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Fig. 9. Ectodermal explants do not require FGF signaling to be neuralized by Noggin. (A) X. laevis embryos were injected in both blastomeres at the two cell stage with β-catenin MO, then re-injected in the animal pole at the 4 cell stage with 1 or 10 pg of noggin mRNA. Embryos were cultured in SU5402 or 0.32% DMSO from the 32 cell stage until stage 8. At stage 8, animal caps were cut, returned to SU5402 or DMSO, and cultured to stage 24. Whole embryos treated with increasing levels of SU5402 from stage 9 to late neurula stages were stained for apoptotic nuclei using TUNEL (B). 93% of embryos treated with 100 μM SU5402 had widespread TUNEL staining (n = 15). Animal caps from β-catenin morphants and control embryos injected with noggin mRNA were analyzed by in situ hybridization for expression of nrp (A), or by RT-PCR for expression of nrp, and sox2 (D). Ornithine decarboxylase (ODC) was used as a loading control. The efficiency of this SU5402 treatment protocol was confirmed by assaying expression of xbra at stage 11 (C). 0% of Su5402 treated embryos expressed xbra (n = 36). Expression of nrp and sox2 was also quantified using quantitative RT-PCR (F, G) and normalized to ODC expression. Animal caps cut from SU5402 treated, noggin-injected embryos still express nrp and sox2 at comparable levels to animal caps from noggin injected embryos treated only with DMSO. This is also true of animal caps cut from noggin-injected, β-catenin-injected, SU5402-treated embryos. |
