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Fig. 1. NC fate map. (A) Stage 10 embryos were injected with small quantities of the lipophilic marker DiI to label prospective neural crest cells and the surrounding tissues. (B-E) Representative examples of the labelled cells contributing to the cephalic NC (B), trunk NC (C), midbrain (D) and posterior intermediate mesoderm (E). Broken lines outline the neural tube (white), notochord (yellow) and somites (green). (F,G) DLMZ becomes IM and underlies the NC. (F) DiI was used to label the DLMZ of a stage 10 embryo. At the neurula stage (stage 16), the embryos were fixed and in situ hybridisation against Snail2 was performed. (G) Section showing fluorescence in the IM. (G′) Same section as in G showing Snail2 expression (G′) Merge of G and G′. (H) The position of each labelled cell was mapped onto a photo of a stage 10 embryo. Each colour corresponds to the key shown in A. (I) A summary of fate map at stage gastrula stage. (J) Summary of the position of NC in relation to IM at the neurula stage. |
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Fig. 4. NC and cement gland have different sensitivity to Wnt inhibition. Embryos were injected at the eight-cell stage embryo with the indicated mRNA, fixed between stages 16-18 when the expression of Snail2 was analysed. Right side of the embryo corresponds to the injected side. (A) Control embryo. (B) Embryo injected with 1 ng of dd2 mRNA. (C) Embryo injected with 0.6 ng of dnTCF3. (D) Embryo injected with 0.5 ng of GSK3 mRNA. (E) Embryo injected with 2 ng of GSK3 mRNA. (F) Embryo injected with 0.5 ng GSK3 mRNA showing cement gland expansion (arrowhead); (F′) fluorescence overlay showing site of injection. (G) Embryo injected with 2 ng GSK3 showing cement gland expansion (arrow); (G′) fluorescence overlay showing site of injection. (H) Summary of the results in whole embryos. Each experiment was repeated at least three times. |
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Fig. 5. NC maintenance requires activation of Wnt and BMP. (A-E) Explants of the NC and underlying intermediate mesoderm were taken at stage 16, cultured until sibling embryos were at stage 23 and analysed for the expression of the NC marker Snail2. Position of beads is indicated by the blue circle. (A) Diagram of experiments shown in B-E. (B) Control explant cultured in the presence of BSA-soaked bead. (C-E) Explant cultured in the presence of beads soaked with Dkk1 (C), Noggin (D), BMP4 (E). (F-I) Explant of the NC alone were taken at stage 16 and cultured until sibling embryos were at stage 23, then analysed for expression of Snail2. (F) Diagram of experiments shown in G-I. (G) Control explant. (H) Explant from embryos previously injected with β-cateninGR were cultured with or without dexamethasone. (I) Explant cultured in the presence of BMP. For each condition, the percentage of conjugates expressing Snail2 is summarised in the graphs. Each experiment was repeated at least three times. (J,K) IM was dissected from stage 16 embryos and the expression of Wnt8 (J) and BMP4 (K) was analysed. (L-Q) Analysis of NC markers and inducers in stage 16-18 neurula embryos as indicated. (L) Double stating against Snail2 and 12-101 antigen (a muscle specific monoclonal antibody) (Kintner and Brockes, 1984). (M) Double in situ hybridisation against Snail2 and Wnt8. (N) BMP4 expression. (O-Q) Sections of equivalent embryos to those shown in L-N. n, notochord; S, somites; IM, intermediate mesoderm; NC, neural crest. (O) Snail2/12-101 staining showing that it is the IM and not the somite the tissue that underlay the NC. (P) Wnt8 is expressed in the IM. (Q) BMP4 is expressed in the ectoderm next to or within the NC. |
