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Dev Biol
2006 Aug 01;2961:177-89. doi: 10.1016/j.ydbio.2006.04.452.
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Apelin, the ligand for the endothelial G-protein-coupled receptor, APJ, is a potent angiogenic factor required for normal vascular development of the frog embryo.
Cox CM
,
D'Agostino SL
,
Miller MK
,
Heimark RL
,
Krieg PA
.
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The peptide growth factor apelin is the high affinity ligand for the G-protein-coupled receptor APJ. During embryonic development of mouse and frog, APJ receptor is expressed at high levels in endothelial precursor cells and in nascent vascular structures. Characterization of Xenopus apelin shows that the sequence of the bioactive region of the peptide is perfectly conserved between frogs and mammals. Embryonic expression studies indicate that apelin is expressed in, or immediately adjacent to, a subset of the developing vascular structures, particularly the intersegmental vessels. Experimental inhibition of either apelin or APJ expression, using antisense morpholino oligos, results in elimination or disruption of intersegmental vessels in a majority of embryos. In gain of function experiments, apelin peptide is a potent angiogenic factor when tested using two in vivo angiogenesis assays, the frog embryo and the chicken chorioallantoic membrane. Furthermore, studies using the mouse brain microvascular cell line bEnd.3 show that apelin acts as a mitogenic, chemotactic and anti-apoptotic agent for endothelial cells in culture. Finally, we show that, similar to a number of other angiogenic factors, expression of the apelin gene is increased under conditions of hypoxia. Taken together, these studies indicate that apelin is required for normal vascular development in the frog embryo and has properties consistent with a role during normal and pathological angiogenesis.
Fig. 2. In situ hybridization analysis of APJ and apelin expression during Xenopus development. (A) In the Xenopus embryo at the early tailbud stage (stage 32) APJ
expression marks developing blood vessels throughout the embryo. The aortic arch region (aa) and the posterior cardinal vein (pcv), ventral vascular plexus (plex) and
branching intersegmental vessels (isv) are indicated. The region of expression in the tailbud does not appear to be associated with vascular tissue. (B) At stage 32,
apelin expression is detected in the anterior region of the posterior cardinal vein, the extending intersegmental vessels and the developing blood vessels surrounding the
eye. Apelin expression is also detected in the ventral neural tube (nt). (C) At stage 35, APJ transcripts are detected throughout the developing vasculature, including the
assembling vascular plexus in ventrolateral regions of the embryo and distinctly in the intersegmental vessels. (D) At stage 35, apelin expression is visible in the
branching intersegmental vessels but expression in the vessels surrounding the eye is now absent. No apelin transcripts are detected in the plexus region. (E) Transverse
section through anteriortrunk region of stage 32 embryo stained for apelin transcripts. Note prominent expression in the posterior cardinal veins. (F) Transverse section
through trunk of stage 34 embryo stained for apelin. In addition to posterior cardinal veins, expression is visible in the hypochord (hc) and in paired regions within the
ventral neural tube (nt). (G) Longitudinal section through ventral neural tube of stage 34 embryo stained for apelin. Anterior is to the left. Note that expression is not
continuous but appears to mark paired motor neurons (Saha et al., 1997) (H).
Fig. 3. Apelin is an angiogenic factor in the frog embryo. Porous beads treated with apelin, control peptide or VEGF, or cells expressing apelin protein, were implanted
into frog embryos at stages 246, and vascular structure was assayed by in situ hybridization using the vascular-specific erg marker at stage 34. (A) Whole frog
embryo showing location of apelin-soaked bead (arrow) and outgrowth of vascular tissue towards bead. (B, C) Enlarged views of vascular growth in the vicinity of the
apelin bead. (D, E) Ectopic vascular cells in the vicinity of cell implants expressing apelin. Arrows indicate the position of the cell implants. (F) A bead soaked with
mutated apelin peptide produces no ectopic marker expression. (G) A bead treated with VEGF(165) also produces vascular growth in the Xenopus embryo. (H) In situ
hybridization analysis for VEGF transcripts shows that VEGF expression is not detected in the vicinity of the apelin bead 24 h after implantation but is detected within
the somites as previously reported (Cleaver et al., 1997).
Fig. 4. Antisense morpholino inhibition of apelin/APJ signaling in the frog embryo leads to vascular patterning defects. (A) Control experiments were carried out as
previously described (Small et al., 2005) to demonstrate effective inhibition of translation. Briefly, fusion transcripts contained 5′ UTR sequences of the target mRNAs
upstream of the EGFP coding region. Fluorescence was visualized under UVillumination. (B) Embryos were injected with antisense morpholino into one cell of the
two-cell embryo. The uninjected side of the embryo serves as a stage-matched control. In all cases, structure of vascular tissue was assayed by in situ hybridization
using probe for erg transcripts. (B, C) Control and injected side respectively of an embryo treated with apelin antisense morpholino (apelin MO1). Note reduction in
intersegmental vessels on the MO-treated side (dashed arrows), while the majority of vascular structures appear to be unaffected. (D, E) Control and treated side
respectively of an embryo injected with a second apelin antisense MO (apelin MO2), again showing disruption of intersegmental vessels in the injected side. (F, G)
Control and treated side respectively of an embryo injected with APJ antisense MO. Once again, growth of intersegmental vessels is disrupted on the injected side
(dashed arrows). (H) Coinjection of APJ mRNA partially rescues intersomitic defects generated using APJ MO1. Rescue with both 200 and 400 pg of APJ mRNA is
statistically significant using the Chi-squared test.
Fig. 5. Apelin is an angiogenic factor in the chicken chorioallantoic membrane
(CAM) assay. (A) Photograph of CAM treated with buffer only. (B) Photograph
of representative CAM treated with VEGF and (C) representative CAM treated
with apelin. (D) Angiogenic activity was quantitated by counting the number of
vascular branch points resulting after each treatment. The bar indicates the
average of 3 different experiments, each counting at least 7 treated CAMs. The
standard error is indicated. Both VEGF and apelin stimulate approximately 2.5-
fold increase in angiogenic growth in the CAM assay.
At stage 35, APJ transcripts are detected throughout the developing vasculature, including the assembling vascular plexus in ventrolateral regions of the embryo and distinctly in the intersegmental vessels.
At stage 35, apelin expression is visible in the branching intersegmental vessels but expression in the vessels surrounding the eye is now absent. No apelin transcripts are detected in the plexus region.
In the Xenopus embryo at the early tailbud stage (NF stage 32) aplnr
(apelin receptor) expression marks developing blood vessels throughout the embryo. The aortic arch region (aa) and the posterior cardinal vein (pcv), ventral vascular plexus (plex) and branching intersegmental vessels (isv) are indicated. The region of expression in the tailbud and tail tip does not appear to be associated with vascular tissue.
In the Xenopus embryo at the early tailbud stage (stage 32) APJ expression marks developing blood vessels throughout the embryo. The aortic arch region (aa) and the posterior cardinal vein (pcv), ventral vascular plexus (plex) and branching intersegmental vessels (isv) are indicated. The region of expression in the tailbud does not appear to be associated with vascular tissue.
