A positive-feedback-based bistable |[lsquo]|memory module|[rsquo]| that governs a cell fate decision

doi:doi:10.1038/nature02089

Nature 426, 460-465 (27 November 2003) | doi:10.1038/nature02089; Received 9 April 2003; Accepted 18 September 2003

There is a Corrigendum (30 August 2007) associated with this document.

A positive-feedback-based bistable 'memory module' that governs a cell fate decision

Wen Xiong¹ & James E. Ferrell, Jr¹

Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California 94305-5174, USA

Correspondence to: James E. Ferrell, Jr¹ Email: james.ferrell@stanford.edu

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The maturation of Xenopus oocytes can be thought of as a process of cell fate induction, with the immature oocyte representing the default fate and the mature oocyte representing the induced fate^1,². Crucial mediators of Xenopus oocyte maturation, including the p42 mitogen-activated protein kinase (MAPK) and the cell-division cycle protein kinase Cdc2, are known to be organized into positive feedback loops³. In principle, such positive feedback loops could produce an actively maintained 'memory' of a transient inductive stimulus and could explain the irreversibility of maturation^3,^4,^5,⁶. Here we show that the p42 MAPK and Cdc2 system normally generates an irreversible biochemical response from a transient stimulus, but the response becomes transient when positive feedback is blocked. Our results explain how a group of intrinsically reversible signal transducers can generate an irreversible response at a systems level, and show how a cell fate can be maintained by a self-sustaining pattern of protein kinase activation.

Immature Xenopus oocytes are arrested in a G2-like phase of the cell cycle. In response to steroid hormones, the oocyte is released from its G2 arrest, undergoes germinal vesicle breakdown (GVBD), completes meiosis I, proceeds directly into meiosis II, and then arrests again in the metaphase of meiosis II. Early work indicated that at some point in this process, the oocyte irrevocably commits to maturation^7,^8,⁹. We corroborated these observations by using low concentrations of the steroid hormone progesterone and thorough washing procedures to minimize the possibility that commitment was an artefact of inadequate washing. We found that oocytes generally committed to maturation after 2–4 h of progesterone treatment and before (and in one experiment just before) GVBD (Fig. 1a–c). This timing agrees well with studies of the acquisition of 'maturation inertia' in Rana temporaria oocytes treated with gonadotropin⁷. Oocytes remained arrested in their mature, GVBD state for up to several days after progesterone removal. Thus, transient treatment with progesterone results in a sustained cellular response.

Figure 1: Cell fate commitment during oocyte maturation.

a–c, Timing of commitment versus maturation from three independent experiments. d, View of the signal transduction network that culminates in Xenopus oocyte maturation.

High resolution image and legend (99K)

At a biochemical level, oocyte maturation is controlled by p42 MAPK and cyclin B/Cdc2 (Fig. 1d). Both of these protein kinases are activated during maturation: p42 MAPK through phosphorylation by MAPK kinase (MEK), and cyclin B/Cdc2 through dephosphorylation by Cdc25. In addition, both kinases seem to be actively maintained in their high-activity, M-phase states. For example, in mature oocytes where p42 MAPK activity is high and unchanging, the two phosphorylation events that activate p42 MAPK still turn over rapidly with half-lives (t_1/2) of about 5 min (ref. 10). EDTA-quenching experiments suggest that phosphorylation events turn over rapidly (t_1/2 < 10 min) in other members of the MAPK cascade, Mos and MEK, and also in Cdc25 and Wee1 (data not shown). This raises the issue of how these intrinsically reversible signalling proteins can 'remember' a transient stimulus and convert it into an irreversible response.

An attractive possibility is that the irreversibility is produced by positive feedback, and indeed numerous positive feedback loops have been identified in the p42 MAPK/Cdc2 network of an oocyte. For example, Mos activates p42 MAPK through the intermediacy of MEK, and active p42 MAPK feeds back to promote the accumulation of Mos (refs 11–13 and Fig. 1d). This protein-synthesis-dependent positive feedback loop is strong enough to allow p42 MAPK to show an all-or-none, bistable response to progesterone or microinjected Mos¹⁴. If protein synthesis is blocked (and thus the positive feedback is blocked), however, the response becomes graded¹⁴.

