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.

Main text

In the vertebrate embryo, the formation of the large axial vessels, namely the dorsal aorta (DA) and the cardinal vein (CV), establishes a first circulatory loop and thereby the core of the developing cardiovascular system. Angioblasts are initially specified in the lateral plate mesoderm and migrate between the somites towards the midline, where they coalesce and assemble the DA and the CV underneath the notochord (NC) (Figure 1A,B–F, Video 1).

Figure 1

Angioblast migration to the midline is not regulated by Vegf.

(A–F) Schematic (A) and confocal (B–F) in vivo time-lapse imaging of angioblasts (green) and their migration in a Tg(fli1a:EGFP) embryo, injected with H2B-mCherry mRNA (purple, all nuclei), dorsal views at indicated time points. Arrows indicate the initial migration to the midline, and the coalescing into the dorsal aorta (DA). (G–J) Loss of Vegf-signaling components by vegfaa/vegfab double morpholino (MO) injection (H) or in vegf receptor 2 (kdrl) mutants (J) does not affect angioblast migration. Confocal projections of Tg(fli1a:EGFP) embryos in dorsal views at 17 hpf. For each condition, n > 15.

DOI: http://dx.doi.org/10.7554/eLife.06726.003

DOI: http://dx.doi.org/10.7554/eLife.06726.004

(A–J) Vegfa signaling was impaired using either ligand MOs (vegfaa/vegfab MO), chemical inhibition of Vegfr2 signaling (2.5 μM SLKB1002 [Zhang et al., 2011]) or mutations in the vegfr2 gene (kdrl). Confocal projections of Tg(fli1a:EGFP) embryos in dorsal views at 17 hpf (A, B, C, G, H, I), 24 hpf (F, J, K, L) and 28 hpf (D, E). At 17 hpf, angioblasts in control embryos as well as Vegfa signaling deficient embryos arrived at the midline (A, B, C, G, H, I). Control embryos showed sprouting of the intersegmental vessels (D, F, K) whereas Vegfa signaling deficient embryos lacked the intersegmental vessels (E, J, I).

DOI: http://dx.doi.org/10.7554/eLife.06726.005

(A, B) Embryos treated with 100 μM Cyclopamine (CyA) from 11 hpf to 18 hpf to block Sonic hedgehog signaling showed no defects in medial angioblast migration. Confocal projections of Tg(fli1a:EGFP) embryos in dorsal views at 18 hpf. c-d) vegfaa expression in the somites is abolished in embryos treated with 100 μM Cyclopamine (CyA) from 11 hpf to 18 hpf.

DOI: http://dx.doi.org/10.7554/eLife.06726.006

Video 1.

Angioblast migration between 12.5 hpf and 16 hpf observed by time-lapse imaging using Tg(fli1a:EGFP) to visualize angioblasts.

Angioblasts migrate to the midline in Wt embryos.

DOI: http://dx.doi.org/10.7554/eLife.06726.007

Time-lapse movie showing angioblast (green) migration to the midline analyzed using Tg(fli1a:EGFP) embryos injected with H2B-mCherry mRNA (purple, all nuclei).

It has been previously proposed that this process is regulated by Vascular endothelial growth factor A (VEGF-A) (Coultas et al., 2005; Verma et al., 2010; Gore et al., 2012), a master regulator of vascular growth in the embryonic and adult organism. In Xenopus embryos, transcripts for the vegfa gene are expressed in the midline and migrating angioblasts were guided towards sites of ectopic Vegfa expression (Cleaver and Krieg, 1998). Global inactivation of the genes encoding VEGF-A or the corresponding receptor VEGFR2 in mice led to strong vascular defects affecting the whole vascular network including the DA (Shalaby et al., 1995; Ferrara et al., 1996). Based on these data it was concluded that in vertebrates VEGF-A signaling regulates angioblast migration to the midline (Coultas et al., 2005; Verma et al., 2010; Gore et al., 2012). To get direct, mechanistic and dynamic insight into angioblast migration and formation of the great vessels, we decided to investigate these processes in zebrafish embryos.

As previously published (Nicoli et al., 2008), expression analysis of vegfaa, the gene for the main VEGF-A ortholog in zebrafish, showed the presence of transcripts in the somites between 12 and 15 hr post fertilization (hpf), that is the stage when angioblasts migrate to the midline (see Figure 1B–F). To 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 (Lawson et al., 2002), 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.

Figure 1—figure supplement 1.

Inhibition of Vegfa signaling resulted in normal angioblast migration to the midline, but impaired sprouting angiogenesis.

(A–J) Vegfa signaling was impaired using either ligand MOs (vegfaa/vegfab MO), chemical inhibition of Vegfr2 signaling (2.5 μM SLKB1002 [Zhang et al., 2011]) or mutations in the vegfr2 gene (kdrl). Confocal projections of Tg(fli1a:EGFP) embryos in dorsal views at 17 hpf (A, B, C, G, H, I), 24 hpf (F, J, K, L) and 28 hpf (D, E). At 17 hpf, angioblasts in control embryos as well as Vegfa signaling deficient embryos arrived at the midline (A, B, C, G, H, I). Control embryos showed sprouting of the intersegmental vessels (D, F, K) whereas Vegfa signaling deficient embryos lacked the intersegmental vessels (E, J, I).

DOI: http://dx.doi.org/10.7554/eLife.06726.005

Figure 1—figure supplement 2.

Abrogation of Shh signaling abolishes vegfaa expression, but does not influence angioblast migration.

(A, B) Embryos treated with 100 μM Cyclopamine (CyA) from 11 hpf to 18 hpf to block Sonic hedgehog signaling showed no defects in medial angioblast migration. Confocal projections of Tg(fli1a:EGFP) embryos in dorsal views at 18 hpf. c-d) vegfaa expression in the somites is abolished in embryos treated with 100 μM Cyclopamine (CyA) from 11 hpf to 18 hpf.

DOI: http://dx.doi.org/10.7554/eLife.06726.006

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 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).

Figure 2—figure supplement 1.

Expression of aplnra, aplnrb, apln and ela coincides with the formation of the DA and PCV.

