Fig. 6. Regulation of the ATH5 promoter during retinogenesis. (A) Retinal cells isolated at stages 22 (E3) to 37 (E12) were transfected with an ATH5-promoter/CAT-reporter plasmid singly or in combinations with ATH5 and/or NGN2 expression vectors. Cells were assayed for CAT activity 24 hours after transfection. ATH5 transcription is passing through three phases in the course of retinogenesis. During the first phase (HH22-HH24), the promoter is weakly active and responds strongly to NGN2 overexpression, except in the presence of ATH5. During the second phase (HH25-HH30), upregulation of promoter activity coincides with a transient increase in ATH5 mRNA (curve). ATH5 and NGN2 both enhance promoter activity and ATH5 becomes dominant over NGN2. The third phase (HH34 and beyond) sees a decrease in ATH5 mRNA and is marked by the inability of either proneural protein to transactivate the promoter. (B) Retinal cells isolated at stages 22-23 and 29-30 were transfected with an ATH5-promoter/lacZ-reporter plasmid singly or in combinations with NGN2 or ATH5 expression vectors. lac⁺ cells were counted after 24 hours in culture. The number of lac⁺ cells obtained upon transfection with a control SV40-promoter/lacZ-reporter plasmid at each stage is set at 100 and cell numbers are given relative to this value. (C) Schematic representation of promoter activity as revealed by X-gal and CAT assays. Approximately 30% of cells express ATH5 at stages 22-23 and 29-30. The horizontal arrows indicate average promoter activity as measured by CAT assay, the open arrowhead marks the threshold for X-gal detection. At stage 22-23, promoter activity is low and only one in 30 ATH5-expressing cells is detected by X-gal. Overexpression of NGN2 increases promoter activity 10-fold but only six out of 30 ATH5-expressing cells are stained with X-gal. At stage 29-30, the whole population of ATH5-expressing cells is stained with X-GAL upon ATH5 overexpression. (D) At stage 22-23, most cells are weakly stained with X-GAL (arrowheads in a). Overexpression of NGN2 strongly enhances promoter activity (b) and the number of X-gal stained cells. At stage 29-30, cells display strong promoter activity (c) and overexpression of ATH5 enhances staining intensity (d).