsequent removal of agarose from the head/tail region). Medium and agarose were supplemented with 19.2 mg/l Tricaine and 30 mg/l Phenylthiourea. For time-lapse imaging, embryos were kept in a 28.5˚C heated chamber surrounding the microscope stage. All fluorescent images were acquired using an upright Leica Sp5 DM 6000 or a Zeiss LSM 780 Confocal microscope. Confocal stacks and confocal movies were assembled using Imaris Software (Bitplane, Switzerland).

Transplantation experiments
Donor embryos were injected with ctr MO or aplnra/b MO and labeled by the injection of 0.1 ng mini-Ruby (Tetramethylrhodamine, Invitrogen/Life Technologies). At sphere stage, cells were removed from donor embryos and transferred to wild type hosts using a glass capillary. Transplanted angioblasts were identified by transgenic EGFP expression together with mini-Ruby stain. By counting the number of EGFP positive donor angioblasts in the midline vs all EGFP positive donor angioblasts (100%), the percentage of donor angioblasts in the midline was determined for each individual embryo.

Figure 5 Continued
The following source data is available for figure 5: Source data 1. Statistical analysis and single values for the number of angioblasts and embryos analyzed. DOI: 10.7554/eLife.06726.016