Effects of Shh and Noggin on neural crest formation demonstrate that BMP is required in the neural tube but not ectoderm

To define the timing of neural crest formation, we challenged the fate of p resumptive neural crest cells by grafting notochords, Sonic Hedgehog- (Shh) or Noggin-secreting cells at different stages of neurulation in chick embr yos. Notochords or Shh-secreting cells are able to prevent neural crest for mation at open neural plate levels, as assayed by DiI-labeling and expressi on of the transcription factor, Slug, suggesting that neural crest cells ar e not committed to their fate at this time. In contrast, the BMP signaling antagonist, Noggin, does not repress neural crest formation at the open neu ral plate stage, but does so if injected into the lumen of the closing neur al tube. The period of Noggin sensitivity corresponds to the time when BMPs are expressed in the dorsal neural tube but are down-regulated in the non- neural ectoderm, To confirm the timing of neural crest formation, Shh or No ggin were added to neural folds at defined times in culture, Shh inhibits n eural crest production at early stages (0-5 hours in culture), whereas Nogg in exerts an effect on neural crest production only later (5-10 hours in cu lture). Our results suggest three phases of neurulation that relate to neur al crest formation: (1) an initial BMP-independent phase that can be preven ted by Shh-mediated signals from the notochord; (2) an intermediate BMP-dep endent phase around the time of neural tube closure, when BMP-4 is expresse d in the dorsal neural tube; and (3) a later premigratory phase which is re fractory to exogenous Shh and Noggin.