(A–D) In situ hybridizations detecting expression of aplnra (A), aplnrb (B), apln (C) and ela (D) at the indicated time points (12 hpf, 18 hpf shown in dorsal views, 20 hpf in lateral views). aplnrs are expressed in the migrating angioblasts and continue to be expressed during formation of the DA and PCV. At 12 hpf, aplnrb is strongly expressed in the angioblasts (prior to migration, arrowheads) and within the lateral plate mesoderm and at the notochord somite boundary. aplnra expression is strong in the paraxial mesoderm (A). Both receptors are expressed in the angioblasts at 18 hpf (A, B, arrowheads) and in the developing axial vessels at 20 hpf (Arrowheads point to the DA, arrows to the PCV). Prior to angioblast migration apln and ela are both expressed by the notochord, with ela showing a much stronger expression (C, D). After angioblasts reached the midline (18 hpf, 20 hpf) only apln is still expressed by the notochord, whereas notochord derived ela expression is strongly reduced to absent. (E) Knockdown of aplnr does not influence the specification of angioblasts. In situ hybridizations detecting expression of the angioblast marker etv2 at 13.5 hpf (dorsal views).

DOI: http://dx.doi.org/10.7554/eLife.06726.010

Figure 2.

Ela/Apelin receptor - signaling guides angioblast migration to the midline.

(A) Angioblasts have migrated to the midline at 17 hpf in wild type, apln mutant or apln MO injected zebrafish embryos. MO-mediated knockdown of apelin receptor a or b partially inhibited midline migration, while simultaneous loss of both apelin receptor genes completely abolished midline migration of angioblasts. Likewise, embryos with homozygous mutations in the ligand ela display impaired migration of angioblasts. Arrows indicate aberrant positions. (B, C) Mutations in apln receptor genes impair angioblast migration. Analysis of the offspring of aplnra;aplnrb double heterozygous parents resulted in four phenotypic categories (normal, mild, strong, stronger). (B) Genotyping of individual embryos revealed an additive effect, while mild phenotypes were observed when one receptor gene was homozygously mutant, phenotypic strength increased with additional loss of functional receptor genes (c; Ra, aplnra; Rb, aplnrb). (D, E) Ela deficiency can partially be compensated by Apln. Analysis of the offspring of apln;ela double heterozygous parents resulted in four phenotypic categories (normal, mild, strong, severe). (D) Genotyping of individual embryos revealed a dose dependency, with increasing phenotypic strength correlating with additional loss of apln alleles in ela mutant embryos. (E) ela; apln double mutants phenocopied apln receptor deficiency. Angioblasts (green) were labeled by Tg(fli1a:EGFP) expression, scale bars represent 30 μm.

DOI: http://dx.doi.org/10.7554/eLife.06726.008

DOI: http://dx.doi.org/10.7554/eLife.06726.009

(A–D) In situ hybridizations detecting expression of aplnra (A), aplnrb (B), apln (C) and ela (D) at the indicated time points (12 hpf, 18 hpf shown in dorsal views, 20 hpf in lateral views). aplnrs are expressed in the migrating angioblasts and continue to be expressed during formation of the DA and PCV. At 12 hpf, aplnrb is strongly expressed in the angioblasts (prior to migration, arrowheads) and within the lateral plate mesoderm and at the notochord somite boundary. aplnra expression is strong in the paraxial mesoderm (A). Both receptors are expressed in the angioblasts at 18 hpf (A, B, arrowheads) and in the developing axial vessels at 20 hpf (Arrowheads point to the DA, arrows to the PCV). Prior to angioblast migration apln and ela are both expressed by the notochord, with ela showing a much stronger expression (C, D). After angioblasts reached the midline (18 hpf, 20 hpf) only apln is still expressed by the notochord, whereas notochord derived ela expression is strongly reduced to absent. (E) Knockdown of aplnr does not influence the specification of angioblasts. In situ hybridizations detecting expression of the angioblast marker etv2 at 13.5 hpf (dorsal views).

DOI: http://dx.doi.org/10.7554/eLife.06726.010

Targeted indel mutations in the aplnra gene were induced using TALEN mutagenesis, and in the aplnrb and apln genes were induced by engineered sgRNA:Cas9. The wild type nucleotide sequences are shown at the top with the TALEN (A) or CRISPR (B, C) target sites highlighted in yellow and the spacer (A) or PAM (B, C) sequences highlighted in red. (A) aplnra coding sequence and TALEN mutagenesis target site. An 8 base pair insertion, shown as lower case letters in blue leads to a premature stop and a shortening of the Aplnra protein to 13 AA. (B) aplnrb coding sequence and CRISPR mutagenesis target site. A 4 base pair insertion, shown as lower case letters in blue leads to a premature stop and a shortening of the Aplnrb protein to 65 AA. (C) apln coding sequence and CRISPR mutagenesis target site. An 1 baise pair loss / 9 base pair insertion, shown as lower case letters in blue causes a frameshift leading to a premature stop codon and a shortening of the Apln protein to 11 AA.

DOI: http://dx.doi.org/10.7554/eLife.06726.011

Video 2.

Angioblast migration between 12.5 hpf and 16 hpf observed by time-lapse imaging using Tg(fli1a:EGFP) to visualize angioblasts.

No migration to the midline, but minor movements and strong filopodia formation can be visualized in Aplnra/b double deficient embryos (aplnra/b MO).

DOI: http://dx.doi.org/10.7554/eLife.06726.012

Figure 2—figure supplement 2.

Generation of zebrafish aplnra, aplnrb and apln mutants.

Targeted indel mutations in the aplnra gene were induced using TALEN mutagenesis, and in the aplnrb and apln genes were induced by engineered sgRNA:Cas9. The wild type nucleotide sequences are shown at the top with the TALEN (A) or CRISPR (B, C) target sites highlighted in yellow and the spacer (A) or PAM (B, C) sequences highlighted in red. (A) aplnra coding sequence and TALEN mutagenesis target site. An 8 base pair insertion, shown as lower case letters in blue leads to a premature stop and a shortening of the Aplnra protein to 13 AA. (B) aplnrb coding sequence and CRISPR mutagenesis target site. A 4 base pair insertion, shown as lower case letters in blue leads to a premature stop and a shortening of the Aplnrb protein to 65 AA. (C) apln coding sequence and CRISPR mutagenesis target site. An 1 baise pair loss / 9 base pair insertion, shown as lower case letters in blue causes a frameshift leading to a premature stop codon and a shortening of the Apln protein to 11 AA.

