DEV025890 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Rostral neural plate border cells are specified as neural crest cells at the late blastula stage. Consecutive sections showing expression of molecular markers in stage XI B, stage 2 RB and stage 4 RB explants. (A) Stage XI B explants (n>30) cultured for 36 hours generated Sox1⁺ and Sox2⁺ neural cells in the medial region of the explants and Ker⁺ epidermal cells in the lateral part of the explants, but no, or only a few, Snail2⁺ and HNK-1⁺ neural crest cells. (B) Stage 2 RB explants (n>30) cultured for 40 hours generated a few Snail2⁺ premigratory and many HNK-1⁺ migratory neural crest cells, but almost no Sox1⁺, Sox2⁺ neural cells or Ker⁺ epidermal cells. (C) Stage 4 RB explants (n>30) cultured for 22 hours generated Sox2⁺ and Ker⁺ placodal cells, but no Sox1⁺ and only a few Snail2⁺ and HNK-1⁺ cells. Scale bar: 100 µm.

• Supplemental Figure S2 -

  Fig. S2. In the absence of BMP activity, Wnt signals induce caudal character in prospective neural cells. Consecutive sections showing expression of molecular markers in explants cultured for 28 hours. (A) Stage 2 M explants (n=9) generated Sox1⁺ and Otx2⁺ cells, but no Gbx2⁺ cells. (B) Stage 2 M explants cultured together with Wnt3A and Noggin (n=14) generated Sox1⁺ and Gbx2⁺ cells, but no Otx2⁺ cells. Scale bar: 100 µm.

• Supplemental Figure S3 -

  Fig. S3. At the late blastula stage, prospective neural cells are specified as dorsal forebrain cells in response to low levels of Wnt activity. Consecutive sections showing expression of molecular markers in explants cultured for 48 hours. (A) Stage 2 M explants (n>30) generated BF1⁺ and Pax6⁺ cells, but no Nkx2.1⁺ cells. (B) Stage 2 M explants cultured together with mFrz8CRD (n=20) generated BF1⁺ and Nkx2.1⁺ cells, but no Pax6⁺ cells. Scale bar: 100 µm.

• Supplemental Figure S4 -

  Fig. S4. BMP4-induced Smad1/5/8 phosphorylation is blocked by Noggin and increased by higher concentrations of BMP4. Western blot analysis of phosphorylated Smad (p-Smad1/5/8) from stage 2 M explants cultured for 1 hour, alone or together with different combinations of BMP4 (5-10 ng/ml), Noggin or mFrz8CRD. p-Smad1/5/8 was induced by BMP4 (5 ng/ml), which was blocked by exposure to Noggin. BMP4 protein (10 ng/ml) induced a large relative increase in p-Smad1/5/8.

• Supplemental Figure S5 -

  Fig. S5. BMP activity is upregulated in neural plate border cells and Wnt activity is downregulated in rostral neural plate border cells between late blastula and late gastrula stages. Relative Bmp4, Dlx5 and Sp5 mRNA levels measured by quantitative real-time PCR in explants before culture. (A) mRNA expression of Bmp4 and of the BMP target gene Dlx5 were upregulated 6.0-fold and 14.7-fold, respectively, in stage 4 relative to stage 2 prospective neural plate border explants. Bars represent mean ± s.e.m. of three independent experiments. (B) Stage 2 prospective neural plate border cells express Sp5 mRNA, a Wnt target gene, which was downregulated 2.5-fold in stage 4 prospective rostral neural plate border cells and upregulated 3.1-fold in stage 4 prospective caudal neural plate border cells. Bars represent mean ± s.e.m. of five independent experiments.

• Supplemental Figure S6 -

  Fig. S6. Prospective neural plate border cells exhibit neural character during the Wnt-dependent early phase of specification. Consecutive sections showing expression of molecular markers in explants cultured for 8 hours. (A) Stage 2 M explants (n=8) generated Sox2⁺ and Otx2⁺ cells. (B) Stage 2 M explants cultured together with Wnt3A (n=8) generated Sox2⁺ and Otx2⁺ cells. (C) Stage 2 B explants (n=8) generated Sox2⁺ and Otx2⁺ cells. (D) Stage 2 M explants cultured together with BMP4 (5 ng/ml) (n=8) generated no Sox2⁺ or Otx2⁺ cells. Scale bar: 100 µm.

• Supplemental Figure S7 -

  Fig. S7. At the late blastula stage, BMP activity is higher in prospective epidermal cells than in prospective neural plate border cells. Relative Bmp4 and Dlx5 mRNA levels measured by quantitative real-time PCR in explants before culture. The levels of mRNA expression of Bmp4 and of the BMP target gene Dlx5 were 4.1-fold and 3.5-fold higher, respectively, in stage 2 L than in stage 2 B explants. Bars represent mean ± s.e.m. of five independent experiments.

• Supplemental Figure S8 -

  Fig. S8. Wnt-regulated temporal control of BMP exposure mediates the induction of neural plate border cells. Consecutive sections showing expression of molecular markers in explants cultured for 28 hours. (A) Stage 2 B explants (n>30) generated Snail2⁺ and HNK-1⁺ cells, but no, or only a few, Sox2⁺ and Ker⁺ cells, and no Sox1⁺ cells. (B) Stage 2 B explants cultured together with BMP4 (5 ng/ml) for the first 10 hours of culture (n=9) generated Ker⁺ cells, but no, or only a few, Sox1⁺, Sox2⁺, Snail2⁺ or HNK-1⁺ cells. (C) Stage 2 M explants (n>30) generated Sox1⁺ and Sox2⁺ cells, but no Snail2⁺, HNK-1⁺ or Ker⁺ cells. (D) Stage 2 M explants cultured together with Wnt for the entire culture period and BMP4 (5 ng/ml) for the last 18 hours (n=19) generated Snail2⁺ and HNK-1⁺ cells, and a few Ker⁺ cells, but no Sox1⁺ or Sox2⁺ cells. Scale bar: 100 µm.