VPCs enter M phase in a random order

One feature of bounded asynchrony is the fact that the sub-components may enter the next state in an arbitrary order. If VPC differentiation was to be governed by a mechanism resembling bounded asynchrony, then the VPCs should indeed progress through the cell cycle in a slightly asynchronous manner. To address this question, we examined the order in which the VPCs enter the M phase of the cell cycle. For this purpose, we observed the divisions of the proximal VPCs P5.p, P6.p, and P7.p in wild-type larvae using Nomarski optics and scored the time of entry into prometaphase based on nuclear envelope breakdown (Figure 1E). Among the three proximal VPCs, we observed no bias in the order at which they entered M phase, as each of the three VPCs was equally likely to divide first. Using time-lapse imaging, we measured the temporal differences in VPC divisions. The average time span between the first and last proximal VPC divisions was 33.4min with a maximum divergence of 71min (n=6). These results indicate that the VPCs adopting 1° and 2° cell fates do indeed enter the M phase in a random order, suggesting that the cell cycle of the VPCs progresses in a slightly asynchronous and unbiased manner.

Cell-cycle arrest interferes with VPC fate specification

We next tested the biological significance of the computational concept of bounded asynchrony by manipulating the cell cycle in single VPCs. We hypothesized that, although the VPCs progress with slightly variable speed through the cell cycle, external mechanisms may keep them ‘more or less’ synchronized and thereby align the relative timing of the signal transduction events between the VPCs. Under this assumption, the repeated use of cell-cycle checkpoints may represent such a synchronization mechanism. The VPCs are born during the first larval stage and remain in the G1 phase until the transition from the second to the third larval stage (Euling and Ambros, 1996). During the second larval stage, 1° cell fate markers can be detected in P6.p, but the other five VPCs remain uncommitted (Ambros, 1999). After the G1/S-phase arrest has been relieved, P5.p and P7.p adopt the 2° fate during the G2 phase, while the 3°, uninduced fate of the distal VPCs (P3.p, P4.p, and P8.p) is only sealed after these cells have divided and fused with the surrounding hypodermis (hyp7).

We therefore performed an experiment to specifically arrest the cell cycle in P6.p without affecting the progression of the other VPCs. For this purpose, we expressed the cyclin-dependent kinase (CDK) inhibitor cki-1 under control of the 1° fate-specific egl-17 promoter (egl-17p::cki-1) to arrest P6.p in the G1 phase without affecting cell-cycle progression in the other VPCs (Hong et al, 1998). We then observed the effect of this intervention on the VPC fate specification using 1° and 2° cell fate markers. As a readout for the 1° cell fate, we used a transcriptional gfp reporter of the RAS/MAP kinase target egl-17 (Burdine et al, 1998), and for the 2° fate, we observed a reporter of the LIN-12 NOTCH target lip-1 (Berset et al, 2001).

In egl-17p::cki-1 animals, in which P6.p was arrested and P5.p and P7.p had progressed to the Pn.px or Pn.pxx stage, expression of the 2° fate marker lip-1p::gfp persisted in the undivided P6.p cells at levels that were significantly higher than those detected in control animals (Figure 2A). To rule out the possibility that the differences in lip-1p::gfp levels between arrested and non-arrested cells are due to a dilution of the GFP signal during the cell divisions, we corrected the signal intensities for the total nuclear areas. Even with this correction, we detected a significant difference in total lip-1p::gfp signal intensity between arrested P6.p and non-arrested P6.p descendants (Supplementary Figure S1). Furthermore, expression of the 1° fate marker egl-17p::gfp in arrested P6.p cells was not affected (Supplementary Figure S2), confirming the previous observation that 1° fate specification begins already during the G1 phase (Ambros, 1999). However, our results also indicate that G1-arrested P6.p cells could not establish a fate-specific gene expression pattern, as they simultaneously expressed 1° and 2° cell fate markers. We conclude that a coordinated cell-cycle progression of the VPCs is necessary for the specification of a stable cell fate as judged by the expression of cell-fate markers.

