Figure 3

Dkk3 is a positive regulator of Wnt signaling in Müller glia and a negative regulator in COS7 cells. (A) Wnt signaling was induced by Wnt3a conditioned media in the MIO-M1 human Müller glia cell line, measured using the TOP-FLASH Wnt luciferase reporter assay. The mutated control reporter FOP-FLASH did not show a response to Wnt3a. Induction is expressed as fold change of conditioned media containing Wnt3a to conditioned media not containing Wnt3a. (B) Luciferase reporter assays in cells transfected with Dkk1, Dkk3 and a control gene (GFP). Dkk3 increased Wnt3a-induced Wnt signaling whereas Dkk1 decreased Wnt signaling in the MIO-M1 human Müller glia cell line (black bars). Wnt signaling was measured using the TOP-FLASH luciferase assay. Wnt signaling is shown relative to GFP-expressing cells. In the COS7 cell line (grey bars), Dkk1 reduced Wnt signaling and Dkk3 had a small but not significant inhibitory effect. (C) Primary Müller glia cultured from the mouse retina that were treated with conditioned media with or without Dkk3 had baseline luciferase levels. Incubation with Wnt3a showed a significant induction of Wnt signaling that was greater when Dkk3 was present (Dkk3+Wnt3a) than when Wnt3a was added alone without Dkk3 (Control+Wnt3a). * = p < 0.05, *** = p < 0.001, relative to GFP-expressing cells. Mean ± standard deviation is shown.