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. 2020 Sep 21;9:e55589. doi: 10.7554/eLife.55589

Figure 1. Apelin signaling promotes endothelial sprouting.

Visualization of apelin and apelin receptor b expression using transgenic reporter lines. Confocal projection images of the trunk region of zebrafish embryos. (A) TgBAC(apln:EGFP) expression is detectable in growing ISVs at 30 and 54 hpf. Arrowheads point to strong apln expression in tip cells, while arrows point to weak apln expression in stalk cells. (B) TgBAC(aplnrb:aplnrb-EGFP) expression is detectable in sprouting ECs (arrowheads) at 26 hpf and is clearly present in the ISVs and DLAV at 54 hpf. (C) Inactivation of Apelin ligand and receptor genes impairs angiogenesis. Confocal projection images of the blood vasculature in the trunk region of Tg(fli1a:EGFP) embryos. apln -/-; apela -/- as well as aplnra -/-; aplnrb -/- embryos exhibit a reduction in vascular sprouting at 28 and 54 hpf. Arrowheads point to stalled ISVs. Scale bars: A’, 10 µm; A’’, B’, C’, C’’, 20 µm; B’’, 15 µm.

Figure 1.

Figure 1—figure supplement 1. Expression of apelin ligand and receptor genes by in situ hybridization.

Figure 1—figure supplement 1.

Expression of apelin ligand and receptor genes at 24 and 32 hpf. apln is expressed in ISVs (arrowheads). apela is not detected in blood vessels. aplnra is expressed in the posterior cardinal vein. aplnrb is expressed in the posterior cardinal vein and ISVs (arrowheads). Scale bars: 500 µm; insets, 100 µm.
Figure 1—figure supplement 2. Generation of the TgBAC(apln:EGFP) and Tg(aplnrb:aplnrb-TagRFP-sfGFP) reporter lines.

Figure 1—figure supplement 2.

BAC engineering was used to generate the apln:EGFP and aplnrb:aplnrb-TagRFP-sfGFP constructs. The clone RP71-2G21 was engineered to replace the ATG of apln with an EGFP cassette. The clone CH211-102K8 was engineered to replace the stop codon of aplnrb with a TagRFP-sfGFP cassette. To enhance the frequency of insertion into the genome, tol2 arms were added to the backbone of the BAC.
Figure 1—figure supplement 3. Quantification of angiogenic defects.

Figure 1—figure supplement 3.

Confocal projection images of the trunk region of Tg(fli1a:EGFP) embryos at 30 hpf. Analysis of the offspring of apln +/-; apela+/- (n = 85) and aplnra +/-; aplnrb +/- (n = 71) fish resulted in different phenotypic categories according to the extent of ISV development. Genotyping of single embryos revealed an additive effect for apln and apela ligands and aplnra and aplnrb receptors. Mild phenotypes correlated with loss of single ligand or receptor genes, while deficiency in apln resulted in stronger phenotypes than deficiency in apela. aplnrb deficiency resulted in stronger phenotypes than deficiency in aplnra. Deficiencies in both ligands or both receptors resulted in more severe phenotypes. Scale bar: 40 µm.