DOI: http://dx.doi.org/10.7554/eLife.06726.011

Excel table showing the phenotype categories and the number of embryos for each genotype in these phenotypic categories (for the apln/ela double mutant analysis).

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 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). 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 mutant embryos failed to migrate to the midline, ta 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 midline and almost absent in noto mutant embryos (Figure 3B). We next wanted to test whether midline ela expression would be sufficient to restore angioblast migration in ta 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 embryos (Figure 3C), demonstrating that midline Ela expression is not only necessary, but sufficient to regulate angioblast migration during vasculogenesis.

Figure 3.

Notochord (NC) ela expression is sufficient to guide angioblasts to the midline.

(A) Angioblasts fail to migrate to the midline position in the NC mutants noto and ta. Confocal projections of Tg(fli1a:EGFP) embryos in dorsal view at 17 hpf (scale bars: 20 μm). (B) In situ hybridization in 17 hpf old zebrafish embryos showing ela expression from the NC and somites (white arrows). NC deficiency abolishes or strongly reduces ela expression in noto or ta mutant embryos. (C) Mosaic expression of Ela in the midline, achieved by injection of shh:ela DNA, led to rescue of angioblast migration in ta mutant embryos (n = 61 embryos, ctr injected n = 32 embryos). Significance was calculated by chi-square (χ2 = 19.65) equivalent to p < 0.001 (p = 9E-06). Scale bars: 30 μm.

DOI: http://dx.doi.org/10.7554/eLife.06726.013

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.

Figure 4.

Ela overexpressing cells attract angioblasts.

(A) F1 embryos from noto or ta parents were injected with 250 pg Tol2 mRNA as well as 10 pg of DNA constructs, in which the heatshock promotor was used to drive either control (ctr, Cherry) or ela expression. Individual Ela overexpressing cells were labeled by IRES mediated RFP expression. Expression was induced by two consecutive heatshocks (incubation at 39°C) at 12 hpf and 14 hpf for 1 hr each. (B–C′) Angioblast migration in noto mutant embryos injected with the ctr (B, B′) or the ela overexpression (C, C′) construct. (D) Quantification of Ela/RFP or ctr (Cherry) positive cells showed significantly more Ela overexpressing cells attracting angioblasts than ctr cells (n = 10 embryos for hs:ela, n = 20 embryos for hs:ctr ; p*** = 0.0001). (E–F′) Angioblast migration in ta mutant embryos injected with the ctr (E, E′) or the ela overexpression (F, F′) construct (G) Quantification of Ela/RFP or ctr (Cherry) positive cells showed significantly more Ela overexpressing cells attracting angioblasts than ctr cells (n = 8 embryos for hs:ela, n = 10 embryos for hs:ctr ; p** = 0.0089). Significance was calculated by chi-square test. Maximum intensity projections give an overview of the analyzed embryos. Single planes visualize the closeness of Ela or ctr (Cherry) expressing cells to angioblasts. White arrows point to Ela overexpressing cells with less than 5 μm distance to angioblasts. <5 μm distance between an Ela/ctr positive cell and an angioblast was counted as ‘attractant’; 5–40 μm distance was counted as ‘not attractant’. Scale bars represent 30 μm.

DOI: http://dx.doi.org/10.7554/eLife.06726.014

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.

Figure 5.

Cell autonomous requirement for Apelin receptor signaling in angioblasts.

(A) Experimental design: mini-ruby injected cells from ctr MO (C) aplnra/b MO (D) or WT (E) Tg(fli1a:EGFP) embryos were transplanted into WT or aplnra/b MO host embryos and scored for their migration to the midline. (B) Quantification: 93.6% 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—source data 1. (C–E) Confocal projections showing representative embryos of the transplantation experiments at 17 hpf. Arrows indicate transplanted angioblasts; asterisks label transplanted cells, which are not angioblasts; dashed lines indicate the midline.

DOI: http://dx.doi.org/10.7554/eLife.06726.015

DOI: http://dx.doi.org/10.7554/eLife.06726.016

Statistical analysis and single values for the number of angioblasts and embryos analyzed.

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.

Materials and methods

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]).

Zebrafish strains used were Tg(fli1a:EGFP) (Lawson and Weinstein, 2002), kdrl (Bussmann et al., 2010), ta (Halpern et al., 1993), noto (Odenthal et al., 1996) and ela (Chng et al., 2013).

Generation of apln, aplnra and aplnrb mutants

For apln and aplnrb Crispr mediated mutagenesis was used. Nls-zCas9-nls mRNA was synthesized as previously described (Jao et al., 2013).

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 aplnand a FokI (New England BioLabs) restriction site was used for genotyping of aplnrb.

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. 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).

mRNA was transcribed using SP6 polymerase (Sp6 mMessage mMachine kit [Ambion]). 100 pg H2B-cherry mRNA (Santoro et al., 2007), 2 ng aplnra MO (Scott et al., 2007), 0.5 ng aplnrb MO (Zeng et al., 2007), 2 ng apln MO (Scott et al., 2007) or 2 ng vegfaa/ 2.3 ng vegfab MO (Ober et al., 2004) were injected. Simultaneous knockdown of aplnra and aplnrb was done by coinjection of 2 ng aplnra MO and 0.5 ng aplnrb MO (referred to as aplnra/b MO). 4 ng of standard control MO (Gene tools, Philomath, Oregon) were injected as control.

Plasmid DNA was injected together with Tol2 mRNA (Kawakami et al., 2004). A 2.2 kb fragment of the shh promoter (Gordon et al., 2013) was used to drive either rfp or ela expression (shh:ctr or shh:ela). The heatshock promoter (hsp70l) was used to drive either Cherry (hs:ctr [Hesselson et al., 2009]) or ela-IRES-RFP (hs:ela).

