Figure 6.

Merlin inhibits Wnt/β-catenin signaling in Xenopus embryos. (a) Animal caps isolated from embryos were injected with 2 pg of Wnt8 (or 10 pg of LRP6ΔN) at one cell stage with or without 500 pg of each Merlin mRNA. Expression levels of Wnt target genes (Siamois, Xnr3) and others were examined by RT-PCR at stage 10.5. ODC (Ornithine decarboxylase 1), a loading control; Ct (Control), animal cap samples obtained from non-injected embryos; WE, whole embryo as a positive control. (b) Merlin mRNA was injected at one cell stage, and total RNA was extracted from the dorsal marginal zone (DMZ) at stage 10.5 to synthesize cDNA for RT-PCR analysis. (c) Formation of secondary embryonic axis by injection of 1 pg of LRP6ΔN mRNA into the ventral side of two blastomeres from four-cell stage embryos was severely reduced by co-injection of 200 pg of Merlin mRNA. (d) Injection of Merlin mRNA into the dorsal side of embryos blocked formation of the embryonic axis (ventralization). (e and f) Animal caps isolated from embryos were injected with 2 pg of Wnt8, 10 pg of LRP6ΔN, or 100 pg of β-catenin S37A at one cell stage with or without 500 pg of each Merlin (SA/SD) mRNA. Expression of Wnt target genes and ODC was examined by RT-PCR at stage 10.5 (left panel). Ectopic expression of Flag-Merlin was examined by western blotting (right panel). (g) Nf2 morpholino (Nf2 MO) was injected into the dorsal or ventral side of two blastomeres from four-cell stage embryos (right panel), and total RNAs were extracted from the DMZ (left panel) or VMZ (middle panel) at indicated stages for RT-PCR. Ct (Control), DMZ, or VMZ samples were obtained from non-injected embryos; AC, animal cap as a negative control for Wnt target gene expression; WE, whole embryo as a positive control. All experiments were performed more than three times, and these data are representative