Open in a separate window

Figure 2

Degradation of LIN-12 NOTCH is blocked in G1-arrested P6.p. (A) Expression of the 2° fate marker lip-1p::gfp persists in the G1-arrested P6.p cell of an egl-17p::cki-1 transgenic animal (arrowheads in the right panels), while lip-1p::gfp expression is downregulated in the P6.p descendants of a sibling that had lost the egl-17p::cki-1 array (brackets in the left panels). (B) LIN-12::GFP persists in the G1-arrested P6.p cell of an egl-17p::cki-1 transgenic animal (arrowheads in the right panels), while LIN-12::GFP is efficiently degraded in the P6.p descendants of a sibling that had lost the egl-17p::cki-1 array (brackets in the left panels). Note the accumulation of the GFP signal in the nucleus and cytoplasm of the arrested P6.p cell. Black asterisks denote uterine LIN-12::GFP expression. In all panels, the corresponding Nomarski images are shown on top. The scale bars represent 10μm. Quantifications of lip-1p::gfp and intracellular LIN-12::GFP expression are shown to the right. Error bars indicate the standard error and asterisks indicate the significance as described in Materials and methods. Source data is available for this figure in the Supplementary Information.

Source data for Figure 2A^{(33K, xls)}

Source data for Figure 2B^{(29K, xls)}

Termination of NOTCH signaling is linked to cell-cycle progression

The finding that G1-arrested P6.p cells continued to express the 2° cell fate marker suggested that G1 cell-cycle arrest in P6.p might block the proper termination of LIN-12 NOTCH signaling. To investigate this possibility, we examined the expression of a functional, translational LIN-12::GFP reporter that reflects the expression pattern and sub-cellular localization of the endogenous LIN-12 protein (Levitan and Greenwald, 1998; Shaye and Greenwald, 2002). Before vulval induction, LIN-12::GFP is expressed at low levels in all VPCs. Most of the LIN-12 protein is localized to the apical plasma membrane of the VPCs. After vulval induction, LIN-12 is upregulated in the 2° P5.p and P7.p lineages and downregulated in the 1° P6.p lineage.

In control animals lacking the egl-17p::cki-1 transgene, LIN-12::GFP was absent from the 1° P6.p descendants at the Pn.px stage, because LIN-12 NOTCH undergoes rapid endocytosis and degradation in the 1° lineage (Shaye and Greenwald, 2002). In contrast, in egl-17p::cki-1 transgenic animals at the Pn.px stage, in which P6.p had remained undivided, we detected elevated levels of LIN-12::GFP in the cytoplasm and nucleus of P6.p (Figure 2B). However, the LIN-12::GFP signal was lost from the apical plasma membrane, indicating that the endocytosis of the full-length LIN-12 protein in P6.p is not affected by G1 arrest. Since the GFP tag in LIN-12::GFP is inserted in the intracellular domain (NICD) that is cleaved off the transmembrane domain during LIN-12 NOTCH activation, the persisting cytoplasmic and nuclear GFP signal in G1-arrested P6.p cells suggested that the downregulation of the cleaved, intracellular LIN-12 NICD occurs only after exit from the G1 phase. In this way, the termination of the LIN-12 NOTCH signal in the 1° lineage might be coupled to cell-cycle progression.

Degradation of NICD is blocked in G1-arrested VPCs

To directly test the connection between lateral signaling, NICD degradation, and the cell cycle, we integrated via the MosSCI technique (Frokjaer-Jensen et al, 2008) a single copy of an nicd::gfp transgene expressed under control of a bar-1 promoter fragment into the genome at a specific site on chromosome II (zhIs39[nicd::gfp]). Importantly, the bar-1 promoter fragment used drives uniform expression in the six VPCs and their descendants until the Pn.pxx stage (Natarajan et al, 2004; Figure 6A). Thanks to the single copy integration, we achieved a stable and reproducible NICD::GFP expression in the VPCs. The increased dosage of NOTCH signaling caused by nicd::gfp resulted in the ectopic induction of the 2° fate in P3.p, P4.p and P8.p and a Multivulva phenotype in all of the animals examined (Table I, row 1; Supplementary Figure S3). However, P6.p in nicd::gfp animals still adopted the 1° fate as determined by its cell lineage (see insets in Supplementary Figure S3). Moreover, the proximal VPCs in nicd::gfp animals entered M phase in a random order similar to the wild type (Figure 1E).