Whole mount in situ hybridization

In situ hybridizations were performed as previously described (Helker et al., 2013) using the following probes: cdh5 (Larson et al., 2004), etv2 (Helker et al., 2013), vegfc (Hogan et al., 2009), vegfaa (Lawson et al., 2002).

apln, aplnra and aplnrb probe templates were amplified from cDNA of 19 somite stage old embryos using the following primers: apln-forward 5′-GAAAGGCCCAAGTCACAGAG-3′ and apln-reverse 5′-GAGTTCACTATCTGATGTCAAACCA-3′, aplnra-forward 5′-GAAAGGCCCAAGTCACAGAG-3′ and aplnra-reverse 5′-GAGTTCACTATCTGATGTCAAACCA-3′, aplnrb-forward 5′-GAAAGGCCCAAGTCACAGAG-3′ and aplnrb-reverse 5′-GAGTTCACTATCTGATGTCAAACCA-3′.

The T7 promotor was added to the 5′- end of the reverse primer in a second round of amplification (T7-apln-reverse 5′-GTAATACGACTCACTATAGGGAGTTCACTATCTGATGTCAAACCA-3′, (T7-aplnra-reverse 5′-GTAATACGACTCACTATAGGGAGTTCACTATCTGATGTCAAACCA-3′, (T7-aplnrb-reverse 5′-GTAATACGACTCACTATAGGGAGTTCACTATCTGATGTCAAACCA-3′).

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.

eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.

Thank you for sending your work entitled "The hormonal peptides Elabela and Apelin guide angioblasts to the midline during vasculogenesis" for consideration at eLife. Your article has been evaluated by Janet Rossant (Senior editor), Tanya Whitfield (Reviewing Editor), and two additional reviewers.

The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.

While the reviewers have all found the work interesting, all three have concerns over the extent that the data support your conclusions, including the challenges that you propose to existing literature. The full reviews are appended below, but a summary of the main points that must be addressed is:

1) Data should be supplied to demonstrate the efficacy of each of the morpholinos used, especially (in the case of the Vegf study) when the authors are proposing that knockdown has no effect on angioblast migration. The authors should also show whether any of the morpholinos cause systemic non-specific effects, such as developmental delay. If mutants are available, it would be helpful to repeat the analysis in mutants.

2) Additional, and quantitative, data are needed to support the assertion that Ela acts as an instructive chemoattractant in this context and not simply as a motogen. As two of the reviewers have pointed out, the dataset is currently too small to be able to reach this conclusion. Further data should be supplied to illustrate this point.

It is vitally important to provide strong support for the two points above, as these are the proposed advances in the understanding of angioblast migration over previously published literature.

3) The relative contributions of Apln and Ela to angioblast migration should be clarified, both in the Results section and in the Abstract and Impact Statement.

4) A reduction in Shh signalling should be demonstrated to support the cyclopamine data.

5) Take care to acknowledge the findings of previously published work e.g. Pauli et al., 2014, and previously published expression patterns, appropriately.

6) Improve quantitation of all experiments: provide cell counts to support image panels; state n numbers wherever % values are shown, and ensure that appropriate statistical tests have been applied.

7) Correct typos, formatting and gene nomenclature throughout.

Reviewer #1:

This is an interesting and well-written short report examining the role of the Apelin receptor signalling pathway in angioblast migration in the zebrafish embryo.

The study challenges the current VEGF model for angioblast migration to the midline, and also proposes that in this context, the ligand Elabela functions as a chemoattractant rather than a motogen, again challenging previous interpretations in the published literature. Although the authors present some interesting preliminary observations to support these challenges, the data need further analysis and quantitation to strengthen the authors' conclusions.

The paper relies on morpholino (MO) knockdown for a number of experiments. Given the current scepticism in the field surrounding the use of MOs, it is particularly important to demonstrate that the MO experiments are justified and rigorous. The authors give references to previously published work for each MO they use, but nevertheless it would be helpful to show data (e.g. antibody or RNA splicing data demonstrating specific knockdown), or give a statement, to comment on the specificity and efficacy of each MO.

Confidence in the MO data is especially important given that the main phenotype that is shown for many of the experiments (a failure of angioblasts to migrate to the midline) could also be explained by a generalised developmental delay. It is therefore important to demonstrate that the MOs do not cause any general developmental delay (e.g. by showing a brightfield image of the whole embryo, and providing quantitative information about, for example, somite number).

The challenge to the VEGF model, a major conclusion of the manuscript, is based on just two data panels (Figure 1C), with no accompanying quantitation. For the vegfaa MO experiment, notwithstanding the caveats mentioned above for MO use, there do appear to be a few green cells beyond the midline, but the picture is so heavily cropped that it is not possible to assess this properly. The authors should provide some quantitation, together with further information to show that the MO is indeed disrupting VEGF signalling, to strengthen their conclusions for this experiment. The authors state that vegfaa is the 'main ortholog' in zebrafish, but it would also be helpful to know whether there are any other vegf genes or isoforms expressed at the relevant time and place, and whether these are affected by vegfaa MO knockdown or not. The mutant VEGFR (kdrl) analysis is more convincing, but again, are there other genes or isoforms that might function redundantly with the kdrl receptor?

The second challenge to existing literature, that Elabela (in this context) acts a chemoattractant rather than a motogen, is also supported only weakly, being based on a single supplementary figure panel (Figure 3–figure supplement 1D) showing one (or possibly two) green angioblasts in an ectopic dorsal position near to the ectopic Elabela-expressing (red) cells. This needs some further examples and quantitiation to support the authors' conclusion.

Quantitation:

The % data shown in Figures 2C, 3C and 4B must be accompanied by corresponding n numbers, preferably shown directly on the graphs.

Figure 4B. A t-test would not be recommended for an experiment involving more than one experimental condition. This analysis should be repeated using ANOVA or an equivalent test. The graph should use a bar to show which columns are being compared (presumably each of the experimental situations with the control).

