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There is evidence in Xenopus and zebrafish embryos that the neural crest/neural folds are specified at the border of the neural plate by a precise threshold concentration of a Bmp gradient. In order to understand the molecular mechanism by which a gradient of Bmp is able to specify the neural crest, we analyzed how the expression of Bmp targets, the Msx genes, is regulated and the role that Msx genes has in neural crest specification. As Msx genes are directly downstream of Bmp, we analyzed Msx gene expression after experimental modification in the level of Bmp activity by grafting a bead soaked with noggin into Xenopus embryos, by expressing in the ectoderm a dominant-negative Bmp4 or Bmp receptor in Xenopus and zebrafish embryos, and also through Bmp pathway component mutants in the zebrafish. All the results show that a reduction in the level of Bmp activity leads to an increase in the expression of Msx genes in the neural plate border. Interestingly, by reaching different levels of Bmp activity in animal cap ectoderm, we show that a specific concentration of Bmp induces msx1 expression to a level similar to that required to induce neural crest. Our results indicate that an intermediate level of Bmp activity specifies the expression of Msx genes in the neural fold region. In addition, we have analyzed the role that msx1 plays on neural crest specification. As msx1 has a role in dorsoventral pattering, we have carried out conditional gain- and loss-of-function experiments using different msx1 constructs fused to a glucocorticoid receptor element to avoid an early effect of this factor. We show that msx1 expression is able to induce all other early neural crest markers tested (snail, slug, foxd3) at the time of neural crest specification. Furthermore, the expression of a dominant negative of Msx genes leads to the inhibition of all the neural crest markers analyzed. It has been previously shown that snail is one of the earliest genes acting in the neural crest genetic cascade. In order to study the hierarchical relationship between msx1 and snail/slug we performed several rescue experiments using dominant negatives for these genes. The rescuing activity by snail and slug on neural crest development of the msx1 dominant negative, together with the inability of msx1 to rescue the dominant negatives of slug and snail strongly argue that msx1 is upstream of snail and slug in the genetic cascade that specifies the neural crest in the ectoderm. We propose a model where a gradient of Bmp activity specifies the expression of Msx genes in the neural folds, and that this expression is essential for the early specification of the neural crest.
Fig. 1. Msx genes are expressed in the neural crest region of Xenopus and zebrafish embryos. (A-G) Xenopus embryos. Stage 13. (H-K) Zebrafish expression. Bud and five-somites stage. Anterior is upwards. (A,B) Bmp4 expression. (C-G) msx1 expression. (H-K) msxb expression. Arrowhead indicates anterior neural fold; asterisk: indicates prospective neural crest. (A) Dorsal view, showing strong expression in the anterior neural fold. (B) Lateral view, showing strong expression in the anterior neural fold, intermediate in the prospective neural crest and weaker in the ventralectoderm. Arrow in B indicates expression in the ventral side. (C) Dorsal view of a double in situ hybridization for msx1 (purple) and XSlug (blue) of a mid/late gastrula stage embryo. Note the overlapping in the expression of both genes in the prospective neural crest region (square) in the whole embryo (C), in the sectioned embryo (F) and in a higher magnification (G). (D) Dorsal view showing the strong expression of msx1 in the neural folds. (E) Lateral view showing expression in the neural folds. (H) Dorsal view showing expression of msxb in the neural plate border. (I) Lateral view showing expression in the neural plate border. (J) Flat mount showing expression in the prospective neural crest region. (K) Higher magnification of J.
Fig. 2. msx1 expression is increased by inhibiting Bmp signaling in Xenopus embryos. One blastomere of an eight- to 16-cell stage embryo was injected with CM-Bmp4 mRNA (A,B) or δBmpr mRNA (D), or a bead soaked with noggin was grafted near the neural fold of a stage 11 embryo (C), and the expression of msx1 was analyzed at stage 17. Anterior is towards the right; the injected side was recognized by FLDx staining and the operated side by the bead, both are indicated by an arrowhead. (A) CM-Bmp4 mRNA (250 pg). Note the stronger expression in the injected side. (B) CM-Bmp4 mRNA (500 pg). Note the stronger and expanded expression in the injected side. (C) Embryo grafted with a noggin soaked with 100 μg/ml of noggin (asterisk). Note the expansion in expression at the grafted side. (D) δBmp4 mRNA (500 pg). Note the stronger and expanded expression in the injected side. (E) Summary of the results. The expression of msx1 was analyzed for each embryo comparing the injected and uninjected side. Total number of embryos is 450. Brackets indicate the domain of msx1 expression at the hindbrain level.
