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Fig. 1. Neural crest marker induction after recombining ectoderm to mesoderm. (A) The DMZ (blue) or the DLMZ (yellow) are dissected at stage 10-10.5 as depicted. (B) Each type of mesoderm explant is recombined to the animal cap ectoderm (AC, red) of stage 8-9 embryos, to form the DLMZ-AC and DMZ-AC recombinants, respectively. (C) RT-PCR analysis of gene expression in the stage 18 recombinants shows that the DLMZ-AC recombinants express a whole range of neural crest markers at stage 18 (Slug, FoxD3, Sox9, Zic5, Snail and Twist, lane 5), whereas the DMZ-AC recombinants express only a subset of them (lane 7). Lanes 1, 2: controls (see Materials and Methods). Lanes 3, 4, 6: isolated AC, DLMZ and DMZ, respectively. (D) In situ hybridization for the four most specific neural crest markers studied (see text) on normal embryos around stage 18 seen in dorsal view, anterior is towards the bottom. Note that Slug and FoxD3 are restricted to the neural crest, whereas Zic5 and Sox9 are also expressed in other areas. |
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Fig. 5. FGF8 induces neural crest in vivo and in vitro. (A-D) In vivo injections of FGF8 mRNA in one of two-cell stage embryos, analyzed by in situ hybridization for Slug (A,B) or MyoD (C,D) at stage 18-20. (A) Control embryos. (B) FGF8 mRNA unilateral injections result in a strong overexpression of Slug on the injected side (yellow arrows) and sometimes in the contralateral side and the anterior neural fold (red arrowheads). (C) Control embryos. (D) FGF8 mRNA injections (injected side indicated by yellow arrowheads) do not expand paraxial mesoderm, they even reduce it in some embryos (embryo on the right) (red arrowhead). (E) FGF8 mRNA is expressed as a ring around the blastopore at stage 11 (top), reinforced dorsally (red arrows). Later on, FGF8 is expressed in the DLMZ and downregulated in the midline (bottom, red arrow). (F) FGF8 mRNA injections induce neural crest markers in animal caps. RT-PCR analysis shows the induction of FoxD3 and Zic5 by 100 pg of FGF8 mRNA, but not of paraxial mesoderm formation. (G) When the caps are analyzed earlier (stage 15), increased doses of FGF8 induce strongly FoxD3, Sox9 and Zic5. By stage 19, FoxD3 and Zic5 expression was not maintained. |
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Fig. 8. Neural crest formation can be experimentally uncoupled from neural plate patterning. (A-D) Injected embryos were analyzed around stage 18 and stained for Slug expression. (A) Control embryo. (B) XFD injections result in gastrulation defects and loss of most Slug staining. (C) NFz8 injections most often produce an abnormally shaped neural plate, gastrulation defects and reduced Slug expression. (D) dnFGFR4a-injected embryos show severe gastrulation defects but still present a strong Slug staining (the right hand embryo is shown in side view). (E,F) Similar injections were analyzed at stage 11.5-12 for Otx2 expression. Otx2 labels the area anterior to the neural crest-forming regions. It is found further from the blastopore as development proceeds (blue bars measure the distance between the posterior part of the Otx2 domain and the blastopore; anterior is indicated by the red star). (E) Stage 11.5 (left) and stage 12 (right) control embryos. (F) NFz8-injected embryos show a strongly reduced posterior neural crest-forming domain. (G) dnFGFR4a injections result either in normal sized posterior domain (left) or strongly reduced ones (right). Both types of embryos will show a strong Slug expression at stage 18 (D). Red stars indicate anterior. |
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Fig. 6. In vivo analysis of neural crest and neural plate patterning after modification of Wnt signaling. (A,B) Control embryos stained for Slug and Krox20 at stage 19. (C,D) Wnt8 mRNA injections (50 pg) are followed by the extension of Slug and Krox 20 domains together, in the posterior parts of the embryos (arrows). (E,F) dnWnt8 mRNA injections (50 pg) result in reduction of both Slug and Krox 20 expression (arrows). |