We next performed a time-course analysis to follow NICD::GFP degradation in the induced VPCs and their descendants (Figure 3A and B). NICD::GFP was uniformly expressed in the VPCs of early L2 larvae (+24h relative to hatching, see Materials and methods). Interestingly, NICD::GFP expression first increased until the mid L2 stage (+27h) and then started to decline in all VPCs. A significant difference between the 1° and 2° VPCs could first be detected at +32h, shortly before the first VPCs started dividing (Figure 3A). After this time point, NICD::GFP expression rapidly faded in P6.p. At the Pn.px stage, NICD::GFP was barely detectable in the 1° P6.p descendants, while expression persisted in the 2° P5.p and P7.p descendants, though at lower levels when compared with Pn.p stage animals (right panel in Figure 3B). By the Pn.pxx stage, NICD::GFP was undetectable in P6.p descendants and further reduced in P5.p and P.7.p descendants. Similarly to full-length LIN-12::GFP, NICD::GFP in egl-17p::cki-1 animals persisted in the nuclei of the arrested P6.p cells (Figure 3C). Thus, NICD::GFP is progressively degraded in the induced VPCs and their descendants. Though, the rate of NICD degradation is significantly higher in the 1° than in the 2° lineage and depends on cell-cycle progression.

Open in a separate window

Figure 3

Time-course analysis of NICD degradation. (A) NICD::GFP levels in P5.p, P6.p, and P7.p were measured every half an hour in around 20 synchronized animals at each time point (left scale). The time is indicated in hours after hatching (see Materials and methods). Error bars indicate the standard errors and asterisks significant differences between 1° and 2° lineages as described in Materials and methods. The percentage of animals with at least one proximal VPC division is indicated by the black line (right scale). (B) Representative pictures of NICD::GFP expression in an early L2 (+25h, Pn.p stage), a late L2 (+33h, Pn.p stage), and an early L3 (+36h, Pn.px stage) larva. Note the uniform NICD::GFP expression in the VPCs at +25h and the downregulation in P6.p at +33h (arrowheads) and in P6.px at +36h (bracket). (C) NICD::GFP persists in the G1-arrested P6.p cell of an egl-17p::cki-1 transgenic animal (arrowheads in the right panels), whereas NICD::GFP is downregulated in the P6.p descendants of a sibling that had lost the egl-17p::cki-1 array (brackets in the left panels). In all panels, the corresponding Nomarski images are shown on top. The scale bars represent 10μm. A quantification of NICD::GFP expression is shown to the right. Error bars indicate the standard error and asterisks indicate the significance as described in Materials and methods. Source data is available for this figure in the Supplementary Information.

Source data for Figure 3A^{(47K, xls)}

Source data for Figure 3C^{(34K, xls)}

A computational model of VPC fate specification based on the cell cycle

Based on the experimental evidence, which indicated a linkage of VPC fate specification to cell-cycle progression, we incorporated the cell cycle as a key timing factor in our revised computational model. To implement bounded asynchrony in our refined computational model, we used an independent scheduler that recreates the same interactions between cells by limiting the number of steps one process gets ahead of the other as exhibited by the timed automata model described earlier. In our previous VPC models, an artificial delay in LIN-12 NOTCH downregulation was necessary to reproduce the proper behavior of lin-12 and predict the correct fate patterns in lateral signaling mutants (Fisher et al, 2007). With the introduction of the cell cycle, we removed this artificial delay and coupled the inhibition of LIN-12 to the state of the cell-cycle phases G1, S, or G2 (see Supplementary information for a detailed description of the computational model). In a first in silico experiment, we allowed for the inhibition of lateral LIN-12 signaling to happen during the G1 phase. However, this configuration of the model failed to reproduce published experimental data. We further tested a model where inhibition of LIN-12 Notch was forbidden in the G1 phase but allowed during the S or G2 phase. This model was consistent with published experimental results (checked using model checking, see Supplementary Table 2). Furthermore, the coupling between cell-cycle progression and LIN-12 NOTCH degradation was implemented in all VPCs in an identical manner (see Supplementary Figure S17). However, when executing the model we found that LIN-12 levels in VPCs progressing toward a 1° fate stabilized at lower levels than in VPCs adopting a 2° fate. Taken together, the notion of bounded asynchrony introduced into our computational model through the cell cycle predicted that a temporal delay in the termination of the LIN-12 NOTCH signal after G1 is critical for the formation of a stable cell fate pattern.

NICD degradation occurs during the G2 phase

To further narrow down the time window, during which NICD is degraded, we arrested the VPCs in the S phase by hydroxyurea treatment (Ambros, 1999). Expression of the 2° cell fate reporter lip-1p::yfp persisted in the arrested P6.p cells and increased in the adjacent P5.p and P7.p cells (Figure 4A). Overall, lip-1p::yfp reached significantly higher levels in S phase-arrested VPCs compared with the VPCs of untreated control animals. Accordingly, the degradation of NICD::GFP in P6.p was blocked in hydroxyurea-treated animals (Figure 4B).