Other positive feedback loops are also present in the signalling network that culminates in oocyte maturation. Cdc2 promotes activation of the Cdc2 activator Cdc25 (refs 15, 16), and promotes inactivation of the Cdc2 inhibitor Myt1 (ref. 17 and Fig. 1d). The p42 MAPK and Cdc2 systems also interact with each other through positive feedback: p42 MAPK acts positively on Cdc2 by promoting the inactivation of Myt1 (ref. 18), and Cdc2 acts positively on p42 MAPK by promoting the accumulation of Mos (ref. 19 and Fig. 1d). Thus, positive feedback is a recurring theme in oocyte signal transduction, and indeed positive feedback and bistability are important in various biological contexts^20,^21,^22,²³.

In theory, a system whose positive feedback is strong enough to produce bistability should show either hysteresis (meaning that it is easier to maintain the system in its on state than to toggle the system from off to on) or, if the feedback is particularly strong, irreversibility (Box 1). We considered that the bistable p42 MAPK/Cdc2 system of the oocyte might possess sufficiently strong positive feedback to allow the system to generate this type of self-sustaining, actively maintained irreversibility.

To test this hypothesis, we set out to determine whether hysteresis or irreversibility is apparent in the oocyte's response to progesterone. This amounts to determining whether the concentration of progesterone needed to induce p42 MAPK phosphorylation and Cdc2 activation differs from that needed to maintain the activities of these kinases. To obtain 'induction' stimulus–response curves, we incubated immature oocytes with different concentrations of progesterone, waited until oocyte maturation had reached a plateau, and then measured the percentage of GVBD (%GVBD), p42 MAPK phosphorylation, Cdc2 activation and progesterone binding. Progesterone-treated oocytes showed dose-dependent increases in all of these responses (Fig. 2a–c, 'induction'), with maximal kinase activation and %GVBD obtained with 600 nM progesterone.

Figure 2: Irreversibility in the biochemical responses of oocytes to progesterone.

GVBD (a), p42 MAPK phosphorylation (b), Cdc2 H1 kinase activity (c) and progesterone binding (d) were assessed at the end of the induction period and the end of the maintenance period. GVBD, MAPK phosphorylation and Cdc2 activity data are from one of three similar experiments. Progesterone binding data (shown as means plusminus s.d.) are from a separate experiment.

High resolution image and legend (43K)

To obtain 'maintenance' stimulus–response curves, we incubated oocytes with 600 nM progesterone, waited until GVBD had reached a plateau (5 h in the experiments shown in Fig. 2), washed the oocytes for 10 h to remove progesterone, and then resuspended the washed oocytes in various concentrations of progesterone. After 5 h of further incubation, we monitored %GVBD, p42 MAPK phosphorylation, Cdc2 activation and progesterone binding. Oocytes were found to maintain maximal p42 MAPK and Cdc2 activities after being washed free of progesterone (Fig. 2b, c, 'maintenance'), and none of the oocytes lost its white dot (Fig. 2a). The progesterone-binding data argued against the trivial explanation that the irreversibility in the p42 MAPK and Cdc2 responses was simply due to a failure to wash the progesterone away adequately (Fig. 2d). Thus, once oocytes are mature, the continued presence of progesterone seems to be unnecessary. Some 'memory' of the progesterone must be maintained either by the p42 MAPK/Cdc2 system or by signal transducers upstream of this system.

To test whether the p42 MAPK/Cdc2 system itself can generate an irreversible response from a transient stimulus, we used a chimaeric protein of Raf and the oestrogen receptor ( Delta Raf:ER), in which an activated Raf1 protein is rendered conditional by fusion to an ER hormone-binding domain²⁴. In oocytes expressing Delta Raf:ER, the steroid hormone oestradiol (which by itself has no effect on maturation, p42 MAPK activation or Cdc2 activation in oocytes) can be used to introduce a stimulus directly into the MAPK cascade, bypassing the progesterone receptor and other upstream signalling proteins.

