Figure 1.

Ectodermal MAPK activity before and during gastrulation. (A) Effects of PD098059 on FGF-induced MAPK activity. Animal caps were isolated at stage 8, aged in VLCMR until stage 11, and treated with 50 μM PD098059. Animal caps were pretreated with PD098059 in dimethyl sulfoxide or dimethyl sulfoxide alone for 30 min before addition of 15 ng/ml Xenopus bFGF + 0.5 mg/ml BSA. After 1 hr of bFGF treatment, the animal caps were lysed and assayed for phosphorylation of the Xnf7 substrate peptide. Pretreatment with PD098059 reduces the amount of substrate phosphorylated by lysates of FGF-treated ectoderm by approximately 90% (n = 4). (B) MAPK activity in ectoderm isolated from midblastula (stage 8) and early gastrula (stage 10) embryos and in neural ectoderm isolated at midgastrula (NP-11). Positive controls include thiophosphorylated MAPK (thio-phos MAPK) and midblastula animal cap ectoderm treated with 100 ng/ml bFGF (FGF). The lysate of FGF-treated ectoderm was also run in the absence of substrate (FGF-no sub). Autoradiograms in A and B show an immunoblot probed with anti-MAPK 42-kDa antibody (Upper) and an autoradiogram of the phosphorylated 2.8-kDa peptide (Lower). A ratio of the intensity of the phosphorylated substrate band to that of the MAPK protein provides a relative measure of MAPK specific activity. Relative specific activity for MAPK in ectoderm increases 6-fold between stage 8 and stage 10, with an additional increase during neural specification. (Mean ± SEM, n = 5).