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Fig. 2. Id3 is required for neural crest precursor formation. (A) Western blot of lysates prepared from control embryos or embryos injected with an epitope tagged Id3 and then further injected with control or Id3 MOs demonstrates successful Id3 depletion. (B) Western blot of lysates from injected embryos demonstrating that although Id3 MOs efficiently deplete Xenopus Id3 protein, they do not deplete the human Id3 used for rescue experiments. The size difference between Xenopus and human Id3 is primarily due to the presence of five rather than six Myc epitopes, respectively. (C,D) In situ hybridization of embryos injected with Id3 MOs shows loss of Slug (C) and Sox10 (D) expression on the injected side of the embryo (arrowheads). (E) Id3-depleted embryos show reduced or absent migratory neural crest cells on the injected side (arrow), as visualized by Twist expression. (F) Loss of Slug expression in Id3-depleted embryos can be rescued by subsequent injection of human Id3, translation of which is not blocked by the MOs. Arrowhead indicates the injected side, red staining is from lineage tracer β-gal. |
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Fig. 5. (A) In situ hybridization showing Sox10 expression just after the onset of neural crest migration. At these stages, Sox10 marks the entire neural crest stem cell population. (B) Double in situ hybridization showing expression of Sox10 (cyan) and N-tubulin (magenta) at stage 28. Sox10 expression at these stages is restricted to glia in the peripheral ganglia and CNS (aqua) as well as to melanoblasts (not shown). (C,D) Forced expression of Id3 leads to the persistence of Sox10 expression in neural crest cells migrating to the pharyngeal pouches on the injected side of the embryo (C, arrows). At this stage, expression of Sox10 has normally been downregulated in all neural crest cells except those committing to a glial or melanocyte fate, as seen on the uninjected side of the embryo (D). (E,F) No significant difference is seen in the number of migratory neural crest precursor cells following forced Id3 expression, as visualized by comparing expressing of Twist on the Id3-injected (E) versus uninjected (F) side of the embryo. (G,H) Overexpression of Slug leads to expanded expression of markers of neural crest precursor cells on the injected side of the embryo (arrowheads) at neural plate stages. (I,J) In contrast to forced Id3 expression, Slug expression does not lead to persistent expression of Sox10 in migrating neural crest cells (arrows indicate location of migratory neural crest cells). |
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Fig. 7. Promotion of neural crest progenitor fate is a conserved activity of Id family proteins. (A,B) Expression of an N-terminally tagged form of Xenopus Id2 phenocopies the effects of Id3, leading to the persistent expression of Sox10 (red arrows) on the injected (A) versus control (B) side of the embryo. (C-F) Expression of N-terminally epitope tagged forms of human Id2 (C versus D) and human Id3 (E versus F) had identical effects. |