Oocytes expressing Delta Raf:ER possessed low but detectable Delta Raf:ER activity in the absence of oestradiol (Fig. 3a, middle, lane 3). In response to oestradiol, there was a prompt 3–5-fold increase in Delta Raf:ER activity (Fig. 3a, middle, lanes 3–5), followed by a slower, additional 3–5-fold increase in Delta Raf:ER protein and activity (Fig. 3a, top and middle, lanes 6–10). The MEK inhibitor PD98059 and the protein synthesis inhibitor cycloheximide blocked the slow increase in Delta Raf:ER protein (data not shown) and activity (Fig. 4a, c), indicating that this component of the Delta Raf:ER activation depends on positive feedback between p42 MAPK and Delta Raf:ER. Within several hours, the oestradiol-induced activation of Delta Raf:ER resulted in complete activation of endogenous p42 MAPK (Fig. 3a, bottom, lanes 9–10).

Figure 3: Irreversibility in the biochemical responses of oocytes expressing Raf:ER to oestradiol.

Figure 3 : Irreversibility in the biochemical responses of oocytes expressing |[Delta]|Raf:ER to oestradiol. Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com

a, Time course of oestradiol-induced Delta Raf:ER and p42 MAPK activation, assessed by ER immunoblot analysis, Delta Raf:ER immune complex kinase assay, and p42 MAPK immunoblot analysis. b–f, Steady-state responses. GVBD (b), Delta Raf:ER activity (c), p42 MAPK phosphorylation (d), Cdc2 H1 kinase activity (e) and oestradiol binding (f) were assessed at the end of the induction period and the end of the maintenance period. GVBD, Delta Raf:ER activity, p42 MAPK phosphorylation and Cdc2 activity data are from one of three similar experiments. Oestradiol binding data (shown as means plusminus s.d.) are from a separate experiment.

High resolution image and legend (80K)

Figure 4: Positive feedback is required for irreversible biochemical responses.

Oocytes expressing Delta Raf:ER were treated by an 18-h incubation with no added oestradiol (- E2); an 18-h incubation with 10 nM oestradiol (+ E2); or a 2-h incubation with 10 nM oestradiol, followed by a 16-h wash (+ E2, washed). a, A sample of oocytes was treated with cycloheximide (CHX, 100 micro g ml^-1) to block protein synthesis and positive feedback. b, A sample of oocytes was injected with a morpholino Mos antisense oligonucleotide (100 ng per oocyte) to block Mos synthesis. c, A sample of oocytes was treated with the MEK inhibitor PD98059 (100 micro M) to block MEK-mediated positive feedback and downstream events.

High resolution image and legend (49K)

We used the Delta Raf:ER-expressing oocytes to look for hysteresis or irreversibility in the response to oestradiol, by using the induction versus maintenance strategy described above. When immature oocytes expressing Delta Raf:ER were treated with increasing concentrations of oestradiol, there was a graded, dose-dependent increase in %GVBD and in steady-state Delta Raf:ER activity, p42 MAPK phosphorylation and Cdc2 activity (Fig. 3b–e, 'induction'). The kinases remained maximally active after the oestradiol was washed away (Fig. 3c–e, 'maintenance'), and every oocyte maintained its white spot after washing (Fig. 3b). The trivial explanation of incomplete removal of oestradiol did not seem to account for the observed irreversibility (Fig. 3f). Thus, the p42 MAPK/Cdc2 system itself can convert a transient stimulus into an irreversible activation response; that is, the system can generate a long-term 'memory' of a transient differentiation stimulus.

