Skip to main content
. 2015 May 27;4:e06726. doi: 10.7554/eLife.06726

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, aplnmu267 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)y1 expression, scale bars represent 30 μm.

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

Figure 2—source data 1. 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).
DOI: http://dx.doi.org/10.7554/eLife.06726.009
elife-06726-fig2-data1.xlsx (50.5KB, xlsx)
DOI: 10.7554/eLife.06726.009

Figure 2.

Figure 2—figure supplement 1. Expression of aplnra, aplnrb, apln and ela coincides with the formation of the DA and PCV.

Figure 2—figure supplement 1.

(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—figure supplement 2. Generation of zebrafish aplnra, aplnrb and apln mutants.

Figure 2—figure supplement 2.

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