Delayed APC/C activation extends the first mitosis of mouse embryos

Abstract

The correct temporal regulation of mitosis underpins genomic stability because it ensures the alignment of chromosomes on the mitotic spindle that is required for their proper segregation to the two daughter cells. Crucially, sister chromatid separation must be delayed until all the chromosomes have attached to the spindle; this is achieved by the Spindle Assembly Checkpoint (SAC) that inhibits the Anaphase Promoting Complex/Cyclosome (APC/C) ubiquitin ligase. In many species the first embryonic M-phase is significantly prolonged compared to the subsequent divisions, but the reason behind this has remained unclear. Here, we show that the first M-phase in the mouse embryo is significantly extended due to a delay in APC/C activation. Unlike in somatic cells, where the APC/C first targets cyclin A2 for degradation at nuclear envelope breakdown (NEBD), we find that in zygotes cyclin A2 remains stable for a significant period of time after NEBD. Our findings that the SAC prevents cyclin A2 degradation, whereas over-expressed Plk1 stimulates it, support our conclusion that the delay in cyclin A2 degradation is caused by low APC/C activity. As a consequence of delayed APC/C activation cyclin B1 stability in the first mitosis is also prolonged, leading to the unusual length of the first M-phase.

Figure 1

Spatiotemporal dynamics of cyclins A2 and B1 in mouse embryos (a) Spatiotemporal dynamics of cyclin A2-YFP in dividing zygotes. Selected images from a time-lapse recording, time shown in hours and minutes counted from the timepoint of nuclear envelope breakdown (NEBD). (b) Spatiotemporal dynamics of cyclin B1-YFP in dividing zygotes. Selected images from a time-lapse recording, time shown in hours and minutes counted from the timepoint of NEBD. (c) Quantification of cyclin A2-YFP degradation in 1- and 2-cell embryos, averaged from 30 and 37 embryos respectively. (d) Quantification of cyclin B1-Ruby degradation in 1- and 2-cell embryos, averaged from 55 and 31 embryos respectively. Scale bars in (a) and (b) are 20 µm. Plots in (c) and (d) show mean values + /− standard deviation.

Figure 2

Role of cyclin A2 in regulation of cyclin B1 degradation (a) Quantification of cyclin B1-Ruby degradation in zygotes injected with increasing concentrations (0, 0.07 μg/μl, 0.2 μg/μl, 0.4 μg/μl) of cyclin A2-YFP mRNA, averaged from 55, 41, 12 and 10 embryos respectively. Plots show mean values +/− standard deviation. Images show representative zygotes injected with increasing concentrations of cyclin A2-YFP mRNA and the same concentration of cyclin B1 mRNA. Over-expression of cyclin A2 was confirmed by an immunostaining (the bottom panel). Scale bar is 100 μm. (b) Quantification of cyclin B1-Ruby and cyclin A2-YFP degradation in a single representative zygote.

Figure 3

Role of SAC in regulation of cyclin A2 and B1 degradation (a) Quantification of cyclin B1-Ruby degradation in control zygotes and zygotes treated with reversine averaged from 55 and 57 embryos respectively. (b) Quantification of cyclin A2-YFP degradation in control zygotes and zygotes treated with reversine averaged from 30 and 31 embryos respectively. Plots in (a) and (b) show mean values +/− standard deviation.

Figure 4

Role of Emi 2 and Plk1 in the regulation of cyclin A2 and B1 degradation (a) Time between NEBD and the onset of cyclin B1-YFP degradation in zygotes injected with Emi2 MO and Emi2–5MP MO, averaged from 28 and 11 embryos respectively. The boxes show medians and the first and third quartiles. The whiskers are set at 1.5*IQR above the third and below the first quartile. Outlier values are marked with dots. (b) Quantification of cyclin B1-Ruby degradation in control zygotes, zygotes injected with wt Plk1-YFP or kd Plk1-YFP, and zygotes treated with BI2536, a Plk1 inhibitor, averaged from 55, 38, 11 and 12 embryos respectively. (c) Quantification of cyclin A2-YFP degradation in control zygotes, zygotes injected with wt Plk1-TagRFP and zygotes treated with BI2536, averaged from 30, 22 and 38 embryos respectively. (d) Quantification of cyclin B1-Ruby degradation in control zygotes and zygotes treated with BI2536 or BI2536 and reversine, averaged from 55, 12, and 15 embryos respectively. Plots in (b–d) show mean values +/− standard deviation.