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Figure 1. Msx1, Pax3, and Slug Expression in the Presumptive Neural Crest Domain and Msx1 Neural Crest-Inducing Activity In Vivo and in Explants(A) Msx1 (a–d) and Pax3 (a′–d′) expression in X. laevis at stages 11 (a and a′), 13 (b and b′), and 16 (c–d′) costained with Krox20 (d–d′, indicates third rhombomere). Arrows in a–a′ indicate the blastopore lips at stage 11. a–a′, lateral views; b–d′, dorsal views.(B) Coexpression of Msx1 and Slug by double ISH (Msx1 [blue]; Slug [red]; a; b, dorsal views; a′–b′, transverse sections).(C) Msx1 mRNA was injected in one blastomere at the four cell stage, LacZ is revealed in pink (yellow arrow points at injected side). The embryos were analyzed at early (stage 13, a, c, e, and g) or late (stage 16, b, d, f, and h) neurula stages by ISH for Slug (a and b), Pax3 (c and d), Twist (e and f), and Sox2 (g and h) (red bar, uninjected neural plate half; yellow bar, width of the neural plate on the injected side). Dorsal views.(D–F) RT-PCR analysis of neural, ectoderm and early NC markers: Pax3 and ZicR1 (D); AP-2, Snail, and Slug (E); FoxD3, Pax3, Zic5, and Sox9 (F) after coinjection of Msx1 and Noggin. (D) Lane 4, Noggin (25 pg); lane 5, Msx1 (500 pg); lane 6, Msx1 (500 pg) + Noggin (25pg). (E) Lane 4, Noggin (25 pg); lane 5, Msx1 (100 pg); lane 6, Msx1 (500 pg); lane 7, Msx1 (100 pg) + Noggin (25 pg); lane 8, Msx1 (500 pg) + Noggin (25 pg). (F) Lanes 1–4, as in (E). Lanes 5–7, Msx1 (100–250–500 pg). Lanes 8–10, Noggin (25 pg) + Msx1 mRNA (100–250–500 pg). |
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Figure 5. Neural Crest Induction by the WNT Pathway Requires Pax3, but Not Msx1 Activity(A) In vivo overexpression of Wnt7b in the neural fold results in Slug and Pax3 in the presence of control or Msx1 MOs. In contrast, Pax3-MO coinjections block Wnt7b activity and result in downregulation of Slug in the neural fold. (MOs are traced with fluorescence, and RNAs are traced with LacZ).(B) RT-PCR analysis of animal caps injected with Wnt7b + Noggin in the presence of control (lane 4), Msx1 (lanes 5 and 7), and Pax3 (lanes 6 and 7) MOs. Msx1-MO does not prevent Pax3 nor Slug induction, whereas Pax3-MO reduces Slug induction.(C) The activity of the canonical WNT pathway is required for activation of Msx1 and Pax3 in the neural folds: β-catenin MO (Supplemental Data) or of GSK3 injections in one cell at the 16 cell stage.(D) Model part 2: activation of Msx1 and Pax3 expression in the embryo requires an active WNT signaling, but WNT pathway depends on Pax3 function only for Slug induction. |
