DEV01675 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Peripehral nerves contain HB9⁺ motor axons. (A) Transverse section of E14.5 HB9-eGFP transgenic embryo is shown. HB9-eGFP⁺ axons are present in spinal cord and project along nerve branching (arrows). (B-E) Whole-mount triple immunofluorescence confocal microscopy with antibodies to GFP (B,C, green), neurofilament (2H3) (B,D, red) and a pan-endothelial marker PECAM1 (B,E, blue) shows some eGFP⁺ motor axons in the 2H3⁺ axon bundles which follow PECAM1⁺ vessels. Scale bars: 100mm. (F) RT-PCR analysis of VEGFA isoforms expression in freshly isolated HB9-eGFP⁺ motoneurons in the spinal cord. EGFP⁺ motoneurons express all VEGFA isoforms. HPRT expression was identified at a constant level. mRNA from E14.5 DRG was used as a positive control.

• Supplemental Figure 2 -

  Fig. S2. Wnt1-Cre and Isl1-Cre are active in the peripheral nerves in the limb skin. Whole-mount triple immunofluorescence confocal microscopy of limb skin was performed with antibodies to b-galactosidase (b-gal) (A,B,G-J, red, arrows), BFABP (C,D,G,H, blue, open arrowheads), and a neuron specific marker bIII tubulin (TuJ1) (E,F,I,J, green, open arrows). The Wnt1-Cre activity is mainly seen in BFABP⁺ Schwann cells, because the b-gal does not diffuse down to the peripheral axons (G versus I, arrows and open arrowheads). No Isl1-Cre activity is seen in BFABP⁺ Schwann cells (B,H,J, open arrows). Scale bars: 100mm.

• Supplemental Figure 3 -

  Fig. S3. No significant defects in arteriogenesis were seen in the major arterial vessels. (A-X) Triple immunofluoresence confocal microscopy of E15.5 trunk sections performed using antibodies to either NP1 (D-F,P-Q, red, arrows) or CX40 (J-L,V-X, red, arrows), together with PECAM1 (A-C,G-I,M-O,S-U, blue), a neuronal class III b-tubulin marker TuJ1 (A-C,G-I,M-O,S-U, green). Development of the carotid artery (A-L, arrows) and thoracic artery (M-X, arrows) in the mutants appears to be normal. Open arrowhead indicates sympathetic ganglion. Scale bar: 100 mm.

• Supplemental Figure 4 -

  Fig. S4. VEGFA is expressed by large-diameter vessels. (A-D) Whole-mount triple labelling with antibodies to VEGFA (A,B, green), PECAM1 (A,C, blue) and peripherin (A,D, red) reveals that VEGFA is expressed by large-diameter blood vessels (arrows) as well as peripherin⁺ nerves (open arrowheads) in the limb skin. (E-H) Triple labelling with antibodies to a smooth muscle cell marker aSMA (E,H, red) in addition to VEGFA (E,F, green) and PECAM1 (E,G, blue) indicates that VEGFA is expressed by both endothelial and smooth muscle cells of large-diameter blood vessels (E-H, arrow; nerve-derived VEGFA expression is indicated by open arrowheads). Scale bars: 100mm.