DEV011452 Supplementary Material

Files in this Data Supplement:

• Supplemental Table 1 - Adobe PDF

• Supplemental Figure S1 -

  Fig. S1. Effects of Sema3A overexpression and silencing in motoneurons in vivo. (A) Global view of in situ hybridization of Sema3A on brachial sections. (B) Schematic transverse section of HH24 chick embryo showing the region (green) targeted in the electroporation experiments. (C) Myc immunostaining showing the localization of Myc-tagged Sema3A protein, and in situ hybridization against Sema3A mRNA showing the level of Sema3A overexpression obtained with Sema3AiresEGFP electroporation. (D) Nrp1 (NP1) immunolabelling on Sema3AiresEGFP embryos showing that Sema3A overexpression efficiently prevents innervation of the dorsal root entry zone by Nrp1⁺ sensory fibers (arrowhead) in the electroporated side. (E) TUNEL on embryos electroporated with Sema3AiresEGFP. EGFP labeling indicates the electroporated side. (F) Anti-Islet1/2 immunostaining on Sema3AiresEGFP embryos showing similar motoneuron localization in electroporated (arrowhead) and non-electroporated sides. Immunostaining at P0 on Sema3AiresEGFP embryos showing similar localization of Schwann cells on the control and experimental sides (arrow). The bracket indicates the electroporated side. (G,H) Effects of control EGFP or Sema3AiresEGFP overexpression on the projection of spinal axons (arrow) towards the aorta at the cervical (G) and brachial (H) levels (aorta is outlined). (I) In situ hybridization of Sema3A and Sema3C mRNAs on transverse sections of pShSema3A-EGFP and pShScramble-EGFP HH25 embryos (bracket, electroporated side). (J) TUNEL on pShSema3A-EGFP embryos. (K) Plot comparing the number of Islet1/2-positive motoneurons in electroporated and non-electroporated ventral horns (33 transverse sections, three embryos). P>0.5 with Wilcoxon test on paired data. Scale bar: 100 µm.

• Supplemental Figure S2 -

  Fig. S2. Ventral spinal cord explants from embryos electroporated with Sema3AiresEGFP, pShSema3A-EGFP or control plasmids. (A) Morphology of axons emerging from non-electroporated, ctrlEGFP and Sema3AiresEGFP ventral spinal cord explants after exposure to Sema3A supernatant. Higher magnification views of the boxed regions (right panel) show collapsed ctrlEGFP⁺ growth cones and non-collapsed Sema3AiresEGFP⁺ growth cones (arrows). (B) Morphology of axons emerging from pShScramble-EGFP and pShSema3A-EGFP ventral spinal cord explants. Higher magnification (right panel) shows similar growth cone morphologies in both conditions, prior to exposure to Sema3A supernatant. Scale bars: 20 µm.