Rif1 prolongs the embryonic S phase at the Drosophila mid-blastula transition
(A) Stills from time-lapse imaging of Rif1-GFP and mCherry-PCNA during cycle 13 after injection of either buffer (control) or dsRNA against the three mitotic cyclins (A, B, and B3) in cycle 10. The knockdown of the cyclins increases the persistence of Rif1 foci, extends S phase (PCNA staining), and blocks progression to cycle 14. The time of the last frame showing a Rif1 focus is indicated as the lifetime of Rif1 foci in (C). (B) Stills from time-lapse imaging of Rif1-GFP and mCherry-PCNA during cycle 14 after injection of either buffer (control) or mRNA encoding the Cdk1 activator Cdc25 (twine) in cycle 13. Artificial activation of Cdk1 during S phase 14 accelerates both the dissociation of Rif1 and late replication. In the control embryo, Rif1 foci were detected 70 min into S phase 14, and prominent foci of PCNA were documented at 74 min. Expression of Cdc25 resulted in the loss of Rif1 foci by 16:00, and the final late-replicating PCNA signal documented was at 18:00 (S7 Movie). (C) Plot of lifetime of Rif1 foci in minutes during either S phase 13 or S phase 14 following the indicated injection (S1 Data). Cdc25, cell division cycle 25; Cdk1, cyclin-dependent kinase 1; dsRNA, double-stranded RNA; GFP, green fluorescent protein; PCNA, proliferating cell nuclear antigen; Rif1, Rap1 interacting factor 1; RNAi, RNA interference.