Worms at the larval L4 stage were incubated on 6 cm RNAi feeding plates for 28-34 hours at 25°C, or for 48-52 hours at 20°C when using the GFP-PSF-1 knock-in worms. Adult worms were then dissected in M9 medium (6 g/l Na2HPO4, 3 g/l KH2PO4, 5 g/l NaCl, 0.25 g/l MgSO4) and embryos were mounted on a 2% agarose pad. Time lapse images were then recorded as described previously17 (link), 18 (link), at 23–24°C using an Olympus IX81 microscope (MAG Biosystems) with a CSU-X1 spinning-disk confocal imager (Yokogawa Electric Corporation), a Cascade II camera (Photometrics) and a 60X/1.40 Plan Apochromat oil immersion lens (Olympus). A single optical section (z-layer) was imaged for each time point. Photobleaching of the female pronucleus in the first embryonic cell cycle was done with a 488nm laser using the ‘iLas2 system’ (Roger Scientific).
Images were captured using MetaMorph software (Molecular Devices) and analysed with ImageJ software (National Institutes of Health). For each timelapse experiment depicted in the figures, the raw images for selected timepoints were rotated in order to orient the anterior of the chosen embryo to the left, and then cropped to focus on a particular nucleus or nuclei, or on the entire embryo. The series of images were then combined into a contiguous sequence, and the images were subjected to Gaussian Blur with a radius of 1 pixel. Subsequently, the levels were adjusted, the pixel density was adjusted to 300 dots per inch and the bit depth was changed from 16-bits to 8-bits per channel. Images were processed in a similar manner in order to generate videos, except that timepoints were not combined into a sequence and the pixel density was not adjusted to 300 dpi.
To generate the data inFigure 3b , the duration of cell cycle phases in the second cell cycle (P1 cell) were measured as follows. Interphase was measured from nuclear formation (appearance of nuclear GFP-PSF-1) until the start of chromosome condensation, which marked the beginning of prophase. The latter phase ended with nuclear envelope breakdown, after which the time of metaphase and anaphase was measured as the period until nuclear envelop reformation.
Images were captured using MetaMorph software (Molecular Devices) and analysed with ImageJ software (National Institutes of Health). For each timelapse experiment depicted in the figures, the raw images for selected timepoints were rotated in order to orient the anterior of the chosen embryo to the left, and then cropped to focus on a particular nucleus or nuclei, or on the entire embryo. The series of images were then combined into a contiguous sequence, and the images were subjected to Gaussian Blur with a radius of 1 pixel. Subsequently, the levels were adjusted, the pixel density was adjusted to 300 dots per inch and the bit depth was changed from 16-bits to 8-bits per channel. Images were processed in a similar manner in order to generate videos, except that timepoints were not combined into a sequence and the pixel density was not adjusted to 300 dpi.
To generate the data in