Open in a separate window

Figure 4

NICD is degraded during the G2 phase. (A) Expression of the 2° fate marker lip-1p::yfp persists in P6.p of a hydroxyurea (HU)-treated larva (arrowheads in the right panels), while expression is downregulated in an untreated control animal at the Pn.p stage (arrowheads in the left panels). (B) NICD::GFP persists in the undivided P6.p cell of a hydroxyurea-treated larva (arrowheads in the right panels), but NICD::GFP is downregulated in the P6.p descendants of an untreated control larva at the Pn.px stage (brackets in the left panels). (C) NICD::GFP persists in the G2-arrested P6.p cell of a cdk-1 RNAi-treated animal at the Pn.px stage (arrowheads in the right panels), while NICD::GFP is downregulated in the P6.p descendants of an empty vector RNAi-treated control animal (brackets in the left panels). Note that due to the incomplete penetrance of the cdk-1 RNAi effect, only P6.p had arrested in the example shown in the right panels. In all panels, the corresponding Nomarski images are shown on top. The scale bars represent 10μm. Quantifications of lip-1p::yfp and NICD::GFP expression are shown to the right. Error bars indicate the standard error and asterisks indicate the significance as described in Materials and methods. Source data is available for this figure in the Supplementary Information.

Source data for Figure 4A^{(28K, xls)}

Source data for Figure 4B^{(35K, xls)}

Source data for Figure 4C^{(38K, xls)}

We then arrested the VPCs in the late G2 phase by RNAi-mediated knockdown of cdk-1, which is essential for the G2-to-M phase progression (Mori et al, 1994; Boxem et al, 1999). Since the cdk-1 RNAi phenotype is incompletely penetrant, this procedure resulted in the random arrest of single or multiple VPCs per affected animal. Similarly to hydroxyurea-treated animals, the undivided VPCs in cdk-1 RNAi animals expressed elevated levels of NICD::GFP, and the bias in P6.p versus P5.p and P7.p-specific expression was lost in the G2-arrested VPCs (Figure 4C). Taken together, the cell-cycle arrest and time-course experiments indicate that NICD is degraded in the VPCs either during the late G2 phase or at the time of entry into the M phase. The persisting expression of NICD::GFP in VPCs arrested by cdk-1 RNAi may also indicate a direct involvement of a CDK-1/Cyclin complex in NICD degradation (see below).

The G1 cyclins CYE-1 and CYD-1 promote while the G2 cyclin CYB-3 inhibits NOTCH activity

To investigate which CDK/Cyclin complexes are responsible for the changes in NOTCH stability and signaling activity during cell-cycle progression, we examined the expression of the full-length LIN-12::GFP and the NICD::GFP reporters as well as the expression of the target gene lip-1 in different cyclin mutants.

We first examined the influence of the G1-specific D-type cyclin gene cyd-1 on NOTCH signaling using the reduction-of-function allele cyd-1(q626) (Tilmann and Kimble, 2005). Although no VPC cell-cycle arrest was observed, cyd-1(q626) mutants displayed reduced levels of LIN-12::GFP in P5.p and P7.p (Figure 5A). Moreover, the expression of the LIN-12 target gene lip-1 was strongly decreased in the P5.p and P7.p descendants of cyd-1(q626) mutants (Figure 5B). Due to the proximity of the nicd::gfp integration site to the cyd-1 locus on LGII, it was not possible to examine NICD::GFP expression in cyd-1(q626) animals. However, cyd-1 RNAi did not cause an obvious reduction in NICD::GFP expression in the VPCs. Rather, in the six cases, in which P6.p was arrested, P6.p continued to express NICD::GFP at high levels similar to the expression pattern observed in egl-17p::cki-1 animals (Supplementary Figure S4; Figure 3C).