To determine whether positive feedback is essential for maintaining this memory, we made use of three ways of blocking feedback from p42 MAPK to Mos. The first feedback inhibitor was cycloheximide. If oocytes are provided with a sufficiently strong maturation stimulus, such as microinjected Mos or cyclin A, protein synthesis is not essential for maturation^25,²⁶; however, protein synthesis is essential for the feedback from p42 MAPK to Mos^11,^12,¹³, and possibly for other interconnected positive feedback loops²⁷. Thus, we examined whether cycloheximide would convert the observed irreversible oestradiol response into a reversible one. In the absence of cycloheximide, oestradiol again caused marked increases in the steady-state activities of Delta Raf:ER, p42 MAPK and Cdc2, and these responses were undiminished after washing (Fig. 4a). In the presence of cycloheximide, the responses of Delta Raf:ER and p42 MAPK to oestradiol were blunted, and the response of Cdc2 was markedly diminished (Fig. 4a). In addition, the responses were no longer irreversible: after the oestradiol was washed away, the activity of Delta Raf:ER, the phosphorylation of p42 MAPK, and the low activation of Cdc2 all decreased to near-basal levels (Fig. 4a). Thus, protein synthesis, which is essential for feedback from p42 MAPK to Mos, is also required for the irreversibility of the oestradiol responses.

The second feedback inhibitor was a morpholino Mos antisense oligonucleotide (Mos-AS), which blocks progesterone-induced Mos synthesis without globally abolishing protein synthesis²⁸. Mos-AS blocked oestradiol-induced Mos synthesis and also abolished the irreversibility of Delta Raf:ER activation and p42 MAPK phosphorylation (Fig. 4b). The low Cdc2 activation induced by oestradiol also became reversible (Fig. 4b). Thus, Mos synthesis is required for the observed irreversibility. Finally, we examined the effects of the MEK inhibitor PD98059, which blocks both downstream effects and feedback effects that depend on MEK activation. PD98059 rendered oestradiol-induced Delta Raf:ER activation reversible and also blocked oestradiol-induced p42 MAPK and Cdc2 activation (Fig. 4c).

Taken together, these findings rule out the possibility that an inability to wash away Delta Raf:ER-bound oestradiol, or an intrinsic defect in the capacity of Delta Raf:ER to be inactivated, can account for the irreversible p42 MAPK and Cdc2 responses seen in Figs 3 and 4. Instead, the irreversibility seems to be actively generated by a bistable, positive feedback system, and compromising the feedback abolishes the irreversibility.

The idea that the irreversibility of the differentiated state might be maintained by feedback loops that generate self-sustaining patterns of gene expression dates back more than 40 years (ref. 4), and the recognition that feedback-enforced patterns of protein activity might also produce a systems-level 'memory' of transient stimuli dates back almost 20 years (ref. 5). Studies of artificial bistable gene expression systems in Escherichia coli and Saccharomyces cerevisiae have shown that bistable systems can in fact function as memory modules, but have also shown that stochastic effects sometimes cause two bistable steady states to equilibrate with each other, undermining any memory^29,³⁰. Our work provides experimental evidence that in a physiological process of cell fate induction, Xenopus oocyte maturation, a bistable signalling system converts a transient stimulus into a reliable, self-sustaining, effectively irreversible pattern of protein activities. It will be interesting to see how common these bistable memory modules are in cell signalling.

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Methods

Oocytes, expression constructs and reagents

Xenopus laevis oocytes were defolliculated by collagenase treatment and stage VI oocytes were manually sorted. We prepared oocyte lysates as described¹⁰.

The high-activity DD form of Delta Raf:ER (in pBabepuro3)²⁴ was a gift from M. McMahon and was subcloned into pSP64(polyA). Delta Raf:ER mRNA was transcribed in vitro using an Sp6 transcription kit (Ambion), and the RNA concentration was determined by gel electrophoresis and staining. Delta Raf:ER mRNA (5 ng) was microinjected with a Nanoinjector (Drummond Scientific), and oocytes were left for protein expression for 2 h before oestradiol was added. We purchased the Mos antisense morpholino oligonucleotide 5'-AAGGCATTGCTGTGTGACTCGCTGA-3' (ref. 28) from Gene Tools LLC.

Oestradiol, progesterone and cycloheximide were from Sigma, and [³H]oestradiol and [¹⁴C]progesterone were from New England Nuclear. The MEK inhibitor PD98059 was from Calbiochem.

