DEV028845 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Time course of VEGF-A expression in the quail neural tube at the limb (thoracic) level. In situ hybridization of transverse cryostat sections of quail neural tubes with an antisense probe to VEGF-A. (A) At HH stage 18, when electroporations were performed, there was some VEGF-A reactivity, with a concentration in the floor plate and the roof plate. (B) At HH stage 23, VEGF-A reactivity was increased and seen prominently in the motoneuron area and the floor plate. (C) At HH stage 25, approximately when the analysis was done, the overall level of VEGF-A reactivity was increased, with some concentration of the signal in the floor plate and the roof plate. Scale bar: 100 µm.

• Supplemental Figure S2 -

  Fig. S2. Expression analysis of VEGF-A isoform transgenes in electroporated neural tubes. Quail neural tubes were electroporated as described on day 3, and harvested on day 5. Total RNA was isolated from three to five pooled neural tubes and processed for RT-PCR using primers that recognize the hVEGF-A transgene (they span the alternative splice sites) or eGFP. Neural tubes were electroporated with: lane 1, eGFP only; lane 2, hVEGF121-GFP; lane 3, hVEGF165-GFP; lane 4, hVEGF189-GFP. The size of the amplification products is shown on the right.

• Supplemental Figure S3 -

  Fig. S3. Radial glia patterning is not perturbed in neural tubes that ectopically express VEGF-A isoforms. (A-R) Quail neural tubes were electroporated with hVEGF121 (A-F), hVEGF165 (G-L) or hVEGF189 (M-R) 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,G,M) to visualize vessels; and transitin (red, D,J,P) to visualize radial glia. (B,E,H,K,N,Q) eGFP expression (green) illustrates the neural tube side expressing ectopic VEGF-A isoforms (left) versus the control contralateral side (right) on adjacent sections; (C,F,I,L,O,R) merge of marker and eGFP channels for each section. Scale bar: 100 µm.