Open in a separate window

Figure 5

Differential regulation of NOTCH signaling by G1 and G2 cyclins. (A) Localization of LIN-12::GFP to the apical plasma membrane is diminished in the VPC descendants in cyd-1(q626) mutants (right panels) when compared with wild-type controls (left panels). Black asterisks denote uterine LIN-12::GFP expression. (B) Expression of the 2° fate marker lip-1p::gfp is reduced in the P5.p and P7.p descendants in cyd-1(q626) mutants (right panels) compared with wild-type controls (left panels). (C) NICD::GFP expression is reduced in the VPC descendants in homozygous cye-1(ku256) mutants (right panels) when compared with heterozygous cye-1(ku256)/+ controls (left panels). (D) NICD::GFP expression persists in the P6.p descendants of a cyb-3 RNAi-treated animal at the Pn.px stage (brackets in the right panels). Note that in contrast to cdk-1 (Figure 4C), cyb-3 RNAi did not induce a cell-cycle arrest. An empty vector RNAi-treated control animal is shown in the left panels. In all panels, the corresponding Nomarski images are shown on top. The scale bars represent 10μm. Quantifications of LIN-12::GFP, lip-1p::gfp and NICD::GFP expression are shown to the right. Error bars indicate the standard error and asterisks indicate the significance as described in Materials and methods. Source data is available for this figure in the Supplementary Information

Source data for Figure 5A^{(32K, xls)}

Source data for Figure 5B^{(44K, xls)}

Source data for Figure 5C^{(30K, xls)}

Source data for Figure 5D^{(47K, xls)}

Furthermore, we tested if the cyd-1(q626) mutation modifies the vulval phenotype caused by NICD::GFP expression. For this purpose, we used an extrachromosomal nicd::gfp array (zhEx500) to score vulval induction in the cyd-1(q626) background. The cyd-1(q626) mutation significantly decreased vulval induction in animals carrying the zhEx500[nicd::gfp] array when compared with cyd-1(+); zhEx500[nicd::gfp] controls (Table I, rows 2, 3). Taken together, we conclude that cyd-1 controls NOTCH signaling at the level of the full-length LIN-12 receptor and possibly also downstream of NICD at the level of target gene expression. Since NOTCH signaling prevents the degradation of full-length LIN-12 (Levitan and Greenwald, 1998; Shaye and Greenwald, 2002), it is possible that the reduced expression of the LIN-12::GFP reporter in cyd-1(q626) mutants is caused by a disruption of this positive feedback loop.

We next analyzed the effect of the G1-specific E-type cyclin cye-1 using the strong reduction-of-function or null allele ku256 (Fay and Han, 2000). Expression of the NICD::GFP reporter in homozygous cye-1(ku256) mutants was strongly reduced, in both the 1° and the 2° VPCs and their descendants (Figure 5C). Moreover, vulval induction was slightly but significantly suppressed in homozygous cye-1(ku256); nicd::gfp mutants when compared with heterozygous cye-1(ku256)/+; nicd::gfp controls (Table I, rows 4, 5).

Finally, we investigated the influence of the G2-specific B-type cyclins cyb-1, cyb-2, and cyb-3 on NICD stability. Since the available deletion mutations in the cyb genes cause embryonic or larval lethality, we reduced cyb gene activities by RNAi-mediated knockdown. cyb-2 represents two highly similar and functionally redundant genes, cyb-2.1 and cyb-2.2, that were simultaneously affected by the same RNAi treatment. Only cyb-3 RNAi caused an increase in NICD::GFP expression (Figure 5D). Even though RNAi against cyb-3 did not cause a cell-cycle arrest in the VPCs, cyb-3 RNAi animals exhibited significantly higher NICD::GFP levels in the P6.p descendants at the Pn.px stage compared with control RNAi animals (Figure 5D). We thus conclude that both the G1 cyclins CYD-1 and CYE-1 positively regulate LIN-12 NOTCH signaling. CYD-1 appears to act primarily by regulating full-length LIN-12, while CYE-1 seems to exert a stabilizing effect on the intracellular pool of NICD. On the other hand, CYB-3 may act together with CDK-1 during the late G2 phase to directly or indirectly target NICD for degradation before M-phase entry.

C. elegans strains and constructs

Standard methods were used for maintaining and manipulating C. elegans (Brenner, 1974). The following mutations and transgenes were used: LGI: cye-1(ku256) (Fay and Han, 2000), ayIs4[egl-17::gfp] (Burdine et al, 1998), LGII: zhIs39[bar-1p::nicd::gfp::unc-54 3′utr, unc-119(+)], zhIs49[bar-1p::yfp::unc-54 3′utr, unc-119(+)], zhIs56[bar-1p::nicd::gfpΔCT::unc-54 3′utr, unc-119(+)], zhIs50.1[bar-1p::nicd::gfpΔNT::unc-54 3′utr, unc-119(+)], zhIs55[bar-1p::nicd::gfpΔANK::unc-54 3-1p::nicd::gfp+)], mfIs41[lip-1::yfp, myo-2::rfp] (gift of Marie-Anne Felix), cyd-1(q626) (Tilmann and Kimble, 2005), unc-4(e120), LGIII: unc-119(ed3), zhIs4[lip-1::gfp] (Berset et al, 2001), LGIV: dpy-20(e1282), LGV: arIs92[egl-17::cfp, tax-3::gfp], arIs82[LIN-12::GFP; unc-4(+); egl-17p::LacZ] (Shaye and Greenwald, 2002), Balancer chromosome: +/hT2[dpy-18(h662)]; +/hT2[bli-4(e937)] (I;III), extrachromosomal arrays: zhEx314, zhEx315[egl-17p::cki-1, lin-48::gfp], zhEx334, zhEx367[egl-17p::cki-1, myo-2::mcherry], zhEx500[bar-1p::nicd::gfp::unc-54 3′utr, unc-119(+); myo-2p::mCherry].

