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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). |
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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. |
