DEV019893 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Effect of FoxM1-MO on translation of ectopic FoxM1 mRNA. One-cell embryos were co-injected with FoxM1-Myc₂ mRNA (100 pg) and either control MO or FoxM1-MO (36 ng), cultured, collected at stage 13, and analyzed by immunoblotting with anti-Myc antibody or anti-ERK1 antibody (for loading control).

• Supplemental Figure S2 -

  Fig. S2. Effects of BMP inhibition or BMP signaling on cell proliferation and expression of cell-cycle regulators in animal caps. (A) Animal caps were isolated from late blastula embryos pre-injected with either Noggin mRNA (100 pg), Chordin mRNA (200 pg), or dnBMPR mRNA (500 pg) at the one-cell stage. They were cultured until sibling embryos reached stage 20 and then analyzed by either RT-PCR or immunoblotting. (B) Animal caps were isolated from late blastula embryos pre-injected with Noggin mRNA (100 pg) and either distilled H₂O, CA-Alk2 mRNA (200 pg), or BMP4/BMP7 mRNAs (4.6 ng each) at the one-cell stage. CA-Alk2 is a constitutively active form of BMP7 receptor (Suzuki et al., 1997). Note that the forced activation of BMP signaling by CA-Alk2 or BMPs inhibited the induction of FoxM1 and N-CAM by Noggin, while activating E-keratin for epidermalization.

  Suzuki, A., Kaneko, E., Ueno, N. and Hemmati-Brivanlou, A. (1997). Regulation of epidermal induction by BMP2 and BMP7 signaling. Dev. Biol. 189, 112-122.

• Supplemental Figure S3 -

  Fig. S3. Double staining of nuclei and N-tubulin mRNA in the neural plate. Embryos injected with FoxM1-MO (18 ng) at one blastomere at the two-cell stage were cultured until stage 14 and subjected to WISH for staining of N-tubulin mRNA. The embryos were cryosectioned, stained for their nuclei with Sytox Green (Invitrogen), and mounted for confocal microscopy. The top panel is a differential interference contrast image (N-tubulin expression), whereas the middle panel is a fluorescence image (nuclei). In these panels, the dorsal region is up, and the FoxM1-MO-injected side is on the right; the boxed areas are enlarged in the bottom panels. On the FoxM1-MO- uninjected side, N-tubulin was expressed in a population of cells in the sub-surface layer of the neuroectoderm (the staining in the box), as described previously (Chalmers et al., 2002). On the injected side, however, N-tubulin expression was not detected at all in the corresponding region, while the cell number was only moderately reduced (by ∼25%).

  Chalmers, A. D., Welchman, D. and Papalopulu, N. (2002). Intrinsic differences between the superficial and deep layers of the Xenopus ectoderm control primary neuronal differentiation. Dev. Cell 2, 171-182.

• Supplemental Figure S4 -

  Fig. S4. Temporal expression pattern of Cdc25B protein and effect of Cdc25B-MO on Cdc25B translation. (A) Embryos at the indicated stages were analyzed by immunoblotting with anti-Cdc25B antibody (upper panel); note that Cdc25B proteins are detected from stage 10 (instead of stage 9 for Cdc25B transcripts; see Fig. 4A). (B) One-cell embryos were injected with either control MO or Cdc25B-MO (36 ng), cultured, collected at stage 11, and analyzed by immunoblotting with anti-Cdc25B antibody or anti-ERK1 antibody (for control) (lower panel).

• Supplemental Figure S5 -

  Fig. S5. Temporal expression patterns of neural specification and differentiation markers. Embryos at the indicated stages of development were analyzed by RT-PCR. Expression of Sox2 was detected at low levels at the onset of gastrulation (st. 10), increased at the mid-gastrula stage (st. 11), and was maintained during the neurula stages (st. 13-16). Expression of N-CAM and N-tubulin began at the initial and early neurula stages (st. 13 and 14), respectively, and significantly increased thereafter.