DEV01823 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Expression of Pax3, Zic1 and Foxd3 in early Xenopus embryos. Spatiotemporal expression of Pax3 (A,D), Zic1 (B,E) and Foxd3 (C,F) at the mid-gastrula stage (stage 11.5; A-C), the late gastrula stage (stage 13; D-F). Arrowhead, dorsal blastopore lip. (G) Western blot detection of the protein products of HA-tagged Pax3-GR and Zic1-GR at substantial levels in the injected embryos during stages 10.5-15. HSP70 was used as a loading control. (H) Percentages of ectopic Foxd3 expression induced by Pax3 and Zic1 misexpression in the ventral ectoderm. Pax3 or Zic1 mRNA, or both, were injected into the ventral-most animal blastomeres at the 16-cell stage.

• Supplemental Figure 2 -

  Fig. S2. MOs specifically inhibit the translation of Pax3 and Zic1 in the animal cap. All animal blastomeres of eight-cell stage embryos were injected with Pax3A-FLAG (50 pg/cell), Pax3B-FLAG (50 pg/cell) or Zic1-HA (50 pg/cell) mRNA, with or without Pax3A-MO (10 ng/cell), Pax3B-MO (10 ng/cell) and Zic1-MO (10 ng/cell), or control MOs (‘5-mis’ stands for control MOs with a 5-base mismatch) as indicated above the lanes. Animal cap explants were prepared at stage 9, and subjected to western blot analysis with FLAG and HA antibodies. ‘CDS’ indicates that synthetic mRNA contained only the coding sequence, wheresas ‘+5˘UTR’ means that the 5˘UTR, which is the target of the MOs, was included.

• Supplemental Figure 3 -

  Fig. S3. Co-activation of Pax3 and Zic1 in concert with Wnt signaling is essential for neural crest determination of the ectoderm in vivo. (A-F) Synthetic mRNA was injected into the ventral animal blastomeres at the eight-cell stage. Embryos were harvested at stage 15 for in situ hybridization with Foxd3 (A,C,E) and Slug (B,D,F) probes. A higher magnification view is shown at the right half of each panel. Pax3 and Zic1 mRNA injection with lacZ mRNA (A-F), dnTCF3 (50 pg/cell; C-F), and wild-type TCF3 (50 pg/cell; E,F).

• Supplemental Figure 4 -

  Fig. S4. Induction of Pax3, Zic1 and Foxd3 expression by FGF8 signals. For in vivo analysis, Fgf8b (0.1 pg DNA/cell) was injected into a dorsal blastomere at the 16-cell stage (A-C). For animal cap assay: Chd (50 pg DNA/cell) and Fgf8b (1 pg DNA/cell; D,G,I); Chd (50 pg DNA/cell), Fgf8b (1 pg DNA/cell) and Pax3-MO (10 ng/cell; E,J); and Chd (50 pg DNA/cell), Fgf8b (1 pg DNA/cell) and Zic1-MO (10 ng/cell; F,H), were injected into all animal blastomeres at eight-cell stage. Samples were harvested at stage 15 for in situ hybridization with Foxd3 (A,D-F), Pax3 (B,G,H) and Zic1 (C,I,J) probes.

• Supplemental Figure 5 -

  Fig. S5. Zic1-MO does not inhibit the expression of other Zic genes. Zic1-MO (20 ng/cell) was injected unilaterally into the animal blastomeres at the eight-cell stage. Embryos were harvested at stage 15 for in situ hybridization with Zic2, Zic3 and Zic5 probes. Note that Zic1-MO injection, which strongly suppresses Foxd3, showed little effect on the expression of other Zic genes, except that moderate expansion was seen in the neural crest regions (arrow).