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???displayArticle.abstract??? Angioblasts are precursor cells of the vascular endothelium which organize into the primitive blood vessels during embryogenesis. The molecular mechanisms underlying patterning of the embryonic vasculature remain unclear. Mutational analyses of the receptor tyrosine kinase flk-1 and its ligand vascular endothelial growth factor, VEGF, indicate that these molecules are critical for vascular development. Targeted ablation of the flk-1 gene results in complete failure of blood and vascular development (F. Shalaby et al. (1995) Nature 376, 62-66), while targeted ablation of the VEGF gene results in gross abnormalities in vascular patterning (P. Carmeliet et al. (1996) Nature 380, 435-439; N. Ferrara et al. (1996) Nature 380, 439-442). Here we report a role for VEGF in patterning the dorsal aorta of the Xenopus embryo. We show that the diffusible form of VEGF is expressed by the hypochord, which lies at the embryonic midline immediately dorsal to the location of the future dorsal aorta. We find that, initially, no flk-1-expressing angioblasts are present at this location, but that during subsequent development, angioblasts migrate from the lateral plate mesoderm to the midline where they form a single dorsal aorta. We have demonstrated that VEGF can act as a chemoattractant for angioblasts by ectopic expression of VEGF in the embryo. These results strongly suggest that localized sources of VEGF play a role in patterning the embryonic vasculature.
Fig. 1. In situ hybridization reveals expression of VEGF in the hypochord and flk-1 in migrating endothelial precursor cells. (A) Section through the posteriortrunk of an early tailbudembryo (stage 30) showing VEGF expression in the somites and in the hypochord (arrow). (B) Higher magnification of the hypochord (arrow) stained for VEGF expression. The notochord is indicated (n). (C) Section through the trunk of an early tailbudembryo (stage 28) showing expression of flk-1 in groups of endothelial precursor cells in the mesenchyme of the lateral plate mesoderm. Flk-1 transcripts are located at the position of the developing cardinal veins, prior to blood vessel formation. (D) Section positionally equivalent to C through the trunk of a mid-tailbudembryo (stage 33) showing migrating angioblasts (arrowheads) that express flk-1. Position of the posterior cardinal veins is indicated (arrows). (E) High magnification view of the dorsal aorta (stage 35) stained for flk-1 expression. Hypochord is indicated (arrow). (F) Schematic representation of tissue layers and expression domains of VEGF and flk-1. Note that the hypochord lies immediately dorsal to endodermal tissue and at some distance from lateral plate mesodermtissue. Bar, 40 μm (A,C,D) and 20 μm (B,E).
Fig. 2. Lineage tracing of lateral plate mesoderm cells showing migration to the position of the dorsal aorta. (A) Embryo labeled with DiI at the position of the posterior cardinal vein (stage 28). (B) Cryostat section, viewed under fluorescence microscopy, of a tailbudembryo (stage 33) which was labeled with DiI in the lateral plate mesoderm at stage 25. The arrowhead marks the position of DiI labeling and the arrow indicates a cell that has migrated towards the midline near the notochord (n). (C) Cryostat section showing a high magnification view of the dorsal aorta of a later tailbudembryo (stage 35) in which a DiI-labeled cell has become incorporated into the endothelial layer (arrow). Notochord is indicated (n). Bar, 150 μm (A), 20 μm (B,C).
Fig. 3. Removal of lateral plate angioblasts eliminates dorsal aorta formation. (A) Schematic representation of microdissection experiment. Lateral plate mesoderm at the position of the posterior cardinal veins is removed, without damaging underlying endoderm or midline structures. Notochord (n), somite (s), endoderm (e) and mesoderm (m) are indicated. (B) Section through the unmanipulated region of the trunk of a stage 35 embryo, showing X-msr expression in the differentiated blood vessels. The posterior cardinal veins are evident laterally on each side of the embryo (arrows), and the single dorsal aorta (arrowhead) is located immediately under the hypochord. The position of the notochord is indicated (n). (C) Section through the dissected region of the trunk (more posterior) of the embryo shown in B. Posterior cardinal veins and dorsal aorta are not detectable by X-msr staining. Note the absence of damage to the midline structures as the hypochord, notochord and underlying endoderm are undisturbed. (D) Section through embryo where the lateral plate mesoderm on one side only was dissected. Note the absence of the posterior cardinal vein on the manipulated side of the embryo and the abnormality in the dorsal aorta at the midline (narrow arrowhead). The posterior cardinal vein on the opposite side is undisturbed (arrow). Bars, 25 μm (B-D).
Fig. 4. The hypochord expresses transcripts for the small, diffusible form of the VEGF protein. (A) Schematic depiction of the dissection of a stage 32 embryo to separate the hypochord from the somites. (B) RT-PCR analysis demonstrates that transcripts encoding the small, diffusible form of VEGF are preferentially expressed in the hypochord relative to the intermediate or large forms. Positions of the PCR products representing the large, medium and small transcripts are indicated to the right of the figure. Total amount of RNA in the hypochord and somite fractions was estimated using the ubiquitous EF1-α sequence.
Fig. 4. The hypochord expresses transcripts for the small, diffusible form of the VEGF protein. (A) Schematic depiction of the dissection of a stage 32 embryo to separate the hypochord from the somites. (B) RT-PCR analysis demonstrates that transcripts encoding the small, diffusible form of VEGF are preferentially expressed in the hypochord relative to the intermediate or large forms. Positions of the PCR products representing the large, medium and small transcripts are indicated to the right of the figure. Total amount of RNA in the hypochord and somite fractions was estimated using the ubiquitous EF1-α sequence.
