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Abstract
During the early development of Xenopus laevis embryos, the first mitotic cell cycle is long (∼85 min) and the subsequent 11 cycles are short (∼30 min) and clock-like. Here we address the question of how the Cdk1 cell cycle oscillator changes between these two modes of operation. We found that the change can be attributed to an alteration in the balance between Wee1/Myt1 and Cdc25. The change in balance converts a circuit that acts like a positive-plus-negative feedback oscillator, with spikes of Cdk1 activation, to one that acts like a negative-feedback-only oscillator, with a shorter period and smoothly varying Cdk1 activity. Shortening the first cycle, by treating embryos with the Wee1A/Myt1 inhibitor PD0166285, resulted in a dramatic reduction in embryo viability, and restoring the length of the first cycle in inhibitor-treated embryos with low doses of cycloheximide partially rescued viability. Computations with an experimentally parameterized mathematical model show that modest changes in the Wee1/Cdc25 ratio can account for the observed qualitative changes in the cell cycle. The high ratio in the first cycle allows the period to be long and tunable, and decreasing the ratio in the subsequent cycles allows the oscillator to run at a maximal speed. Thus, the embryo rewires its feedback regulation to meet two different developmental requirements during early development.
Figure 2. Stronger Tyr 15 phosphorylation in the first cycle results in a longer interphase.(A) Time courses of levels of cyclin B1, Cdk1 activity, and Cdk1 Y15 phosphorylation. The cyclin B1 and pY15–Cdk1 concentrations were measured by quantitative Western blotting, and the Cdk1 activity was measured by histone H1 kinase assay. The original blots are shown in Figure S1D,E. Each point represents a single embryo. For cycles 2–4, relative timing of individual embryos was corrected to the most recent observed cell division, as indicated by the gray bars (see Materials and Methods). (B) Time courses of levels of cyclin B1, hyperphosphorylated Cdc25C, and pSer287–Cdc25C. M-phase and interphase durations are inferred from dynamics of hyperphosphorylated Cdc25C and pSer287–Cdc25C. (C) Evidence for two expressed cyclin B1 genes. Cyclin B1 antibodies [69] recognized two closely spaced cyclin B1 bands, which could be individually knocked down using two different morpholino oligonucleotides. (D) Knocking down cyclin B1-α or cyclin B1-β lengthens the periods of cycles 2–5.
Figure 3. Inhibiting Cdk1 Y15 phosphorylation affects the duration of only the first cycle.(A) The period of the first three cycles from individual embryos treated with PD0166285. All periods are subtracted by the median value of the control of the same cycle to emphasize the differences. The three black lines correspond to 25, 50, and 75 percentile of the population. (B) Time courses of levels of cyclin B1, Cdk1 activity, and pY15–Cdk1 in control embryos and embryos treated with 50 µM PD0166285. (Inset) Higher magnification of pY15–Cdk1 traces from 2nd to 4th period.
Figure 4. Multiple mechanisms decrease the Wee1/Cdc25 ratio during the transition into the second cycle.(A) Schematic depiction of the decrease of Mos/MEK/MAPK activity, and the increase of Cdc25A concentration, during the first cycle. (B) Cdc25A is absent from one-cell embryos but is present in 2- and 4-cell embryos. The accumulation of Cdc25A is blocked by injection of a Cdc25A morpholino oligonucleotide. The control morpholino is designed with a scrambled sequence of the Cdc25A morpholino. (C) Ablating Cdc25A synthesis causes a small increase in the length of the second through fifth cycles. Fertilized eggs were injected with a Cdc25A morpholino or a scrambled control morpholino. Error bars depict median and 25th and 75th percentiles. (D) Ablating Cdc25A synthesis causes a small increase in pY15 Cdk1 levels in the second and third cycles. The blue points are pY15–Cdk1 levels for embryos injected with a scrambled control morpholino, whereas the red points denote embryos injected with a Cdc25A morpholino. The black points are from uninjected embryos taken during the first cycle. (Inset) Higher magnification of pY15–Cdk1 traces for the 2nd and 3rd periods. (E) The MEK inhibitor U0126 accelerates the postfertilization inactivation of p42-MAPK in a dose-dependent fashion. (F) The period of the first three cycles from individual embryos treated with DMSO or U0126 (150 µM). Error bars depict median and 25th and 75th percentiles. (G) The pY15–Cdk1 level in the first and second cycle. The blue trace denotes the DMSO-treated embryos, and the red trace denotes the U0126-treated embryos. (H) Western blots of Cdc25A, Cdc25C, Myt1, and Wee1A from egg and embryo extracts. (I) Time courses of pY15–Cdk1 and Cdk1 activity after addition of 20 nM δ65–cyclin B1 to egg and embryo extracts. Data are taken from four experiments. Error bars are standard errors of the mean.
