Fig. 3. Requirement of FoxM1 for neuronal differentiation but not specification. (A) Xenopus embryos pre-injected with control MO or FoxM1-MO (36 ng) together with or without FoxM1 mRNA (200 pg) at the one-cell stage were cultured until st. 33 and photographed (left panels). For RT-PCR analysis, embryos were collected at st. 28 (right panel). (B) Embryos injected with lacZ mRNA (100 pg) and either control MO or FoxM1-MO (18 ng) at one blastomere at the two-cell stage were cultured until st. 14 for WISH analysis of Xngnr1, N-tubulin and MyoD, or until st. 16 for analysis of Sox2 and N-CAM. In each panel, the injected side (β-Gal, red) of the embryo is on the right (dorsal view, anterior up). (C) Animal caps from the late blastula embryos pre-injected with control MO or FoxM1-MO (36 ng) at the one-cell stage were cultured with or without Activin (200 pM) until sibling control embryos reached st. 20 and then analyzed by RT-PCR. (D) Animal caps pre-treated with Noggin mRNA (100 pg) and either control MO or FoxM1-MO (36 ng) were cultured and analyzed as in C. (E) Animal caps pre-treated or not with Noggin mRNA (100 pg) were incubated for the indicated times and analyzed by RT-PCR. Whereas FoxM1 expression in control animal caps decreased after 2 hours of incubation, that in Noggin-treated animal caps remained constant until 12 hours of incubation, indicating that the induction of FoxM1 expression by Noggin began after 2 hours of incubation. Note that Sox2 began to be expressed coincidently with FoxM1, whereas expression of N-CAM and N-tubulin began after FoxM1, in Noggin-treated animal caps.