Reviewer #2:

Helker C. et al. demonstrate the essential role for Elabela (Ela)-Apelin (Apln) receptor (Aplnr) signaling in the initial assembly of angioblasts as a vascular cord in the midline. Unexpectedly, vegfa is not required as a chemoattractant for angioblast migration, while Elabela, a secretary peptide from the notochord, functions as an angioblast chemoattractant. The data has been convincingly demonstrated using several gene mutants (krdl-/-, elabr13-/-, apln mu267-/-, and noto) and their morphant.

The objectives written and the findings shown in the present manuscript are clear; however, there are several points should be addressed. There are several statements that are not supported by their results in the present study.

Major concerns:

1) Requirement of Apln for angioblast migration:

The authors claim that not only Ela but also Apln guides angioblasts to the midline in the Results section and in the Title. In Figure 2A, the data of apln clearly show that Apln is not required for angioblast migration. However, the authors point to the requirement of Apln using the embryos of the ela and apln. On the contrary, they state the impact of Ela (instead of Apln)-Aplnr signaling in the Impact Statement. This interpretation might puzzle the readers. Therefore, they need to re-interpret the data and re-write the manuscript including the Title.

2) Vegf or Vegfa:

The authors use "Vegf" and "Vegfa" in the Abstract and main text. This inconsistency should be corrected not to mislead the readers. Are other vegf members except Vegfa are thought to regulate angioblast migration in the previous papers? According to their results, the conclusion might be that Vegfa-Vegfr2 signaling is required for angioblast migration toward the midline. Therefore, they should rewrite the manuscript by keeping the consistency when they use the similar words.

Reviewer #3:

Helker and colleagues provide evidence for new roles of previously identified small peptides Apelin and Elabela in guiding angioblasts towards the embryonic midline during zebrafish embryogenesis. Previous studies implicated Vascular endothelial growth factor in convergence of angioblasts towards the embryonic midline, where they coalesce to build the first embryonic vessels. The authors first present evidence from zebrafish against the requirement for Vegf in angioblast migration towards the midline. Rather, they proposed, Apelin and Elabela, have partially redundant function to guide angioblasts to the midline by acting through the Apelin receptors. Apelin, Apelin GPCR Receptor axis has been previously implicated in cardiac progenitor cell convergence and cell fate specification, by Scott et al., and Zeng et al., in 2007. Elabela acting through Apelin receptors has been shown to be required for gastrulation cell movements and both heart and endoderm development by Chng et al., 2013 and Pauli et al., 2014. However, neither Apelin nor Elabela have been proposed to affect gastrulation cell movements acting as guidance molecules, with Elabela being considered a motogen. The authors employ morpholino oligonucleotides and mutations to show that Apelin and Elabela expressed in the notochord have partially redundant function in angioblast migration. Moreover, apelin receptors a and b expressed in angioblasts also have redundant function in this process. Therefore the proposed role of Apelin/Elabela/Apelin receptor signaling to guide angioblasts towards the embryonic midline would be a significant advance. However, the evidence in support of these conclusions are not compelling. There are also problems with the results and their interpretations. These problems should be addressed before the manuscript becomes suitable for publication.

The most novel conclusion of this work is that Elabela/Apelin/Apelin receptor axis has an instructive role in angioblast migration, by guiding them to the midline. The key line of evidence is that when cells ectopically expressing Ela were present in Ela mutant: "We found that individual Ela overexpressing cell were indeed capable of attracting angioblast migration to ectopic locations […]. our data therefore demonstrate, that Ela acts as a chemoattractant for angioblasts during midline migration".

The presented data is not compelling. The authors observed occasional overlap between angioblasts and these Ela expressing cells. An alternative interpretation is that Ela expressed in ectopic location increased motility of angioblasts and some of them wondered in ectopic locations.

The conclusion about Vegf signaling not being required in angioblast migration is based on the observation that "neither Vegf ligand depletion (vegfaa MO) nor a null mutation in the gene encoding the VEGFR2 ortholog (kdrl) interfered with angioblast migration in zebrafish embryos." However, there is no data showing the degree of loss-of-function for morpholino or for kdrl mutant.

Likewise, when the authors inhibit Shh signaling using cyclopamine, no quantitative data is presented documenting the degree of Shh signaling inhibition. Reduced expression of vegf is shown by in situ hybridization, but this is not a quantitative results. Such body of evidence is not sufficient to support the authors' conclusion regarding:

Does mutation in apelin gene affect elabela expression and vice versa?

Do mutations in kdrl affect phenotypes of apelin, elabela or apelin receptor single mutants?

The authors conclude that simultaneous knockdown of aplnra and b affects angioblast cell movement but not specification, based on morpholino interference. Whereas previously published morpholinos are used, appropriate experiments showing efficiency and specificity should be presented. Given that mutations for these receptors are available, they should be preferably employed.

Similar concerns are regarding apelin morpholino and apelin mutant experiments. Does apelin mutation lead to nonsense mediated degradation of apelin mRNA?

The authors conclude that angioblast migrate normally in apelin mutants, however, in Figure 2 it appears that ca. 30% of apelin mutants showed mild angioblast migration phenotype. This along with a strong enhancement of the phenotype of elabela mutant indicates that apelin is also required for angioblast migration.

In the Results the authors write that cardiomyocyte deficiency observed in aplnr deficient embryos could not be phenocopied by loss of apelin function. This is not correct; Scott et al., and Zeng et al. reported reduction of cardiomyocyte formation in apelin morphant embryos.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "The hormonal peptide Elabela guides angioblasts to the midline during vasculogenesis" for further consideration at eLife. Your revised article has been favorably evaluated by Janet Rossant (Senior editor) and Tanya Whitfield (Reviewing Editor). The manuscript is much improved and most of the reviewers' recommendations have been addressed. Mutant phenotypes have been used to back up the morpholino data, and most of the requested control data have been provided to demonstrate the efficacy of morpholinos and compounds used. Description of relative roles for Apln and Ela in the migration of angioblasts is now much clearer. However, there are still some minor issues concerning the description of the experimental design and quantitation of the data that should be addressed before publication, as outlined below:

1) The lack of a generalised developmental delay (control for somite number) should be stated explicitly in the text as well as in the Materials and methods, so that it can be ruled out as a possible explanation for a delay or failure of angioblasts to migrate to the midline in MO-injected or mutant embryos.

