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Figure S2. Inhibition of versican leads to failure in NC migration, related to Fig. 2. (A) Western blot of extracts from wild-type (wt) embryos and embryos
injected at the two-cell stage with 30 ng VsMO in each blastomere, harvested during NC migration. Lysates were probed with antibodies specific for the
versican V0–1 and the V3 isoforms. β-Actin was used as a loading control. (B–E) Versican MO inhibits NC migration in vivo. Lateral view of stage 27 embryos,
anterior to the left, showing that in comparison with control NC migration (B, arrows), versican depletion can result in mild (C) or severe (D) inhibition
of NC migration (red arrowheads). (E) Quantification of NC migration inhibition in versican-depleted embryos (VsMO; dark brown represents mild phenotype
[32%], light brown represents severe phenotype [49%, total n = 214]) compared with control (CoMO; dark brown represents mild phenotype 5%,
n = 124). (F) Lateral view of control or VsMO-injected 32 Xenopus embryos at stage 32 revealing melanocytes (arrows) and NC derivative population.
(G) The mean number of melanocytes is significantly reduced upon VsMO injection. Bars, SD. (H) Cartilage, another NC derivative, is affected in VsMOinjected
embryos at stage 42 (ventral view). (I and J) Contact inhibition of locomotion is unaffected in NC explants in a confrontation assay. Clusters without
CIL are expected to mix and form a larger overlap area (overlapping index [OI] ∼0.5), and the lack of overlap is indicative of the presence of CIL (Becker
et al., 2013). Bar, mean; error, SD; n = 23 pair of clusters per condition. (K and L) Coattraction of NC clusters explanted from two embryos onto fibronectin
substrates is unaffected by VsMO in a CoA assay (Carmona-Fontaine et al., 2011). (M) Quantification of migration index as MI = d/D. Migration index in
control host embryos with VsMO-injected NC grafts and control NC in VsMO-injected host embryos show that versican depletion in the surrounding tissues
leads to significant reduction in NC migration. Bar, mean; error, SD. (N and O) Rescue of NC migration by versican protein. (N) NC expressing GFP were
grafted into a VsMO-injected embryo, which impairs NC migration. In some embryos, microbeads soaked with versican protein were grafted at the border
of the presumptive NC streams. (O) Migration index in embryos with beads soaked in PBS (n = 4) or versican (n = 5). Bar, mean; error, SD. Note that grafts
of versican-soaked beads at the stream borders lead to a significant rescue of NC migration. (P) Placode localization is affected by VsMO injection, shown
in ISH of a representative stage 25 embryo using Eya1/FoxI1C (88%, n = 24). ***, P < 0.001; **, P < 0.01; ns, not significant. |
