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Fig. 3. Xctr1 regulates Ras-ERK signaling. (A) Coprecipitation of Xctr1, Laloo, and the docking protein, SNT-1/FRS2α. (B) Xctr1 knockdown inhibits Laloo-mediated SNT-1 phosphorylation (0.37 ± 0.11-fold reduction by Xctr1MO; n = 3). Signal represents the phosphorylation of up to six tyrosine residues on SNT-1 (10–12). (C) Xctr1 knockdown inhibits ERK phosphorylation (0.62 ± 0.27-fold reduction by MO; n = 4). Effects of injection of Xctr1MO and Xctr1MM on the FGF-mediated dual phosphorylation (dp) of the ERK MAP kinase in animal cap explants. Explants were cultured in the presence of FGF for 2 h before Western blot analysis; inhibition of ERK phosphorylation by Xctr1MO was not apparent for the first hour of FGF stimulation (data not shown). (D) Inhibition of activin-mediated dorsal mesoderm induction in animal cap explants by 1 ng of dominant inhibitory Ras (dnRas) RNA (1). (E) Rescue of Xctr1 morpholino-mediated dorsal mesoderm inhibition by coinjection of 40-pg constitutively active Ras (v-Ras) RNA (1). (F) Injection of 1 ng dnRas RNA does not enhance elongation of animal caps by FGF. Ras inhibition also inhibits elongation in FGF-treated caps from embryos injected with Xctr1MO (data not shown). (G) RT-PCR analysis of Xctr1 expression in early stage Xenopus embryos. (H) Whole-mount in situ hybridization analysis of Xctr1 expression. Stages (Upper) 4, 8 (animal views), 18 (dorsal view), and (Lower) 32 (lateral view) are shown. Pronephric tubules are indicated by arrowhead; 250-ng morpholinos were injected at early cleavage stages as listed, and 10 ng/ml FGF and 0.5 ng/ml activin were added to animal cap explants at stage 8 as listed. |