2) The new data supporting the assertion that Ela acts as a chemoattractant and not a motogen are still based on rather low numbers, and indeed the new data are still presented only as a supplementary figure (Figure 3–figure supplement 1). In the text description (Results, seventh paragraph), n numbers (cells and embryos) must be quoted in the text to support the percentage values, in addition to the information provided in the figure.

The chemoattractive effect of Ela appears to be an important conclusion of the manuscript, but is only based on this supplementary figure. If the data are not strong enough to be shown as a main figure, the conclusion should also be toned down: rather than stating that the data 'demonstrate' that Ela acts as a chemoattractant, wording such as 'the data support the interpretation that Ela is acting as a chemoattractant in this context, but further experiments and higher n numbers would be required to test this more rigorously' should be used. In addition, use of the word 'chemoattractant' should be treated with similar caution throughout the manuscript.

3) Figure 4 and description and Results. Again, n numbers should be stated along with the percentages here, in the text as well as on the figure.

4) What do the error bars represent in Figure 4B?

1) Data should be supplied to demonstrate the efficacy of each of the morpholinos used, especially (in the case of the Vegf study) when the authors are proposing that knockdown has no effect on angioblast migration. The authors should also show whether any of the morpholinos cause systemic non-specific effects, such as developmental delay. If mutants are available, it would be helpful to repeat the analysis in mutants.

We have added mutant analysis, to complement morpholino data, wherever possible.

We generated new mutants for both Apln receptors as well as double mutants and have provided better controls as well as additional pharmacological studies for the analysis of the role of Vegf signaling.

Figure 1 has been changed and is now showing vegfaa/vegfab double MO injections, so that they cannot compensate for each other.

In addition a new supplemental figure, Figure1–figure supplement 1, has been generated (the cyclopamine data are now Figure1–figure supplement 2). We are now showing the later phenotypes of Vegf-signaling deficiency, to demonstrate efficiency of the used reagents. Also inhibition of Vegf-signaling by chemical treatment has been added as an alternative to MO generated data.

Figure 2: new panels have been added and show the phenotype-genotype correlation of single and double apelin receptor mutant embryos.

Figure 2–figure supplement 2: generation of the aplnr mutants has been added.

Any potential effect of developmental delay has been avoided by careful staging of the embryos (i.e. counting of somites to ensure the same developmental stage: now included in the method description).

2) Additional, and quantitative, data are needed to support the assertion that Ela acts as an instructive chemoattractant in this context and not simply as a motogen. As two of the reviewers have pointed out, the dataset is currently too small to be able to reach this conclusion. Further data should be supplied to illustrate this point.

We have added a new series of experiments, ectopically overexpressing Ela or Cherry in noto or ta mutants (to reduce endogenous Ela and Apln in the midline), in which the distance of angioblasts to Ela overexpressing cells (compared to Cherry overexpressing cells) was quantified.

Our data strongly support the role of Ela as a chemoattractant in the context of angioblast migration.

To address this point, a new Figure 3–figure supplement 1 has been added.

It is vitally important to provide strong support for the two points above, as these are the proposed advances in the understanding of angioblast migration over previously published literature.

3) The relative contributions of Apln and Ela to angioblast migration should be clarified, both in the Results section and in the Abstract and Impact Statement.

We have changed the manuscript text accordingly.

4) A reduction in Shh signalling should be demonstrated to support the cyclopamine data.

Block of hedgehog signaling using cyclopamine was originally published in chicken embryos (Incardona JP et al. Development. 1998 Sep;125(18):3553-62).

In zebrafish cyclopamine treatment has been used in many more than 70 publications, most notably it was shown that:

a) 200µM treatment fully phenocopied the shh mutant sonic you (Neumann CJ et al, Development. 1999 Nov;126(21):4817-26).

b) 100µM completely eliminated Hh activity (Chen W et al., Development. 2001 Jun;128(12):2385-96.

c) 25µM cyclopamine completely abolishes Vegf expression in the somites (as also seen in syu and yot mutants) (Lawson ND et al Dev Cell. 2002 Jul;3(1):127-36).

As this analysis of cyclopamine effects has therefore been done extensively and we have used 100µM (far more than N. Lawson, who demonstrated the effect on Vegf expression), we have not added in situs on other downstream markers. We have changed the wording, to state the previously published result more clearly.

However, we have added an alternative experiment showing the requirement for Vegf signaling by treatment with a Vegf receptor 2 inhibitor, hoping that this will be provide a meaningful alternative (see Figure 1–figure supplement 1).

5) Take care to acknowledge the findings of previously published work e.g. , and previously published expression patterns, appropriately.

We have revised the manuscript and added the appropriate references accordingly,

6) Improve quantitation of all experiments: provide cell counts to support image panels; state n numbers wherever % values are shown, and ensure that appropriate statistical tests have been applied.

We have added the n-numbers (cells and embryos) for all quantifications, provided source files, and added statistical analysis as requested.

7) Correct typos, formatting and gene nomenclature throughout.

We have edited the manuscript carefully, with special attention to these issues. It sometimes remains non-intuitive for the reader, that the nomenclature rules are not the same for mouse and zebrafish, so we have chosen zebrafish nomenclature, unless specifically referring to mouse data. As an example: "VEGF" would be the correct mouse specific spelling (see http://www.informatics.jax.org/mgihome/nomen/gene.shtml), "Vegf" would be correct for zebrafish (see ZFIN at https://wiki.zfin.org/display/general/ZFIN+Zebrafish+Nomenclature+Guidelines).

Reviewer #1:

The study challenges the current VEGF model for angioblast migration to the midline, and also proposes that in this context, the ligand Elabela functions as a chemoattractant rather than a motogen, again challenging previous interpretations in the published literature. Although the authors present some interesting preliminary observations to support these challenges, the data need further analysis and quantitation to strengthen the authors' conclusions.

The paper relies on morpholino (MO) knockdown for a number of experiments. Given the current scepticism in the field surrounding the use of MOs, it is particularly important to demonstrate that the MO experiments are justified and rigorous. The authors give references to previously published work for each MO they use, but nevertheless it would be helpful to show data (e.g. antibody or RNA splicing data demonstrating specific knockdown), or give a statement, to comment on the specificity and efficacy of each MO.

