Fig. 4. Pax3 and Zic1 promote neural crest differentiation in Wnt-treated animal cap ectoderm. All animal blastomeres of eight-cell-stage embryos were injected with: Pax3 (50 pg/cell; A,R,S); Pax3 and Wnt3a (50 pg of each/cell; B,C); Zic1 (50 pg/cell; E); Zic1 and Wnt3a (50 pg of each/cell; F,G); Pax3 (50 pg/cell), Wnt3a (50 pg/cell) and Zic1-MO (10 ng/cell; D); Zic1 (50 pg/cell), Wnt3a (50 pg/cell) and Pax3-MO (10 ng/cell; H); Wnt3a (50 pg/cell; inset of B); Pax3, Wnt3a and Bmp4 (50 pg of each /cell; I,J); Zic1, Wnt3a and Bmp4 (50 pg of each/cell; K,L); Pax3, Zic1 and Wnt3a (50 pg of each/cell; M); Pax3, Zic1, Wnt3a and Bmp4 (50 pg of each/cell; N); Chd (50 pg/cell; O); Chd and Bmp4 (50 pg of each/cell; P); or Pax3 (50 pg/cell) and Zic1-MO (10 ng/cell; S). Bmp4 injection was performed by using plasmid DNA (pCS2-Bmp4). Animal cap explants were prepared at stage 9 and harvested at stage 15 for in situ hybridization with Foxd3 (A,B,D-F,H,I,K,M,N), Zic1 (C,J), Pax3 (G,L) or nrp (O,P) probes. (Q) Scheme of the procedure of the animal cap dissociation experiments. (R,S) Animal cap cells were dissociated at stage 9 and harvested for RT-PCR when the siblings reached stage 15. Wnt3a proteins were added at the stage indicated above.