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First published online 3 March 2004
doi: 10.1242/dev.01039

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The neural tube patterns vessels developmentally using the VEGF signaling pathway

Kelly A. Hogan^1,*, Carrie A. Ambler^1,*, Deborah L. Chapman² and Victoria L. Bautch^1,3,

¹ Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280, USA
² Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA
³ Carolina Cardiovascular Biology Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA

View larger version (132K): [in a new window]   Fig. 1. The neural tube directs PNVP formation in vivo. (A) Transverse section through the trunk of a HH stage 24 quail embryo labeled with the QH1 antibody (brown). The arrow indicates a ring of vessels around the neural tube termed the perineural vascular plexus (PNVP). (B) In ovo manipulations placing a mouse neural tube (blue) between the lateral plate and intermediate mesoderm in a quail host. (C) Section of a HH 24 stage quail embryo with an implanted acrylic bead (asterisk) stained with QH1 antibody (purple). Note that a large vessel is positioned near the bead (arrow), but no vascular plexus surrounds the bead. (D) Low magnification of a transverse section through a HH stage 24 quail host with a grafted ROSA+/– mouse neural tube (asterisk), 3 days post-surgery. Chimeric embryos were whole-mount stained for ß-gal (blue), then sectioned and reacted with QH1 antibody (brown). (E) Higher magnification of D. The arrow indicates a host-derived vascular plexus surrounding the grafted neural tube. n5 for all in ovo manipulations. NC, notochord; NT, neural tube. Scale bar: 100 µm in A; 50 µm in C,E; 200 µm in D.

View larger version (105K): [in a new window]   Fig. 2. The multiple neural tubes of Tbx6 mutant embryos each recruit a PNVP. (A,B) Diagram of a transverse section through the caudal region of a wild-type (A) and Tbx6 homozygous mutant (B) embryo depicting the differences in Tbx6 mutant embryos. Note that somitic tissue (yellow) does not form in the Tbx6 mutants, but ectopic neural tubes (blue) form on both sides of the main neural tube (blue). (C,D) Transverse sections through the posterior trunk of 10.5 dpc PECAM-stained wild-type (C) and Tbx6 mutant (D) embryos. The PNVP around the central neural tube is present in both embryos (arrows in C,D), and the ectopic neural tubes (denoted with asterisks in the Tbx6 mutant embryo) are also surrounded by a vascular plexus (arrowheads in D). Scale bar: 70 µm in C,D.

View larger version (101K): [in a new window]   Fig. 3. The neural tube-derived vascular patterning signal induces target cells at a distance. Mouse presomitic mesoderm grafts were placed in quail embryos adjacent to host medial presomitic mesoderm, in a cavity made by removal of quail lateral presomitic mesoderm. Grafts were either Flk1+/– (lacZ in Flk1 locus) (B-D) or wild type (E-G), and after incubation for 48 or 72 hours, chimeric embryos were fixed and stained for ß-gal (B-D) or PECAM (E-G). (A) In ovo manipulations. The orange arrow indicates the medial quail presomitic mesoderm that is situated between the mouse graft (purple rectangle) and the host neural tube (dark blue). (B) Chimera incubated for 72 hours and stained for ß-gal. Arrows indicate mouse graft-derived vascular cells (blue) in the trunk and limb vessels of the quail host. (C,D) Sections through the trunk of the chimeric embryo in B. ß-Gal-positive vessels are highlighted by arrows in the intersomitic and limb vascular beds (C) and the kidney rudiment (D). (E) Chimera incubated for 72 hours and stained for PECAM. Arrows indicate graft-derived vascular cells (brown) in host intersomitic and limb vessels. (F,G) Sections through the neural tube of the chimera in E stained with PECAM antibody (brown) to visualize mouse graft-derived vascular cells, and QH1 antibody (purple) to visualize host vessels. (F) PECAM-positive cells (brown) identified in the ventral region of the PNVP. The inset shows a higher magnification of this vessel (arrow indicates PECAM-positive graft-derived cells). (G) A different region of the neural tube shows PECAM-positive endothelial cells (arrow) in a quail PNVP vessel. Scale bar: 80 µm in C; 40 µm in D,F; 20 µm in G.

