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Fig. 1. Apelin expression precedes angiogenic growth of intersomitic vessels. (A) Apelin-13 is conserved from Xenopus to man. The amino acid sequences were aligned using ClustalW. Invariant residues are shaded black. The conserved, mature apelin-13 peptide is underlined. The predicted sites for signal peptide cleavage (arrow) and proprotein processing (arrowheads) generating apelin-36, apelin-17 and apelin-13 are indicated. (B–S) The expression patterns of apelin and APJ during Xenopus embryogenesis (B–O) and in the mouse embryo (P–S) were determined by whole mount in situ hybridization. Sections of the trunk (N, O) were cut horizontally at the level of the somites. (B) APJ is detected in all blood vessels including the PCV (arrow), the ISVs (arrowheads) and the VVN (asterisk). (C) APJ is present in the PCV (arrow). (D) Various ISVs (arrowheads) expressing APJ can be detected. (E) APJ levels remain high in all vessels including the ISVs (arrowhead), lateral capillaries (open arrowheads) and the PCV (arrow). (F) Apelin transcripts are found in areas of angiogenesis, such as the intersomitic spaces (arrowheads) but not ventrally (asterisk), where the VVN is forming by vasculogenesis (compare with panel B). (G) In the dermatome (arrowhead), apelin marks the intersomitic spaces, where ISVs will start to sprout. (H) Apelin mRNA is found in areas, where newly forming ISVs are detected (arrowheads). Most notably, a further, posterior domain (arrow) expresses apelin, while no ISV has emerged yet (compare with panel D). (I) Apelin stains the dorsal neural tube (arrowhead) and lateral capillaries (arrows) sprouting into the somites. (J) VEGFA is expressed in all somites (bracket) and the pronephric glomerulus (arrowhead). (K, L) Somitic VEGFA expression remains unaltered during intersomitic vessel growth. (M) Downregulation of VEGFA as ISV formation is completed. (N) APJ staining is confined to the ISVs (arrowheads). (O) Apelin stains the dermatome flanking the intersomitic spaces (arrows). The most mature ISVs express apelin (arrowhead), while the others are still negative (open arrowheads). (P) APJ is expressed throughout the developing vasculature including intersomitic vessels (arrowheads). (Q) Apelin expression is restricted to tissue undergoing angiogenesis, such as the tail (arrowhead) and limb buds (asterisks). (R) Tail explant, where APJ expression is detected in all intersomitic vessels (arrowhead). (S) Apelin stains intersomitic vessels, particularly in the leading edges (arrowheads). |
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Supplementary Figure 6. Molecular phenotypes of apelin-MO knockdown
embryos. Apelin-MO oligonucleotides (5 ng) and the lineage tracer nuclear ßgalactosidase mRNA (0.25 ng) were co-injected into single blastomeres of twocell-stage embryos. Injected embryos were raised to stages 33/34 (A), 35/36
(B), 37/38 (D-F) and 40 (C), fixed, and processed for ß-galactosidase activity.
Expression of various marker genes was visualized by whole-mount in situ
hybridization. Embryos with control and injected sides, respectively, are shown
accompanied by enlargements to visualize the developing intersomitic veins.
Scale bars: 400 µm for whole embryo views, 200 µm for close-up view. (A, B)
Impaired development of intersomitic veins (arrowheads) in apelin-MO injected
embryos is demonstrated by staining for Erg (A) and APJ (B). Note that the
vitelline vein network (asterisk) is unaffected. (C) The trunk vasculature is
disorganized in older stage 40 embryos as visualized by staining for Pecam1
expression. Missing intersomitic veins are indicated (arrowheads). (D )
Expression of apelin in the dermatome is not affected in apelin-MO injected
embryos. This suggests that apelin signaling is not necessary to maintain apelin
expression. (E) Erythropoiesis proceeds unaffected in apelin-MO injected
embryos as visualized by α-globin expression. Moreover, the dispersal of the
erythrocytes from the ventral blood islands is unaffected indicating that the
primary vascular plexus has formed normally. (F) Pronephric duct development
(arrow) is unaffected in apelin-MO injected embryos as demonstrated by Gata3
expression. |
