Figure 3

The Pax6 binding site is required for full activity of the putative Er81 promoter. (A) Exogenously expressed Pax6 activated a co-transfected Er81 promoter-reporter construct in HeLa cells in a dosage-dependent manner. (B) Mutation of the Pax6 binding site completely abolished Pax6-dependent luciferase reporter activity of the putative Er81 promoter in HeLa cells. The plasmid combinations used for transfections are indicated. (C) Mutation of the Pax6-binding site led to a significant decrease in Er81 promoter activity in mouse embryonic (E12.5) primary cortical cultures. (D) The putative Er81 promoter exhibited high activity in Neuro-2A cells (compare Er81(wt)-luc construct and control pGL3). Deletion of the Pax6 binding site did not affect activation of the putative Er81 promoter in Neuro-2A cells (compare Er81(wt)-luc and Er81(mut)-luc constructs). (E-K) Pax6 activated an Er81 promoter-reporter construct in ovo. (E) GFP immunoreactivity demonstrates the high efficiency of the electroporation method. (E/F) After co-electroporation of Er81(wt)-Luc and CMV-eGFP, no luciferase-positive cells were detected. (G-I) Co-electroporation of Er81(wt)-Luc and CMV-Pax6 promoted expression from the Er81 promoter-luciferase reporter construct in hindbrain of chick embryos. (K) The table shows the number of embryos used for immunohistochemical analyses and the results obtained with the indicated antibodies. The strength of the immunohistochemical signal is designated as no, weak or strong staining. Note that the images shown in (E-H) represent strong staining for GFP, Luciferase and Pax6 antibodies. (L/N) Consistently, co-electroporation of Er81(wt)-Luc and CMV-Pax6 in isthmic region, where Pax6 does not express, led to up-regulation of the Er81 promoter-controlled luciferase reporter. Arrows in images for the whole embryo indicated a region where plasmids were injected. Luciferase activity is expressed as mean ± standard deviation (error bars) in each assay. Er81(wt)-Luc, wild-type putative Er81 promoter-reporter construct; Er81(mut)-Luc, mutated putative Er81 promoter-reporter construct.