Wnt-regulated temporal control of BMP exposure directs the choice between neural plate border and epidermal fate

doi:10.1242/dev.025890

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First published online December 7, 2008
doi: 10.1242/10.1242/dev.025890

Development 136, 73-83 (2009)
Published by The Company of Biologists 2009

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Wnt-regulated temporal control of BMP exposure directs the choice between neural plate border and epidermal fate

Cédric Patthey, Thomas Edlund^* and Lena Gunhaga^*

Umeå Center for Molecular Medicine, Building 6M, 4th floor, Umeå University, S-901 87 Umeå, Sweden.

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Fig. 1. Chick neural plate border cells are specified as neural crest cells at the late blastula stage. (A) Ectodermal explants were isolated, separated form the hypoblast/endoderm, and cultured in vitro to the developmental equivalent of stage 10 before fixation, freezing and sectioning. (B) Different combinations of molecular markers define specific ectodermal fates. (C) Schematic representations of stage 2 embryos indicating the position at which medial (M, red box), prospective border [RB (rostral neural plate border), B (border) and CB (caudal border), green boxes] and lateral (L, blue box) explants were isolated. (D-H) Consecutive sections showing expression of molecular markers in explants cultured for 28 hours. (D) Stage 2 M explants (n>30) generated Sox1⁺ and Sox2⁺ cells, but no Snail2⁺, HNK-1⁺ or Ker⁺ cells. (E-G) Stage 2 B explants at different rostrocaudal positions (RB, n=23; B, n>30; CB, n=13) generated Snail2⁺ and HNK-1⁺ cells, but no, or only a few, Sox1⁺, Sox2⁺ or Ker⁺ cells. (H) Stage 2 L explants (n>30) generated Ker⁺ cells, but no Sox1⁺, Sox2⁺, Snail2⁺ or HNK-1⁺ cells. Data are represented as the mean ± s.e.m. Scale bar: 100 µm.

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Fig. 2. BMP and Wnt signals are required for the specification of neural crest cells. (A-C) Consecutive sections showing expression of molecular markers in chick explants cultured for 28 hours. (A) Stage 2 B explants (n>30) generated Snail2⁺ and HNK-1⁺ cells, but no, or only a few Sox1⁺, Sox2⁺ or Ker⁺ cells. (B) Stage 2 B explants cultured in the presence of Noggin (n=26) generated Sox1⁺ and Sox2⁺ cells, but no Snail2⁺, HNK-1⁺ or Ker⁺ cells. (C) Stage 2 B explants cultured in the presence of mFrz8CRD (n>30) generated Sox1⁺ and Sox2⁺ cells, but no Snail2⁺, HNK-1⁺ or Ker⁺ cells. Data are represented as mean ± s.e.m. Scale bar: 100 µm. (D,E) Relative Dlx5 and Sp5 mRNA levels measured by quantitative real-time PCR. mRNA expression of the BMP target gene Dlx5 (D) and of the Wnt target gene Sp5 (E) is inhibited in stage 2 B explants cultured in the presence of Noggin (D) or mFrz8CRD (E). Bars represent mean ± s.e.m. of four (D) and five (E) independent experiments.

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Fig. 3. BMP and Wnt signals are required for the generation of epidermal cells. (A-C) Consecutive sections showing expression of molecular markers in chick explants cultured for 28 hours. (A) Stage 2 L explants (n>30) generated Ker⁺ cells, but no, or only a few, Sox1⁺, Sox2⁺, Snail2⁺ or HNK-1⁺ cells. (B) Stage 2 L explants cultured in the presence of Noggin (n>30) generated Sox1⁺ and Sox2⁺ cells, but no Snail2⁺, HNK-1⁺ or Ker⁺ cells. (C) Stage 2 L explants cultured in the presence of mFrz8CRD (n>30) generated Sox2⁺ and Ker⁺ cells, but no Sox1⁺, Snail2⁺ or HNK-1⁺ cells. (D) Stage 2 L explants cultured in the presence of both Noggin and mFrz8CRD (n=18) generated Sox1⁺ and Sox2⁺ cells, but no Snail2⁺, HNK-1⁺ or Ker⁺ cells. Data are represented as the mean ± s.e.m. Scale bar: 100 µm. (E) Relative Sp5 and Dlx5 mRNA levels measured by quantitative real-time PCR. The mRNA expression of the Wnt target gene Sp5, but not of the BMP target gene Dlx5, is inhibited in stage 2 L explants cultured in the presence of mFrz8CRD. Bars represent mean ± s.e.m. of five independent experiments. ^*P<0.05, ^**P<0.01; two-sided, one-sample t-test.