Figure 5. Constructing an ODE model of the embryonic cell cycle.(AâC) Ultrasensitive negative feedback in embryo extracts. Interphase embryo extracts were treated with PD0166285 and various concentrations of âµ65âcyclin B1, yielding a graded range of Cdk1 activities as assessed by histone H1 phosphorylation (A) and an all-or-none response in the degradation of securin-CFP, an APC/CCdc20 substrate (B). The inferred stimulus/response curve for securin degradation as a function of Cdk1 activity (C) was highly ultrasensitive, with a best-fit Hill exponent of 464 and 90% confidence interval of (55,539). Data are taken from five experiments. (D) Calibrating the positive feedback strength by varying the Wee1 versus Cdc25 activity ratio (r). Several assumed values of r are shown. A value of corresponds well to the bistability observed in experiments on Xenopus egg extracts and a physiologically strength of positive feedback [22]. After the first cycle, r decreases to approximately 1/32; see Figure S3. (E) Oscillations at various assumed positive feedback strengths. (F) Modeling the transition from the first cycle to the subsequent cycles by adjusting only the positive feedback strength (r). Compare to Figure 2A.
Figure 6. Modeled robustness and tunability from negative-feedback-only versus positive-plus-negative feedback.(A) Robustness score of the oscillator assuming various degrees of ultrasensitivity in the negative feedback loop (n â=â 4, 9, or 36; see Text S2), and various values of r. (B, C) Tunability. Each of the modelâs parameters was varied up and down by 32-fold, starting with a value of r that made the model run like a negative-feedback-only oscillator (r â=â 1/32, panel B) or a positive-plus-negative feedback oscillator (r â=â 1/2, panel C). The bars show the maximum increases (B) and decreases (C) in period that resulted. The green bars correspond to parameters related to the positive feedback, the red bars to negative feedback, and the yellow bars represent cyclin synthesis.
Figure 7. Shortening of the first cycle period significantly reduces embryo viability, and cycloheximide rescues viability.(A–C) Application of PD0166285 during the first cycle causes a loss of viability, whereas later treatment does not. (A) Changes in the length of the first cycle in response to two concentrations of PD0166285. (B) Kaplan–Meier survival curves. (C) Survival at 44 h postfertilization. The data in (A) and (C) are from four experiments, whereas the data in (B) are from one representative experiment. (D–F) Cycloheximide (CHX, 0.25 µg/mL) partially rescues the effects of PD0166285 (30 µM) on viability. (D) Changes in the length of the first cycle in response to PD0166285 ± CHX. (E) Kaplan–Meier survival curves. (F) Survival at 44 h postfertilization. The data in (D) and (F) are from four experiments, whereas the data in (E) are from one representative experiment. (G) Photographs of drug-treated and control embryos at various times after fertilization. The embryos were placed in the same petri dish after the inhibitors were washed out at the completion of the first cycle. The arrows designate three PD-treated embryos that have discoordinated cell divisions as early as a few hours postfertilization; the other PD-treated embryos are grossly normal until the midblastula transition (bottom panel). The incubation temperature was 18° for the experiments in (A–F), and 23° for the experiment in (G).
Figure 1. The embryonic cell cycle oscillator consists of interlinked positive-and-negative feedback loops.Cyclin BâCdk1 inhibits its inhibitory kinases Wee1 and Myt1, forming a double negative feedback loop, which in many respects is equivalent to a positive feedback loop. Cyclin BâCdk1 activates its activating phosphatase Cdc25, forming a positive feedback loop. Active cyclin BâCdk1 also activates the E3 ubiquitin ligase APC/CCdc20, which targets cyclin B for degradation. The Cdk1âAPC/CCdc20 circuit is therefore a negative feedback loop.
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