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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).
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