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Additional Supporting Information may be found in the online version of this article.

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DVDY_23817_sm_SuppFig1.tif11225KSupp. Fig. S1. Slit molecules are expressed by migratory neural crest. In situ hybridization was performed on chicken embryos HH17 (A, C, E, G, I, K, M, O), HH20 (B, D, F, H), and HH20 (J, L, N) for Slit1, Slit2 (A–H), and Robo1, Robo2 receptors (I–O). Slit2 or Slit1 expression was observed in the dorsal neural tube (arrows A–C, E–H). Robo1 or Robo2 expression was observed in the caudal neural tube (arrows I, K). Robo1 and Robo2 expression was observed in the dorsal root ganglia (arrows J, L, N). Robo1 and Robo2 expression was identified in migrating trunk neural crest cells via cross-sections (arrows M, O).
DVDY_23817_sm_SuppFig2.tif10129KSupp. Fig. S2. Slit LOF enhances neural crest migration. Embryos (HH16) electroporated with Translational Slit2 morpholinos showed more FITC-positive cells delaminating from the neural tube (arrows in B, D) compared with control morpholinos (arrows in A, C). E, F: Midtrunk/forelimb sections showing more FITC and HNK1 migrating trunk neural crest cells in TrnS2-MO (arrow in F) compared with control (arrow in E). G, H: Neural tubes electroporated with Slit2-MO showed that neural crest cells had an even more predominantly migratory/elongated shape (H) compared with control morpholinos (G).
DVDY_23817_sm_SuppFig3.tif1609KSupp. Fig. S3. Blocking of Slit molecules enhances neural crest cell migration. Chicken embryos HH13–14 were cultured ex ovo in the presence of control media, media conditioned with HEK-293 cells or RoboN media, media conditioned with HEK-293 cells that express RoboN, a soluble form of Robo that blocks Slit (A, B). Sympathetic cells were observed already condensing and of larger size in RoboN embryos compared with control (arrows A, B). Similarly, neural tubes were cultured overnight in the presence of HEK293 control, Slit2, or RoboN conditioned media (C–E). Neural crest cells migrated a farther distance in the presence of Slit2 or RoboN. Measurement corresponds to 100 or 200 μm for comparison.
DVDY_23817_sm_SuppFig4.tif3830KSupp. Fig. S4. Slit2 molecules affects the expression of epithelial markers in a neural crest–derived cell line. SpL201, a Schwann cell precursor immortalized cell line, was transfected with control GFP or Slit2 and stained with acetylated tubulin or actin. Slit2 induced cytoskeletal re-arrangements in these neural crest–derived cells as in neural crest cells. Red shows acetylated tubulin or actin (A–D, M–P). E–L: Acetylated tubulin or actin single channel. Acetylated tubulin was organized differently after Slit2, either very dispersed or condensed around the nucleus (arrowheads in I, J) compared with a clear presence of MTOC in control transfected cells (arrows in E). Actin cytoskeleton show classic stress fibers of migratory cells in control panel (arrows in G, H), while Slit2-expressing cells showed fewer stress fibers or none at all (arrowheads in K, L).
DVDY_23817_sm_SuppFig5.tif3020KSupp. Fig. S5. Slit2 RT-PCR after SpS2-MO. RT-PCR from chicken neural tubes after electroporation with control morpholinos (SpCM) or Slit2 morpholino (SpS2M). Asterisk marks a smaller band corresponding to the spliced Slit2 mRNA after losing exon 19, which results in a 165-bp deletion. The primers were designed to sit on either side of the deletion. Thus SpS2MO will generate an 882-bp fragments instead of the full length 1,047 bp of Slit2 mRNA.
DVDY_23817_sm_SuppMovie1.mov2338KSupp. Movie S1. Control GFP neural crest cells. Neural tubes were cultured after electroporation with control GFP plasmid and visualized for ∼3 hr. Cells migrated away from the neural tube extending in classic mesenchymal fashion.
DVDY_23817_sm_SuppMovie2.mov1751KSupp. Movie S2. Slit2-GFP neural crest cells. Neural tubes were cultured after electroporation with Slit2-GFP plasmid and visualized for ∼3 hr. Cells migrated away from the neural tube but several times during the movie the cells stopped and rounded up, then stretched and continued migrating.

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