Fig. 8. ATH5 expression is upregulated during the last S phase. (A) Retinal cells isolated at stages 22-23 and 28-29 were transfected with an ATH5-promoter/lacZ-reporter plasmid singly or in combinations with NGN2 or ATH5 expression vectors and pulse-labelled with [³H]-thymidine at the end of a 24-hour culture period. (Left) The number of lac⁺ cells counted when the reporter plasmid was transfected alone is set at 1. (Right) At stage 22-23, overexpression of NGN2 enhances promoter activity in proliferating cells (a) and increases the pool of nonradioactive cells whose ATH5 promoter is upregulated (b). At stage 28-29, lac⁺ cells whose promoter is strongly upregulated are unlabelled (d). The detection of double-labelled cells (c) and their increased number upon ATH5 overexpression indicate that ATH5 promoter activity is upregulated during the S phase. (B, left) A retina at stage 29-30 was pulse-labelled for 45 minutes with BrdU and chased for 15 minutes. Transverse sections were hybridized with an ATH5-specific riboprobe. Most BrdU-positive cells are in S phase and their nuclei are located on the vitreous side (vi) of the pz. ATH5 transcripts accumulate on the ventricular side (ve) of the pz in the region where cells are in the G1 and G2 phases of the cell cycle. A few BrdU-positive nuclei are located in this region (arrowheads). (Right) Schematic of mitosis in the pz. Scale bar: 40 µm.