Fig. 2. Wnt signaling is not required for neural crest induction by the DLMZ. (A) The activity of different Wnt antagonists is monitored by analyzing cement gland formation in stage 20 injected embryos: Wnt antagonism results in a striking enlargement of the cement gland [top panels, control embryos; middle panels, NFz8 mRNA treated (800 pg); bottom panel, GSK3 mRNA treated (800 pg)]. (B-E) Slug expression in explants. (B) DLMZ, (C) AC, (D) AC-DLMZ control recombinants, (E) NFz8 (800 pg) injected AC-DLMZ. The top four recombinants in E are also stained for ß-galactosidase activity. Strong Slug expression is found in the ectoderm of about half of the recombinants in D and E. (F) RT-PCR analysis after injecting increasing amounts of NFz8: lanes 3-6 and 7-10, 0-400-800-1600 pg of NFz8 mRNA injections in AC and AC-DLMZ, respectively. 400-800 pg injections do not block response to DLMZ signals. Lanes 1 and 2, controls (see Materials and Methods); n.i., non-injected. (G) GSK3 (800 pg) or dnTCF3 (1 ng) mRNA injections do not prevent the ectoderm to form neural crest in response to the DLMZ. (H) Xdd1 (1 ng) injections do not prevent neural crest marker induction either. (I) The AC+DLMZ recombinants elongate in the same way as controls even in presence of 400 pg of NFz8 (middle) but their elongation is abolished by injections of 1600 pg of NFz8 in the ectoderm (bottom).