This movie illustrates the early division up to the four-cell stage of a C. elegans embryo depleted of npl-4 by RNAi expressing H2B::GFP by DIC and fluorescence microscopy. GFP-tagged histone H2B allows chromosome segregation to be followed. In wild-type, DNA replication generates two sets of sister chromatids that segregate toward opposite spindle poles during mitosis. The first C. elegans embryonic division of the P0 zygote is asymmetric and generates an anterior AB cell, and a smaller posterior P1 cell. These cells have different developmental fates and division timing, with AB dividing approximately 2 min before P1. In npl-4(RNAi) embryos, the movie shows chromosome bridges during mitosis as well as a delay in chromatin condensation, especially in the P1 cell. The movie corresponds to experiments shown in Fig. 3A and Fig S5 and Movie 8 of Mouysset et al. Movie is CIL 25635 and original data is CIL35149.
RNA interference was performed using the feeding method. L4 larvae were placed on IPTG-containing plates seeded with Escherichia coli [HT115(DE3)] expressing double-stranded RNA. Eggs from unc-119(ed3) ruls32[unc-119(+) pie-1::GFP::H2B]III with npl-4(RNAi) were extruded in M9 buffer from dissected adult worms and mounted on 2% agarose pads. Recordings were acquired in 20- or 10-second intervals (2 × 2 binning) with an Orca ER 12-bit digital camera (Hamamatsu) mounted on a wide-field microscope (Axioplan2, 40×/1.3 Plan-Apochromat objective; Carl Zeiss) or a spinning disk confocal microscope (Axioplan, 63×/1.4 Plan-Apochromat objective; Carl Zeiss; and Yokogawa disk head). Image processing was done with AxioVision (Carl Zeiss) or MetaMorph software (Universal Imaging).
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