Fig. 5. An endogenous dorsal to ventral gradient of Wnt signaling. Cotransfection pTOPRed, a reporter construct containing synthetic TCF binding sites driving red fluorescent protein, with pCIG was used to investigate the range of ß-catenin signaling in the neural tube. (a) GFP expression from pCIG shows the entire DV axis was transfected. (b) RFP from pTOPRed shows that the reporter is active across the dorsal three quarters of the neural tube and is more active in the dorsal-most quarter. The dorsal to ventral gradient of RFP expression is jagged, likely due to the variable levels of transfection between cells. By comparing the level of transfection of each cell as marked by GFP with the level of ß-catenin signaling as marked by RFP, a gradient of ß-catenin signaling across much of the DV axis is apparent in the merged image (c). (d-f) Transfection of pFOPRed in which the TCF binding sites are mutated shows no activity (e) despite high levels of cotransfection of pCIG (d). (f) Merged pFOPRed and pCIG images.