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First published online 19 November 2003
doi: 10.1242/dev.00878

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Regulation of Msx genes by a Bmp gradient is essential for neural crest specification

Celeste Tríbulo^1,*, Manuel J. Aybar^1,*, Vu H. Nguyen², Mary C. Mullins² and Roberto Mayor^1,3,

¹ Millennium Nucleus in Developmental Biology, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile
² Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6058, USA
³ Department of Anatomy and Developmental Biology, University College London, UK

View larger version (54K): [in a new window]   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).

View larger version (98K): [in a new window]   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 ventral ectoderm. 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.

View larger version (44K): [in a new window]   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 Bmpr 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) Bmp4 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.

View larger version (64K): [in a new window]   Fig. 3. Msxb expression is increased in Bmp/Smad mutant zebrafish embryos. Wild-type and mutant zebrafish embryos were analyzed by whole-mount in situ hybridization for the expression of Msxb at the five-somite stage. Lateral views, anterior is upwards. (A) Wild-type embryos show the characteristic dorsal expression in the embryo. (B) A swr mutant shows an expansion of the Msxb territory in anterior regions. (C) A sbn mutant embryo shows a dramatic ventral expansion in Msxb expression. (D) A snh mutant embryo shows a moderate, lateral expansion in the expression of Msxb, where the two domains of expression can be seen. (E) Flat-mount of a wild-type embryo analyzed for the expression of Krox20 (arrows) and Myod (bracket). (F) Swr mutant showing the ventral expansion of Krox20 (arrows) and Myod (bracket). Note that the Myod expression is disorganized but can be found in dorsal and ventral sides. (G) swr mutant injected with ntl/sptl morpholinos. A complete absence of Myod expression but an expansion in Krox20 (arrows) was observed. (H-J) swr mutant embryos were injected with chordin mRNA and the expression of Msxb was analyzed. Anterior is towards the left. (H) Control uninjected embryos show the characteristic expansion of Msxb expression. (I) Embryos injected with 50 pg of chordin mRNA; note a reduction in Msxb expression. (J) Embryos injected with 200 pg of chordin mRNA exhibit a total inhibition in the expression of Msxb. Each experiment was repeated at least twice, with similar results. Reducing Bmp signaling with Bmpr treatment yielded similar results to those shown here for chordin.

View larger version (38K): [in a new window]   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.

View larger version (93K): [in a new window]   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).

View larger version (45K): [in a new window]   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).