Fig. 1. Wnt signaling components are expressed in the marginal neural retina. (A-D) In situ hybridization of stage 22 (E4) neural retina probed with chicken Wnt2b (A), Fzd4 (B), Fzd5 (C) and LEF1 (D). le, lens. Scale bars: 100 µm. (E) Western blot of the immunoprecipitates from COS7 cells co-transfected with Fzds-CRD and Wnt2b-Fc. The cells were co-transfected with the construct described below, and the cell lysates were immunoprecipitated with protein A. The immunoprecipitates were detected with the antibodies shown at the top. The Wnt2b-Fc binds both mouse Fzd4-CRD and human Fzd5-CRD, whereas the control c-cad7-Fc did not. (F) Stabilization of cytosolic ß-catenin and upregulation of LEF1 mRNA by chicken Wnt2b. Retinal cultures were incubated for 6 hours in the absence or presence of chicken Wnt2b-conditioned medium (CM) or soluble mouse Fzd4 lysate described below. The membrane-associated and cytosolic ß-catenins were then analyzed by western blotting, and the level of LEF1 mRNA expression was analyzed by RT-PCR. mRNA expression of N-cadherin was used as a control for the RT-PCR analysis. Cytosolic ß-catenin and LEF1 mRNA expression were clearly upregulated in the presence of chicken Wnt2b, an effect completely blocked by the addition of mouse Fzd4-CRD. Molecular weight markers are 200 kDa, 116 kDa, 98 kDa, 66 kDa, 45 kDa and 31 kDa. (G) Relative ratio of cytoplasmic ß-catenin to membrane-bound ß-catenin and of LEF1 mRNA to N-cadherin mRNA. The ratio in the control culture medium was normalized to 1.

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