SDS–PAGE and immunoblotting

For most assays, samples were resolved by electrophoresis on 10% low-bis polyacrylamide gels (acrylamide:bisacrylamide 100:1). For the Cdc2/cyclin B histone H1 kinase assays, we used 12.5% polyacrylamide gels (acrylamide:bisacrylamide 29:1). Gels were transferred to poly(vinylidene difluoride) blotting membranes, which were then blocked with 3% nonfat milk in Tris-buffered saline (150 mM NaCl and 20 mM Tris; pH 7.6) and incubated for 2 h with a 1:1,000 dilution of one of the following primary antibodies: anti-MAPK (DC3, raised in our own laboratory), anti-phospho-MAPK ( 9106, New England Biolabs), anti-ER ( SC-543, Santa Cruz Biotechnology) or a 1:500 dilution of anti-MoS ( SC-86, Santa Cruz Biotechnology). Blots were washed three times with Tris-buffered saline plus 0.1% Tween 20 and probed with alkaline-phosphatase-conjugated secondary antibody for detection by CDP-Star (Tropix-Perkin Elmer). For reprobing, blots were stripped in 100 ml of stripping buffer (100 mM Tris-HCl, pH 7.4, 100 mM beta -mercaptoethanol and 2% SDS) at 70 °C for 40 min.

Kinase assays

We prepared immune complexes of Delta Raf:ER by incubating cell lysate with 30 micro l of protein-A–Sepharose (Sigma) pre-coated with 1 micro l of anti-ER antibody ( SC-543, Santa Cruz Biotechnology). The immunoprecipitates were washed once with lysis buffer and once with kinase buffer (20 mM HEPES, 10 mM MgCl₂ and 1 mM MnCl₂; pH 7.4). Raf kinase reactions were done at 30 °C for 30 min in 40 micro l of kinase buffer with 1 mM dithiotheitol, 1 micro M ATP and 10 micro Ci [ gamma -³²P]ATP (Amersham) with 50 ng of purified recombinant GST–MEK (UBI) as a substrate. The reaction was stopped by boiling in sample buffer and resolved by electrophoresis on a 10% 100:1 polyacrylamide gel.

For the histone H1 kinase assay, oocytes were lysed in a volume of 10 micro l per oocyte, and 2 micro l of cleared lysate was added to 8 micro l of EB buffer (80 mM beta -glycerophosphate, 20 mM EGTA and 15 mM MgCl₂; pH 7.3) and mixed with an equal volume (10 micro l) of reaction mixture (6.73 micro l of 2 $times$ H1 kinase buffer, 0.02 micro l of 0.2 mM ATP, 1.0 micro l of 10 mg ml^-1 histone H1, 2.0 micro l of 100 micro M protein kinase inhibitor (Santa Cruz Biotechnology) and 0.25 micro l of 10 mCi ml^-1 [ gamma -³²P]ATP). Reactions were carried out at 30 °C for 15 min and terminated by the addition of 6 $times$ sample buffer (4 micro l) followed by boiling.

Washing procedure and scintillation counting

Oocytes incubated with progesterone or oestradiol were washed free of the steroids by a series of ten 1-h washes in 50 ml of OR2 solution. For some experiments, cycloheximide or MEK inhibitor (PD98059) was included in both the incubation and the washing buffer. Pre- and post-wash binding of progesterone and oestradiol was determined by the inclusion of radiolabelled tracers ([¹⁴C]progesterone, final specific activity 83 micro Ci micro mol^-1, and [³H]oestradiol, final specific activity 25,000 micro Ci micro mol^-1) and quantified by scintillation counting.

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Acknowledgements

We thank M. McMahon for the Delta Raf:ER constructs, and K. Cimprich and members of the Ferrell laboratory for discussions and comments on the manuscript. This work was supported by a grant from the NIH.

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Competing interests statement

The authors declare no competing financial interests.

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Letters to Nature

A positive-feedback-based bistable 'memory module' that governs a cell fate decision