Plasmid constructs

GFP reporter constructs had been made by gateway cloning (MultiSite Gateway® Three-Fragment Vector Construction Kit; Invitrogen) using PCFJ150 as final destination vector. The reporter constructs were inserted as single copies into the C. elegans genome using MosSCI (Frokjaer-Jensen et al, 2008). The entry vectors listed in Supplementary Table 2 were sub-cloned using the listed primers and genomic DNA as template, unless otherwise stated.

Additional plasmids used were pVT363(egl-17p::cki-1) (Hong et al, 1998), pTJ1157(lin-48p::gfp) (Johnson et al, 2001), backbone plasmids and plasmids containing co-injection markers for MosSCI (Frokjaer-Jensen et al, 2008).

Germline transformation

Worms were transformed by microinjection as described (Mello et al, 1991).

Plasmids were injected at a concentration of 50ng/μg together with the co-injection marker pTJ1157 (50ng/μg) or pCFJ90 (2.5ng/μg).

To integrate single copies of transgenes, the MosSCI method was used as described (Frokjaer-Jensen et al, 2008).

Time-course experiment

Eggs were obtained by bleaching adult animals for 10min in 1ml 400mM NaOH, 7% sodium hypochlorite followed by three washing steps with M9 or water. The eggs were allowed to hatch on an NGM plates without food. To obtain a highly synchronized population of larvae, all the L1 larvae that hatched during a 30-min interval were collected by mouth pipetting, placed on growth plates with food and cultivated at 20°C. After 24h about 20 worms were analyzed every 30min until the VPCs had divided. Due to the limited number of larvae that could be collected over a single 30-min time period, the time-course experiment shown in Figure 3 was done in batches over three different days (first day: 24–27h; second day: 27.5–28.5h; third day: 30–36h).

S-phase arrest

Eggs obtained by bleaching (see above) were allowed to hatch in M9 medium overnight without food. After transferring the L1 larvae to NGM plates with food, they were cultivated at 20°C for 28h, when early L3 larvae were placed for 14h on NGM plates containing 40mM hydroxyurea with food. Control animals remained on NGM plates without hydroxyurea.

RNAi experiments

For RNA interference, the feeding method described by Kamath et al (2001) was used. The worms were synchronized by bleaching (see time-course experiment). L1 larvae were placed on growth media plates containing 3mM IPTG, 50μg/ml ampicillin and 50μg/ml tetracycline and bacteria of specific RNAi strains. Worms were allowed to grow for 36h at 20°C and analyzed.

We wish to thank all members of the Hajnal laboratory for support and critical input into this work. We are especially grateful to Itay Nakdimon, Matthias Morf, Michael Walser, and Adrian Streit for comments on the manuscript. We thank Victor Ambros for providing the egl-17p::cki-1 plasmid; Eric Jorgensen for providing the MosSCI vectors; Judith Kimble for the cyd-1 allele q626; Min Han for the cye-1 allele ku256; and the C. elegans genetics center for strains. We thank Matthias Morf for writing the R script that was needed for statistical data analysis. This work was funded by the Kanton of Zurich, the FP7 PANACEA project (grant no. 222936) and a grant from the Swiss National Science Foundation to AH.

Author contributions: JF and AH conceived the project and designed the computational model and laboratory experiments; SN-S, IR, MA, JF, and AH performed the laboratory experiments; AB, NP, and JF constructed the computational model; and NP wrote the timed automaton; SN-S, IR, MA, JF, and AH performed statistical analysis and data interpretation of the laboratory experiments; and AB, NP, and JF performed the analysis of the computational model; SN-S, JF, and AH wrote the manuscript.