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Fig. 4. BMP, but not Wnt, signals play an instructive role in the specification of neural plate border cells. Consecutive sections showing expression of molecular markers in chick explants cultured for 28 hours. (A) Stage 2 M explants (n>30) generated Sox1⁺ and Sox2⁺ cells, but no Snail2⁺, HNK-1⁺ or Ker⁺ cells. (B) Stage 2 M explants cultured together with BMP4 (5 ng/ml) (n>30) generated Ker⁺ cells, but no, or only a few, Sox1⁺, Sox2⁺, Snail2⁺ or HNK-1⁺ cells. (C) Stage 2 M explants cultured together with Wnt3A (n>30) generated Snail2⁺ and HNK-1⁺ cells, but no, or only a few, Sox1⁺, Sox2⁺ or Ker⁺ cells. (D) Stage 2 M explants cultured together with Wnt3A and Noggin (n=23) generated Sox1⁺ and Sox2⁺ cells, but no Snail2⁺, HNK-1⁺ or Ker⁺ cells. (E) Stage 2 M explants cultured together with Wnt3A and BMP4 (5 ng/ml) (n=25) generated Ker⁺ cells, but no Sox1⁺, Sox2⁺, Snail2⁺ or HNK-1⁺ cells. (F) Stage 2 M explants cultured together with BMP4 (5 ng/ml) and mFrz8CRD (n=17) generated Sox2⁺ and Ker⁺ cells, but no Sox1⁺, Snail2⁺ or HNK-1⁺ cells. Data are represented as the mean ± s.e.m. Scale bar: 100 µm.

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Fig. 5. Wnt signaling promotes the generation of neural crest cells by inducing Bmp4 expression. Relative Bmp4 and Sp5 mRNA levels measured by quantitative real-time PCR in stage 2 M chick explants cultured in the absence (M) or presence (M+Wnt3A) of Wnt3A, or in stage 2 B explants cultured in the absence (B) or presence (B+Frz) of mFrz8CRD. Bars represent mean ± s.e.m. of five independent experiments. ^*P<0.05, ^**P<0.01; two-sided, one-sample t-test.

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Fig. 6. Wnt-regulated temporal control of BMP exposure mediates the induction of neural plate border cells. Consecutive sections showing expression of molecular markers in chick explants cultured for 28 hours. The schematics to the left illustrate the periods of exposure to mFrz8CRD (Frz), Noggin and BMP4 during the culture. (A) Stage 2 B explants (n>30) generated Snail2⁺ and HNK-1⁺ cells, but no, or only a few Sox1⁺, Sox2⁺ or Ker⁺ cells. (B) Stage 2 B explants cultured together with mFrz8CRD for the first 10 hours of culture (n=29) generated Sox1⁺ and Sox2⁺ cells, but no Snail2⁺, HNK-1⁺ or Ker⁺ cells. (C) Stage 2 B explants cultured together with mFrz8CRD for the last 18 hours of culture (n>30) generated Sox2⁺ and Ker⁺ cells, but no Sox1⁺, Snail2⁺ or HNK-1⁺ cells. (D) Stage 2 B explants cultured together with Noggin for the first 10 hours of culture (n=17) generated Snail2⁺ and HNK-1⁺ cells, and a few Sox2⁺ and Ker⁺ cells, but no Sox1⁺ cells. (E) Stage 2 B explants cultured together with Noggin for the last 18 hours of culture (n=16) generated Sox1⁺ and Sox2⁺ cells, but no Snail2⁺, HNK-1⁺ or Ker⁺ cells. (F) Stage 2 B explants cultured together with BMP4 (5 ng/ml) for the entire culture period (n=27) generated Ker⁺ cells, but no Sox1⁺, Sox2⁺, Snail2⁺ or HNK-1⁺ cells. (G) Stage 2 B explants cultured together with BMP4 (5 ng/ml) for the last 18 hours of culture (n>30) generated Snail2⁺ and HNK-1⁺ cells, and a few Sox2⁺ and Ker⁺ cells, but no Sox1⁺ cells. Data are represented as the mean ± s.e.m. Scale bar: 100 µm.

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Fig. 7. Early patterning of the chick non-neural ectoderm is regulated by temporal exposure to Wnt and BMP signals. Proposed Wnt and BMP signaling events required for the specification of rostral and caudal neural plate border cells, and of epidermal cells. (A) An early phase of Wnt signals and a late phase of BMP signals specify rostral neural plate border cells of olfactory and lens placodal (OLP) character. (B) An early phase of Wnt signals and a late phase of both Wnt and BMP signals specify caudal neural plate border cells of neural crest (NC) character. (C) An early and a late phase of both Wnt and BMP signals specify cells of epidermal character.

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