Fig. 6. msx1 participates in the early specification of the neural crest. One blastomere of a two-cell stage embryo was injected with 700 pg of msx-GR mRNA (A-E), with 700 pg of HDmsx-GR mRNA (F-J) or with different combinations of both mRNAs (K-P), treated with dexamethasone at stage 12.5. Embryos were fixed at stage 18/19 and the expression of several genes was analyzed. The arrowheads indicate the injected side that contained FLDx (see Materials and methods). Anterior is towards the right. (A-C,F-H) Neural crest markers. (A-C) Notice the expansion of the markers on the side injected with msx-GR. (A) XSlug expression (n=44, 68% of expansion). (B) XSnail expression (n=60; 80% of expansion). (C) foxd3 expression. (n=52, 61% of expansion). (F-H) Note the inhibition in the expression of the neural crest markers injected with HDmsx-GR. (F) XSlug expression (n=57, 65% of inhibition). (G) XSnail expression (n=42, 69% of inhibition). (H) foxd3 expression (n=66, 64% of inhibition). (D) XSox-2 expression in embryos injected with Msx-GR. Note the inhibition in the expression (n=63, 38% of inhibition). (I) XSox2 expression in embryo injected with HDmsx-GR. Note the expansion in the expression the injected side (n=54, 39% of expansion). (E) XK81a expression in embryos injected with Msx-GR. Note the inhibition in the expression (n=57, 28% of inhibition). (J) XK81a expression in embryos injected with HDmsx-GR. Note the expansion in the expression (n=62, 32% of expansion). (K-M) Embryos were injected with 500 pg of HDmsx-GR mRNA and 250 pg of msx-GR (ratio 2:1). Note the partial rescue in the expression of the neural crest markers. (N-P) Embryos were injected with 500 pg of HDmsx-GR mRNA and 500 pg of msx-GR (ratio 1:1). Note the rescue in the expression of the neural crest markers. (G) Summary of the expression of XSlug. The injected and uninjected side was analyzed for each embryo. Number of embryos analyzed for XSlug expression: 215. Note that the strong rescue (73%) was reached with a proportion of 1:1 for the injected mRNAs. Similar values of rescue were obtained for the other neural crest markers (69% for foxd3, total number is 220; 67% for XSnail, total number is 225).
Fig. 7. msx1 lies upstream of XSlug and XSnail in the cascade leading to neural crest development. Embryos were injected in one blastomere at the two-cell stage with different combinations of HDmsx-GR and XSlug-GR (A-G) or XSnail-GR (H-N), induced at stage 12 and the expression of the neural crest markers XSlug (A,D,H,K), foxd3 (B,E,I,L) and XSnail (C,F,J,M) was analyzed at stage 18. Anterior is towards the right. The injected side detected by fluorescein staining is indicated by an arrowhead. (A-C) Embryos were injected with 500 pg of HDmsx-GR mRNA and 500 pg of XSlug-GR (ratio 1:1). Note the rescue in the expression of the neural crest markers. (D-F) Embryos were injected with 250 pg of HDmsx-GR mRNA and 750 pg of XSlug-GR (ratio 1:3). Note the rescue in the expression of the neural crest markers. (G) Summary of the expression of XSlug. The injected and uninjected side was analyzed for each embryo. Number of embryos analyzed for XSlug expression is 225. Note that the strong rescue (65%) was reached with a ratio of 1:1 for the injected mRNAs. Similar values of rescue were obtained for the other neural crest markers (72% for foxd3, total number is 225; 68% for XSnail, total number is 235). (H,I) Embryos were injected with 500 pg of HDmsx-GR mRNA and 500 pg of XSnail-GR (proportion of 1:1). Note the rescue in the expression of the neural crest markers. (K-M) Embryos were injected with 250 pg of HDmsx-GR mRNA and 750 pg of XSnail-GR (proportion of 1:3). Note the rescue in the expression of the neural crest markers. (N) Summary of the expression of XSlug. The injected and uninjected side was analyzed for each embryo. Number of embryos analyzed for XSlug expression is 215. Note that the strong rescue (67%) was reached with a ratio of 1:1 for the injected mRNAs. Similar values of rescue were obtained for the other neural crest markers (51% for foxd3, total number is 202; 62% for XSnail, total number is 215).
Fig. 5.
msx1 fusion proteins and its phenotypic effects. (A) The constructs used to produce the Msx genes fusion proteins are represented in this figure. HD, msx homeodomain. GR, ligand binding domain of glucocorticoid receptor. See Materials and methods for details. (B) Embryos were injected with 700 pg of the indicated constructs, treated with dexamethasone immediately after the injection and the phenotype was analyzed at the tadpole stage. Anterior is towards the right. Top embryo, uninjected control; middle embryo, embryos injected with msx-GR. Note the inhibition of the anterior structures and ventralization of the embryo, similar to the effect of injection Msx genes mRNA (not shown). Bottom embryo, embryo injected with HDmsx-GR (dominant negative). Note that the effect, dorsalization, is similar to the injection of dominant negatives of msx1 (not shown).
Fig. 4.
Msx1 expression is specified by a threshold concentration of Bmp. One-cell stage embryos were injected with a combination of 50 pg of Wnt5a mRNA and different amounts of CM-Bmp4 mRNA, which are indicated in the figure. Animal caps were dissected at stage 9 and the expression of msx-1, slug and histone H4 was analyzed by RT-PCR when sibling embryos reached the neurula stage 16. (A) Embryos and animal cap samples are shown. (B) Quantification of the gel shown in A.