View larger version (73K): [in a new window]   Fig. 4. VEGFA expression correlates with formation of the PNVP. E8.5-9.5 Vegf-lacZ+/– embryos were dissected and processed for either ß-gal or PECAM antibody staining. (A) An embryo at E8.5-9.0 with 12 somites (left), and an embryo at E9.5 with 20-25 somites (right). The lines indicate the level of transverse sections shown in B-J. (B) Posterior section through an E9.5 wild-type embryo ß-gal stained as a negative control. (C,D) Posterior sections through E8.5 Vegf-lacZ+/– embryos stained for ß-gal (C) or PECAM (D). Note the lack of ß-gal staining in the neural tube (asterisk) and the ß-gal reactivity in the underlying endoderm in C, and the lack of vessels around the neural tube in D. (E,F) Anterior sections through E8.5 Vegf-lacZ+/– embryos stained for ß-gal (E) or PECAM (F). Note the diffuse ß-gal reactivity in the neural tube, and the stronger signal from the underlying endoderm in E, and the partial PNVP in F. (G,H) Posterior sections through E9.5 Vegf-lacZ+/– embryos stained for ß-gal (G) or PECAM (H). (I,J) Anterior sections through E9.5 Vegf-lacZ+/– embryos stained for ß-gal (I) or PECAM (J). Note the stronger ß-gal reactivity that is partially localized to the lateral edge of the neural tube in I, and the fully formed PNVP in J. Asterisks indicate the central canal of the neural tube in B-J. Scale bars: 50 µm in B-H; 100 µm in I,J. A is reproduced, with permission, from Kaufman (Kaufman, 1992).

View larger version (135K): [in a new window]   Fig. 5. Presomitic mesoderm explants form VEGF-dependent vascular networks. 8.5 dpc Flt1+/–, eGFP+/– mouse presomitic mesoderm explants were embedded in collagen gels and cultured for 72 hours. Explants were stained for ß-gal activity (A,C,E), and the same explants were visualized for eGFP expression (B,D,F). Explants were incubated with basal medium alone (A,B), or with added 30 ng/ml VEGFA (C,D) or 25 ng/ml bFGF (E,F). n7 for all three conditions. The arrows in C indicate the vascular plexus, and the arrowheads in A and E indicate ß-gal positive (blue) presumptive vascular cells. Scale bar: 200 µm for all panels.

View larger version (124K): [in a new window]   Fig. 8. A genetic mutation in the VEGF receptor Flk1 blocks neural tube-derived vascular plexus formation. Mouse presomitic mesoderm explants were cultured for 72 hours in basal medium with either 30 ng/ml VEGFA (A,B) or stage HH10-13 quail neural tubes (C,D). Explants were either Flk1+/– (A,C) or Flk1–/– (B,D), and mouse vascular cells were visualized by ß-gal staining. The arrows (A,C) indicate mouse vascular plexus, and the arrowheads (B,D) point to ß-gal positive (blue) presumptive mouse vascular cells. n7 for all conditions. Scale bar: 200 µm for all panels.

View larger version (116K): [in a new window]   Fig. 6. Neural tube-dependent vascular plexus formation in presomitic mesoderm explants. Flt1+/– mouse presomitic mesoderm grafts (PSM) (8.5 dpc) were co-cultured with HH10-13 stage quail neural tubes (NT) or notochords (NC) in collagen gels for 72 hours. Individual co-cultures were photographed at 0 (A,D), 24 (B,E) or 48 hours (C,F) to demonstrate the growth of the tissues. The same cultures were then fixed at 72 hours and stained for ß-gal (G,H). The purple arrows indicate the respective tissues and the black arrows indicate the vascular plexus that forms in PSM/NT co-cultures. The arrowheads indicate the few ß-gal positive presumptive vascular cells seen in PSM/NC co-cultures. n>10 for PSM/NT co-cultures and n=3 for PSM/NC co-cultures. Scale bars: 350 µm in A-F; 140 µm in G,H.

View larger version (126K): [in a new window]   Fig. 7. Inhibitors of VEGF signaling block neural tube-derived vascular plexus formation. 8.5 dpc Flt1+/–, eGFP+/– mouse presomitic mesoderm grafts were placed beside stage HH 10-13 quail neural tubes in collagen gels and cultured for 72 hours. Panels on the right (B,D,F) show eGFP expression that identifies mouse tissue. Co-cultures were grown in basal medium alone (A,B) or with added 1 µg/ml Flt/Fc (C,D) or 10 µM SU5416 (E,F). n4 for all three conditions. Scale bar: 200 µm for all panels.

View larger version (31K): [in a new window]   Fig. 9. Neural tube patterning of blood vessels – a model. (A) Diagram showing cross-sectional view of a mouse embryo at 8.5 dpc. The boxed area is enlarged in B-D. (B) Initially, the presomitic mesoderm does not contain committed vascular precursor cells. (C) A VEGF-independent signal induces FLK1 expression in a subset of presomitic mesoderm cells. (D) FLK1 expressing angioblasts are now competent to respond to the neural tube-derived VEGFA containing signal. The angioblasts now express additional vascular markers, such as FLT1, and they migrate and assemble the PNVP. Other unidentified signals emanating from the neural tube may also contribute to PNVP patterning in vivo. NT, neural tube; NC, notochord; PSM, presomitic mesoderm; IM, intermediate mesoderm; LPM, lateral plate mesoderm.