March 1, 2009;
Differential requirements of BMP and Wnt signalling during gastrulation and neurulation define two steps in neural crest induction.
The neural crest is induced by a combination of secreted signals. Although previous models of neural crest induction have proposed a step-wise activation of these signals, the actual spatial and temporal requirement has not been analysed. Through analysing the role of the mesoderm
we show for the first time that specification of neural crest requires two temporally and chemically different steps: first, an induction at the gastrula
stage dependent on signals arising from the dorsolateral mesoderm
; and second, a maintenance step at the neurula
stage dependent on signals from tissues adjacent to the neural crest. By performing tissue
recombination experiments and using specific inhibitors of different inductive signals, we show that the first inductive step requires Wnt activation and BMP inhibition, whereas the later maintenance step requires activation of both pathways. This change in BMP necessity from BMP inhibition at gastrula
to BMP activation at neurula
stages is further supported by the dynamic expression of BMP4
and its antagonists, and is confirmed by direct measurements of BMP activity in the neural crest cells. The differential requirements of BMP activity allow us to propose an explanation for apparently discrepant results between chick and frog experiments. The demonstration that Wnt signals are required for neural crest induction by mesoderm
solves an additional long-standing controversy. Finally, our results emphasise the importance of considering the order of exposure to signals during an inductive event.
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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.
Fig. 3. Early Induction of the NC by DLMZ requires Wnt activation and BMP inhibition. (A-F) The DLMZ was dissected from stage 10.5 embryos and conjugated with animal caps taken from embryos injected at the eight-cell stage with 1 ng of dd2 or 0.6 ng of dnTCF3 mRNA together with FLDx. The conjugates were cultured until the equivalent of stage 15 and the expression of Snail2 (B-D) or Sox2 (E) was analysed. (B) Control conjugate of DLMZ with uninjected animal cap. (C) Conjugate of DLMZ with an animal cap injected with dd2 mRNA. (D) Conjugate of DLMZ with animal cap injected with dnTCF3 mRNA. (E) Sox2 expression showing continued presence of neural plate (68%, n=19). FDX injected animal caps also express Sox2 (data not shown, 63%, n=11). (F) Summary of the expression of Snail2 in conjugates. Each experiment was repeated three times with at least 26 explants each. (G-L) The DLMZ was dissected from stage 10.5 embryos injected at the eight-cell stage in the equatorial region with 1 ng of dnWNT8 mRNA or 2 ng of morpholinos against chordin (cho MO), and conjugated with animal caps taken from uninjected embryos. The conjugates were cultured until the equivalent of stage 15 and the expression of Snail2 (H-J) or Sox2 (K) was analysed. (H) Control conjugate. (I) Conjugate containing DLMZ injected with dnWnt8 mRNA. (J) Conjugate containing DLMZ injected with cho MO. (K) Sox2 expression showing inhibition of neural plate by cho MO (0% of expression, n=30), when compared with controls (75% of expression, n=20; not shown). (L) Summary of the expression of Snail2 in conjugates. Each experiment was repeated three times with at least 30 explants each. (M,N) In situ hybridisation of Wnt8 in a stage 10.25 gastrula (M) or in DLMZ (N). (O,P) In situ hybridisation of chordin in a stage 10.25 gastrula (O) or in DLMZ (P); inset in N shows LMZ. Lines in M and O indicate how the DLMZ was dissected.
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.
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.
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