As discussed above, we have now provided mutant data to complement MO data, wherever possible, for the Vegf studies we have used chemical inhibition of Vegfr2 as an alternative.

Confidence in the MO data is especially important given that the main phenotype that is shown for many of the experiments (a failure of angioblasts to migrate to the midline) could also be explained by a generalised developmental delay. It is therefore important to demonstrate that the MOs do not cause any general developmental delay (e.g. by showing a brightfield image of the whole embryo, and providing quantitative information about, for example, somite number).

We had already staged all embryos according to somite numbers, but failed to state this in the manuscript, we have changed the Methods section accordingly.

The challenge to the VEGF model, a major conclusion of the manuscript, is based on just two data panels (), with no accompanying quantitation. For the vegfaa MO experiment, notwithstanding the caveats mentioned above for MO use, there do appear to be a few green cells beyond the midline, but the picture is so heavily cropped that it is not possible to assess this properly. The authors should provide some quantitation, together with further information to show that the MO is indeed disrupting VEGF signalling, to strengthen their conclusions for this experiment. The authors state that vegfaa is the 'main ortholog' in zebrafish, but it would also be helpful to know whether there are any other vegf genes or isoforms expressed at the relevant time and place, and whether these are affected by vegfaa MO knockdown or not. The mutant VEGFR (kdrl) analysis is more convincing, but again, are there other genes or isoforms that might function redundantly with the kdrl receptor?

vegfaa MO experiments have been replaced by combined knockdown of vegfaa and vegfab. In addition we show as a supplemental figure an analysis of all Vegf-signaling deficient embryos at a later developmental time point, where they all display the previously described defects in Vegf-dependent sprouting angiogenesis.

The second challenge to existing literature, that Elabela (in this context) acts a chemoattractant rather than a motogen, is also supported only weakly, being based on a single supplementary figure panel (Figure 3–figure supplement 1D) showing one (or possibly two) green angioblasts in an ectopic dorsal position near to the ectopic Elabela-expressing (red) cells. This needs some further examples and quantitiation to support the authors' conclusion.

As described above a new set of experiments with full quantification has been added as Figure 3–figure supplement 1.

Quantitation:

The % data shown in must be accompanied by corresponding n numbers, preferably shown directly on the graphs.

We have added the n numbers in each graph.

. A t-test would not be recommended for an experiment involving more than one experimental condition. This analysis should be repeated using ANOVA or an equivalent test. The graph should use a bar to show which columns are being compared (presumably each of the experimental situations with the control).

We have done an ANOVA analysis as well as the t-test and added the bars.

Reviewer #2:

Major concerns:

1) Requirement of Apln for angioblast migration:

The authors claim that not only Ela but also Apln guides angioblasts to the midline in the Results section and in the Title. In , the data of aplnmu267-/- clearly show that Apln is not required for angioblast migration. However, the authors point to the requirement of Apln using the embryos of the ela-/- and apln-/-. On the contrary, they state the impact of Ela (instead of Apln)-Aplnr signaling in the Impact Statement. This interpretation might puzzle the readers. Therefore, they need to re-interpret the data and re-write the manuscript including the Title.

We have clarified the inconsistencies, and re-written the manuscript accordingly.

2) Vegf or Vegfa:

The authors use "Vegf" and "Vegfa" in the Abstract and main text. This inconsistency should be corrected not to mislead the readers. Are other vegf members except Vegfa are thought to regulate angioblast migration in the previous papers? According to their results, the conclusion might be that Vegfa-Vegfr2 signaling is required for angioblast migration toward the midline. Therefore, they should rewrite the manuscript by keeping the consistency when they use the similar words.

We now always specify Vegfa, and have re-written the manuscript accordingly.

Reviewer #3:

Helker and colleagues provide evidence for new roles of previously identified small peptides Apelin and Elabela in guiding angioblasts towards the embryonic midline during zebrafish embryogenesis. Previous studies implicated Vascular endothelial growth factor in convergence of angioblasts towards the embryonic midline, where they coalesce to build the first embryonic vessels. The authors first present evidence from zebrafish against the requirement for Vegf in angioblast migration towards the midline. Rather, they proposed, Apelin and Elabela, have partially redundant function to guide angioblasts to the midline by acting through the Apelin receptors. Apelin, Apelin GPCR Receptor axis has been previously implicated in cardiac progenitor cell convergence and cell fate specification, by Scott et al., and Zeng et al., in 2007. Elabela acting through Apelin receptors has been shown to be required for gastrulation cell movements and both heart and endoderm development by and . However, neither Apelin nor Elabela have been proposed to affect gastrulation cell movements acting as guidance molecules, with Elabela being considered a motogen. The authors employ morpholino oligonucleotides and mutations to show that Apelin and Elabela expressed in the notochord have partially redundant function in angioblast migration. Moreover, apelin receptors a and b expressed in angioblasts also have redundant function in this process. Therefore the proposed role of Apelin/Elabela/Apelin receptor signaling to guide angioblasts towards the embryonic midline would be a significant advance. However, the evidence in support of these conclusions are not compelling. There are also problems with the results and their interpretations. These problems should be addressed before the manuscript becomes suitable for publication.

The most novel conclusion of this work is that Elabela/Apelin/Apelin receptor axis has an instructive role in angioblast migration, by guiding them to the midline. The key line of evidence is that when cells ectopically expressing Ela were present in Ela mutant: "We found that individual Ela overexpressing cell were indeed capable of attracting angioblast migration to ectopic locations" […] our data therefore demonstrate, that Ela acts as a chemoattractant for angioblasts during midline migration".

The presented data is not compelling. The authors observed occasional overlap between angioblasts and these Ela expressing cells. An alternative interpretation is that Ela expressed in ectopic location increased motility of angioblasts and some of them wondered in ectopic locations.

As described above, we have now added a new figure, in which distances between angioblast and control or ela expressing cells were measured. This analysis now shows a clear attractant role of Ela. By using noto or ta mutants, we have abolished ela and apln expression in the midline and could therefore score all angioblasts and not only those at very ectopic locations.

