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The cues and signaling systems that guide the formation of embryonic blood vessels in tissues and organs are poorly understood. Members of the Eph family of receptor tyrosine kinases and their cell membrane-anchored ligands, the ephrins, have been assigned important roles in the control of cell migration during embryogenesis, particularly in axon guidance and neural crest migration. Here we investigated the role of EphB receptors and their ligands during embryonic blood vessel development in Xenopus laevis. In a survey of tadpole-stage Xenopus embryos for EphB receptor expression, we detected expression of EphB4 receptors in the posterior cardinal veins and their derivatives, the intersomitic veins. Vascular expression of other EphB receptors, including EphB1, EphB2 or EphB3, could however not be observed, suggesting that EphB4 is the principal EphB receptor of the early embryonic vasculature of Xenopus. Furthermore, we found that ephrin-B ligands are expressed complementary to EphB4 in the somites adjacent to the migratory pathways taken by intersomitic veins during angiogenic growth. We performed RNA injection experiments to study the function of EphB4 and its ligands in intersomitic vein development. Disruption of EphB4 signaling by dominant negative EphB4 receptors or misexpression of ephrin-B ligands in Xenopus embryos resulted in intersomitic veins growing abnormally into the adjacent somitic tissue. Our findings demonstrate that EphB4 and B-class ephrins act as regulators of angiogenesis possibly by mediating repulsive guidance cues to migrating endothelial cells.
Fig. 1. Expression of Xenopus EphB4 in the
embryonic vasculature. Transcripts for EphB4
(A,B), Msr (C), EphB1 (D), EphB2 (E), and
EphB3 (F) were detected by whole-mount in
situ hybridization of stage 34 (A,C-F) and 36
(B) embryos. Lateral views are shown with
anterior to the left. (A) Expression of EphB4 in
the pronephric sinus (arrow) and the posterior
cardinal vein (arrowheads) of the embryonic
trunk. (B) Close-up view of the trunk region to
illustrate EphB4 expression in the intersomitic
veins (arrows), the vascular vitelline vein
network (arrowhead), the pronephric sinus (ps)
and the posterior cardinal vein (pcv).
(C) Staining with the vascular marker Msr
visualizes the vascular vitelline network (vvn),
the common cardinal vein (ccv), pronephric
sinus (ps), the posterior cardinal vein (pcv), and
the intersomitic veins (iv). (D-F) EphB1,
EphB2 and EphB3 transcripts are prominently
detected in the head (fb, forebrain; mb,
midbrain; e, eye; ov, otic vesicle; hm, head
mesenchyme), in specific visceral arches (v1,
v4), the cement gland (cg), and the pronephric
region (pr), but not in intersomitic veins. Scale
bars: 500 mm (A,C-D); 200 mm (B).
Fig. 2. EphB4 signaling is required for directed
growth of intersomitic veins. One blastomere of 2-cell
stage embryos was injected with RNA encoding
tmEphB4 (500 pg; A-D) together with 100 pg RNA of
the lineage tracer nuclear b-galactosidase (nucbgal).
RNA for tmEphB1 (1000 pg; D) was injected as a
control. Embryos were fixed at stage 36, stained for
nucbgal and hybridized with Msr. Lateral views are
shown with anterior to the left. (A,B) Control (A) and
injected side (B) of an embryo. Apart from an
aberrant intersomitic vein projecting into an adjacent
somite (arrow), the vasculature including the aortic
vessels (arrowheads) on the injected side appears
overall normal. (C,D) Close-up views of injected
sides of embryos. Arrows indicate aberrant
projections of intersomitic veins in embryos. A
capillary that appears dilated is shown with an
arrowhead. Dorsal growth of intersomitic veins is normal in embryos overexpressing tmEphB1. Scale bars: 400 mm (A,B); 200 mm (C,D).
Fig. 3. Ephrin-B ligand expression in the trunk of Xenopus embryos.
Whole-mount in situ hybridization of stage 32-33 Xenopus embryos
with antisense RNA probes for ephrin-B1 (A,C), and ephrin-B2 (B).
Lateral views (A,B) and a horizontal 50 mm vibratome section (C)
are shown with anterior to the left. Brackets indicate width of a
somite. Arrows indicate ephrin-B1 staining in the somites. Scale
bars: 400 mm (A,B); 100 mm (C).
Fig. 4. Ectopic expression of ephrin-B ligands leads to
aberrant intersomitic vein projections. One blastomere of 2-
cell stage embryos was injected with either 250 pg of ephrin-
B2 (A-D) or with 500 pg of ephrin-B3 RNA (E,F). RNA
encoding nucbgal was coinjected as a lineage tracer.
Embryos were fixed at stage 36, stained for nucbgal and
hybridized with Msr. (A,B) Control (A) and injected side (B)
of an embryo expressing ephrin-B2 ectopically. The posterior
cardinal vein appears dilated and the intersomitic veins
(arrowhead) have failed to grow dorsally. (C,D) Close-up
view of control (C) and ephrin-B2 injected (D) trunk regions.
Frequently, intersomitic veins are found projecting either
abnormally into adjacent somites (arrow) or displaying
reduced growth (arrowhead). (E,F) Trunk region of control
(E) and injected (F) side of an embryo expressing ephrin-B3
ectopically. Intersomitic veins are disorganized, penetrate
aberrantly into the neighboring somites (arrow), and
occasionally form loop-like structures (arrowhead). Scale
bars: 400 mm (A,B); 200 mm (C-F).
Fig. 5. Molecular control of intersomitic vein
development. (A) EphB4/ephrin-B signaling
controls angiogenic growth of intersomitic
veins in Xenopus. The panel depicts
schematically the growth of an intersomitic
vein (red) by sprouting angiogenesis from the
posterior cardinal vein (blue). Ephrin-B1 and
ephrin-B2 are expressed in the somites
(yellow), whereas EphB4 receptors are present
on the posterior cardinal vein and intersomitic
veins. Interactions between EphB4 receptors
and ephrin-B ligands restrict intersomitic veins
from inappropriate growth into adjacent somitic
tissues. Disruption of EphB4 signaling or
ectopic expression of ephrin-B ligands leads to
aberrant projection of intersomitic veins.
(B) Tentative hierarchy of genes regulating intersomitic vein development in vertebrates. Key steps in intersomitic vein formation are shown
together with the developmental block observed in Xenopus or mouse embryos that are disrupted for the indicated gene functions.
EphB2/EphB3 refers to double mutant mice. Ephrin-B1 is also implicated based on its somitic expression and the phenotype observed in
overexpression experiments.
ephb2 (EPH receptor B2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 34, lateral view, anteriorleft, dorsal up.
ephb4 (EPH receptor B4) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 34, lateral view, anteriorleft, dorsal up.
