Visualizing replication forks by GFP–CDC-45 localization. (A) Images from time-lapse sequences taken by a spinning-disk confocal microscope of wild-type, mcm-7 RNAi, and pol-α RNAi embryos expressing GFP–CDC-45 (left images in each panel) and mCherry-Histone (right images in each panel). Cell cycle stages are indicated. Wild-type, mcm-7 RNAi, and pol-α RNAi embryos are shown in the left, middle, and right panels, respectively. Asterisks indicate the position of the polar body. Red arrowhead shows GFP–CDC-45 colocalizing with chromatin. Bar, 10 µm. The top diagrams immediately below the time-lapse series show enrichment of nuclear GFP–CDC-45 relative to the cytoplasm. The bottom diagrams indicate the relative amount of nuclear GFP–CDC-45 (fluorescence intensity multiplied by nuclear volume). The blue lines are smoothings of the data by averaging 10 successive time points. (B) Estimate of S phase length in wild-type and pol-α RNAi embryos by quantification of the period in which GFP–CDC-45 is enriched on chromatin by >20% of its maximum. n = 5. Error bars represent SD. See also Video 3 for wild type.