The conclusion about Vegf signaling not being required in angioblast migration is based on the observation that "neither Vegf ligand depletion (vegfaa MO) nor a null mutation in the gene encoding the VEGFR2 ortholog (kdrl) interfered with angioblast migration in zebrafish embryos." However, there is no data showing the degree of loss-of-function for morpholino or for kdrl mutant.

As described above this has been added, in Figure 1–figure supplement 1.

Likewise, when the authors inhibit Shh signaling using cyclopamine, no quantitative data is presented documenting the degree of Shh signaling inhibition. Reduced expression of vegf is shown by in situ hybridization, but this is not a quantitative results. Such body of evidence is not sufficient to support the authors' conclusion regarding:

The role of Shh (and cyclopamine treatment) on regulating Vegfaa has been published previously (with all controls). We regret that we have not expressed that well enough in the previous manuscript and have changed the wording. Additional experiments (chemical inhibtion of Vegfr2) have been added.

Does mutation in apelin gene affect elabela expression and vice versa?

Do mutations in kdrl affect phenotypes of apelin, elabela or apelin receptor single mutants?

The authors conclude that simultaneous knockdown of aplnra and b affects angioblast cell movement but not specification, based on morpholino interference. Whereas previously published morpholinos are used, appropriate experiments showing efficiency and specificity should be presented. Given that mutations for these receptors are available, they should be preferably employed.

We have by now generated mutant in the aplnr genes and were now able to analyze also double deficient embryos. Our mutant analysis confirms our morpholino generated data.

Similar concerns are regarding apelin morpholino and apelin mutant experiments. Does apelin mutation lead to nonsense mediated degradation of apelin mRNA?

The authors conclude that angioblast migrate normally in apelin mutants, however, in it appears that ca. 30% of apelin mutants showed mild angioblast migration phenotype. This along with a strong enhancement of the phenotype of elabela mutant indicates that apelin is also required for angioblast migration.

In the Results the authors write that cardiomyocyte deficiency observed in aplnr deficient embryos could not be phenocopied by loss of apelin function. This is not correct; Scott et al., and Zeng et al. reported reduction of cardiomyocyte formation in apelin morphant embryos.

Scott et al claim: "Midline expression of the Agtrl1b ligand Apln suggests that it may provide a signal for migration of the myocardial progenitors. However, injection of three independent apln MOs (designed against the 5'UTR and the splice donor site in intron 1) caused severe defects in body axis elongation and morphogenesis (Figure S3D), but did not recapitulate the grn myocardial phenotype, as significant myocardium was formed in the morphants (Figure S3E). The MOs appear to be functional, as coinjection of the 5'UTR-targeted MO was sufficient to reverse the inhibition of cardiomyogenesis caused by apln RNA levels far exceeding those normally found in vivo (Figure S3F). While exogenous apln can therefore recapitulate the grn phenotype, and Apln can act as a ligand to stimulate Agtrl1b activity, an apln mutant will be required to definitively test whether Apln is the in vivo signal for Agtrl1b during myocardial progenitor development.”

Zeng et al. are not as direct in their statements, but they claim: "However, the amount of cell death did not correlate with the severity of heart loss: we observed a level of cell death in agtrl1b morphants (where heart was strongly reduced) that was comparable to that in apelin morphants (with much milder reduction of the heart field, Figure S4 and data not shown).”

However, we have changed our wording to a less explicit: “Previous studies have identified a requirement for Apln receptors in myocard 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 (Charo et al., 2009; Scott et al., 2007; Zeng et al., 2007).”

[Editors' note: further revisions were requested prior to acceptance, as described below.]

1) The lack of a generalised developmental delay (control for somite number) should be stated explicitly in the text as well as in the Materials and methods, so that it can be ruled out as a possible explanation for a delay or failure of angioblasts to migrate to the midline in MO-injected or mutant embryos.

We have changed the text accordingly and pointed this out.

2) The new data supporting the assertion that Ela acts as a chemoattractant and not a motogen are still based on rather low numbers, and indeed the new data are still presented only as a supplementary figure (Figure 3–figure supplement 1). In the text description (Results, seventh paragraph), n numbers (cells and embryos) must be quoted in the text to support the percentage values, in addition to the information provided in the figure.

The chemoattractive effect of Ela appears to be an important conclusion of the manuscript, but is only based on this supplementary figure. If the data are not strong enough to be shown as a main figure, the conclusion should also be toned down: rather than stating that the data 'demonstrate' that Ela acts as a chemoattractant, wording such as 'the data support the interpretation that Ela is acting as a chemoattractant in this context, but further experiments and higher n numbers would be required to test this more rigorously' should be used. In addition, use of the word 'chemoattractant' should be treated with similar caution throughout the manuscript.

We had discussed already previously, whether we should include this figure into the main manuscript figures and have now added it as Figure 4. Figure 4 then has been relabeled to become Figure 5. We have also toned the wording down a little bit (at the position indicated and in the final summary). However, we believe our data not to be based on low numbers as we repeated the same experiment in the 2 notochord mutants and thereby generated separate data sets, each of them alone was statistically significant, so theoretically either of them would have already been sufficient.

3) and description and Results. Again, n numbers should be stated along with the percentages here, in the text as well as on the figure.

We have changed the manuscript in the Results section accordingly. They were already indicated in the figure and stated in the figure legend in b.

4) What do the error bars represent in ?

Now Figure 5B. They actually represent the SEM, however we did not phrase this clearly. We have changed the manuscript accordingly and added the excel sheet used for calculation as a source file. We have referred to these source data in the figure legend of Figure 5.

As an example: transplants of ctr. MO in Wt was analyzed in 7 embryos (with totally 39 cells transplanted), of these in 5 embryos 100% of the transplanted cells migrated to the midline, in 1 embryo 75%, and in one embryo 80%, resulting in an average percentage of 93.57%.

Alternatively, it would also have been possible to calculate total numbers (i.e. 2 cells out of 39 cells did not migrate to the midline), but we wanted to reflect that there was not much variation between the individual embryos analyzed.