Quantitative analysis of angiogenic sprouting into the developing neural tube reveals stereotypical ingression points. Unperturbed quail embryos and embryos whose neural tubes were electroporated with eGFP control DNA at HH stage 16-18 were serially sectioned at stage HH 25-26 through the upper limb. Every sixth 12 μm section was stained for QH1 (red). Fourteen images were analyzed for each embryo as described in the Materials and methods. (A) Unperturbed quail embryo section stained with QH1 to illustrate blood vessel analysis strategy. (B) Total number of angiogenic sprouts within the left (gray) and right (black) neural tube halves of five unperturbed embryos. There were concentrations of ingression points between 0-20° (ventral ingression points) and 70-110° (medial ingression points). (C) Representative image of a quail neural tube electroporated with eGFP DNA (electroporated side to the right). (D) Total number of ingressing angiogenic sprouts within the neural tubes of five control embryos electroporated with eGFP DNA (green); untransfected control contralateral neural tube side (black). Scale bar: 100 μm.

Ectopic expression of heparin-binding VEGF isoforms induces supernumerary vessel ingression points into the developing neural tube. Quail neural tubes were electroporated with hVEGF121-GFP, hVEGF165-GFP or hVEGF189-GFP DNAs (green, panels B, F, J) on day 3 (HH 16-18) and harvested 48 hours later (HH 25-26). Transverse sections were stained with QH1 antibody (red, panels A,E,I) to visualize vessels, and five embryos from each group were analyzed as described (panels D,H,L; green lines, total ingression points for ectopic VEGF-expressing sides of neural tubes at each 10° of arc; black lines, total ingression points for contralateral control sides of the neural tubes at each 10° of arc). C, G and K are a merge of red (QH1) and green (eGFP) channels. (A-C) Quail neural tubes electroporated with hVEGF121 DNA displayed a grossly normal distribution of angiogenic ingression points along the dorsoventral axis of the ectopic VEGF-expressing side of the neural tube (arrows in A,C). (D) The quantitative analysis showed no change in the distribution of ingression points for sprouts between the control (black) and VEGF121-expressing (green) sides of the neural tube, and a slight increase in the frequency of ingression points in the medial region of the VEGF-expressing side of the neural tubes (n=5 embryos). (E-G) Quail neural tubes electroporated with hVEGF165 DNA had ectopic dorsal sprouts (arrows in E,G). (H) The quantitative analysis showed increased distribution and frequency of vessel ingression points in the dorsal region of the hVEGF165-expressing side of the neural tube (green), where ectopic expression is localized (n=5 embryos). (I-K) Quail neural tubes electroporated with hVEGF189 DNA had ectopic dorsal sprouts (arrows in I,K). (L) The quantitative analysis showed increased distribution and frequency of vessel ingression points in the dorsal region of the hVEGF189-expressing side of the neural tube, where ectopic expression is localized (n=5 embryos). Scale bar: 100 μm.

Localized ectopic expression of heparin-binding VEGF-A isoforms in the developing neural tube correlates with supernumerary vessel ingression points. Neural tubes processed for QH1 (red) and eGFP (green, reporter for ectopic VEGF-A isoform expression) were examined for the relationship between vessel ingression points and ectopic VEGF-A isoform expression. (A-C) Lower power views to show location of normal (A) or supernumerary (B,C) vessel sprout ingressions on the dorsoventral axis of the neural tube. (D-F) Higher magnification of the boxed areas in A-C. Several eGFP-positive cells that ectopically express heparin-binding hVEGF165 or hVEGF189 are close to the supernumerary vessel sprouts (arrows in E,F), whereas numerous eGFP-positive cells that ectopically express hVEGF121 (arrowheads in D) do not induce supernumerary vessel ingression points. Scale bar: 100 μm in A-C; 50 μm in D-F.

Neural patterning is not perturbed in neural tubes that ectopically express VEGF-A isoforms. (A-U) Quail neural tubes were electroporated with (A-G) hVEGF121, (H-N) hVEGF165 and (O-U) hVEGF189 on day 3 (HH 16-18), and harvested 48 hours later (HH 25-26). Neural tubes were sectioned and adjacent sections were stained with antibodies to: QH1 (red, A,H,O,C,J,Q) to visualize vessels; Pax7 (purple, D,K,R) to visualize dorsal neural precursors; Pax6 (orange, E,L,S) to visualize medial neural precursors; MNR2 (yellow, F,M,T) to visualize ventral motoneuron precursors; and Tuj1 (blue, G,N,U) to visualize differentiated neurons. (B,I,P) eGFP expression (green) illustrates the neural tube side expressing ectopic VEGF-A isoforms (left) versus the control contralateral side (right); (C-G,J-N,Q-U) merges of marker and eGFP channels for each section. Scale bar: 100 μm.

VEGF signaling from the neural tube is required for blood vessel ingression. Quail neural tubes were electroporated with sFlt1-GFP and analyzed as previously described. (A-C) No medial vessel ingression and little ventral vessel ingression was seen in areas of the neural tube that were eGFP positive. (D-G) Neural patterning is not detectably perturbed on the electroporated side of the neural tube (left) based on Pax7 (D, purple), Pax6 (E, orange), MNR2 (F, yellow) and Tuj1 (G, blue) expression patterns. (H) Quantitative analysis of five electroporated neural tubes showed no medial and few ventral vessel ingression points in areas of localized sFlt1 expression (green), compared with the control contralateral side (black) (n=5 embryos). Scale bar: 100 μm.