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The receptor tyrosine kinase, Flk-1 or VEGFR-2, and its ligand, vascular endothelial growth factor (VEGF) are required for the development of the embryonic vasculature. Targeted disruption of either gene in mice results in the failure of vascular system formation. The Xenopus homologues of flk-1 and VEGF have been cloned and their expression has been examined throughout early embryonic development. These studies indicate that flk-1 is expressed in groups of endothelial precursor cells which will form the major blood vessels of the embryo, including the posterior cardinal veins, the dorsal aorta, the vitelline veins, and the endocardium. VEGF expression is found in tissues adjacent to the mesenchyme containing the flk-1-expressing endothelial precursors. Expression of both flk-1 and VEGF is transient, appearing as the primary vascular plexus is forming and declining steadily after the onset of functional embryonic circulation. After establishment of the primary vascular structures, flk-1 expression is also observed in the intersegmental veins which form by an angiogenic mechanism. Overall, these results support a role for VEGF/flk-1 signaling in both vasculogenesis and angiogenesis in the Xenopus embryo. When VEGF is expressed ectopically in Xenopus embryos by microinjection of either plasmid DNA or synthetic mRNA, large, disorganized vascular structures are produced. This result indicates that ectopic VEGF is capable of altering the architecture of the developing vascular network.
Fig. 4. Whole mount in situ hybridization analysis of flk-1 expression. Embryos in A, B, and F were photographed at 70X, C at 35X, and D and E at 30X. A: Lateral view of a stage 21 embryo showing staining in the endocardial precursor region (small arrow), in the aortic arches (out of the plane of focus, small arrowhead), and in the headmesenchyme underly- ing the prosencephalon (large arrow) and overlying the tip of the notochord (open arrowhead). B: Dorsal view of a stage 23 embryo showing flk-1 expression at the midline in the headmesenchyme (large arrow) and more laterally in the aortic arches (small arrowheads). C: In a stage 27 embryo, flk-1 is prominently expressed in groups of endothelial precursor cells located at the position of the developing cardinal veins (large concave arrow) and of the developing vitelline veins (open arrowhead). Expression is also detected in the endocardium (smallarrow), the aortic arches (small arrowhead) and the headmesenchyme (large arrow). D: Ventral view of a stage 28 embryo showing the ring of flk-1 expression surrounding the region of the ventralblood islands. Arrowheads indicate the vitelline veins and a short arrow indicates the endocardium. E: Staining marks the posterior cardinal veins (large arrow), the common cardinal vein (or the Duct of Cuvier; small arrow), the sinus venosus (open arrow), and the ventralaorta (arrowhead). Note that the cardinal veins show lower levels of flk-1 transcripts anteriorly where the vessels are mature. Posteriorly, the endothelial cells have not yet fully coalesced into mature vessels and are still in a loose association. F: Lateral view of the larval tail of a mid tailbudembryo (stage 34) showing flk-1 staining in the posterior cardinal veins (large arrow), the rectal diverticulum (white arrow), and the dorsal aorta (small arrow).
Fig. 5. Whole mount in situ hybridization analysis of VEGF expression. Embryos in A, B, E, and F were photographed at 70X, C at 30X, and D at 35X. A: Lateral view of a stage 21 embryo showing regions of staining in the aortic arches (out of the plane of focus; arrowhead), in the pharyngeal endoderm (short arrow), in the floor of the neural tube above the tip of the notochord (open arrowhead), and in the archencephalon (long arrow). B: Dorsal view of embryo shown in A. The region of neural staining is located at the midline of the embryo between the eye anlagen (large arrow). Aortic arch staining is indicated by small arrowheads. C: Lateral view of stage 26 embryo. VEGF expression can be seen in the cephalic regions described above, and in the nuclei of the somites (arrowheads) and of the hypochord (large arrow). D: Lateral view of stage 34 embryo. VEGF expression has resolved to sharper domains in the aortic arches (thin arrow) and additional expression can be seen in the developing glomus (open arrowhead). Expression continues in the somites (arrowheads). Note that expression of VEGF in the hypochord (large arrow) is declining in the anterior portion of the embryo. E: Higher magnifica- tion, lateral view of embryo in D showing nuclear staining in the hypochord (large arrow), in the somites (arrowheads), and staining in the glomus (open arrowhead). F: Dorsal view of the somite expression (arrowheads) and of the hypochord nuclei at the midline of the embryo (arrow).
Fig. 6. Transverse sections showing complementarity of flk-1 (A, C, E, G) and VEGF (B, D, F, H) expression patterns. Sections A, B, G, and H were photographed at 100X magnification and sections C at 200X. Dorsal is to the right in A and B. Dorsal is up in all other panels. A: Section through the aortic arch progenitor region of a stage 28 embryo stained for flk-1. Endothelial precursor cells are shown in a punctate pattern in the mesoderm which will form the third aortic arch (arrows) and the anterior portion of the endocardium (arrowhead). B: Section equivalent to A showing staining for VEGF throughout the pharyngeal endoderm which underlies both the aortic arch mesoderm (arrows) and the cardiogenic mesoderm (short arrow). C: Section through the heart of a stage 32 embryo showing flk-1 staining in the endocardium (arrowhead), but not the myocardium. Staining is also visible in the ventral aortae (arrows) which lead from the bulbus cordis (anterior to the heart) to the aortic arches. D: Section equivalent to C showing VEGF expression in the pharyngeal endoderm adjacent to the endocardium (short arrow) and adjacent to the ventral aortae (long arrows). E: Section through the developing kidney of a stage 32 embryo, showing flk-1 staining in the region of the blood sinus which surrounds the developing tubules. F: Section equivalent to E showing VEGF expressing in the developing glomus. G: Section through the posteriortrunk of a stage 30 embryo showing flk-1 in the lateral plate mesoderm, under the somites. These regions of staining mark the developing posterior cardinal veins which have not yet fused into mature vessels. H: Section equivalent to G showing VEGF in the somites (open arrowheads) and in the hypochord (arrow).
Fig. 7. Overexpression of VEGF alters vascular patterning. All em- bryos were photographed at 40X magnification. Anterior is to the right in all panels. A: Lateral view of stage 35 embryo showing the normal pattern of X-msr expression. Stained regions include the posterior cardinal veins and an undefined tissue at the tip of the tail. B,C: Lateral views of posterior portion of VEGF injected embryos, at about stage 33, showing groups of extraneous endothelial cells extending ventrally from the posterior cardi- nal veins. D: Lateral view of the posterior portion of a VEGF injected embryo, at about stage 35, showing large ectopic blood vessels in the posteriorgut that are continuous with the posterior cardinal veins. Additional vessels sprouting anteriorly from this ectopic vascular structure are also visible. E: Ventral view of a control stage 46 embryo stained with benzidine. A single major vessel, the subintestinal vein, is present in this embryonic region (white arrowhead). The heart is indicated by a white arrow. F: Ventral view of a VEGF injected stage 46 embryo stained with benzidine. Injection of VEGF results in substantial alterations to the normal vascular organization, with an increase in the amount and complexity of vascular tissues (black arrowhead). The heart is indicated by a white arrow. G: Lateral view of the tail of a normal stage 46 embryo stained with benzidine. H: Lateral view of the tail of a VEGF injected embryo. Disorganized ectopic vessels are visible in the anterior portion of the tail, above the caudal artery.
