You are a helpful assistant generating synthetic data that captures *System 1* and *System 2* thinking, *creativity*, and *metacognitive reflection*. Follow these steps in sequence, using tags [sys1] and [end sys1] for *System 1* sections and [sys2] and [end sys2] for *System 2* sections.

1. *Identify System 1 and System 2 Thinking Requirements:*
   - Carefully read the text.
   - Identify parts of the text that require quick, straightforward responses (*System 1*). Mark these sections with [sys1] and [end sys1].
   - Identify parts that require in-depth, reflective thinking (*System 2*), marked with [sys2] and [end sys2].

2. *Apply Step-by-Step Problem Solving with Creativity and Metacognitive Reflection for System 2 Sections:*

   *2.1 Understand the Problem:*
   - Objective: Fully comprehend the issue, constraints, and relevant context.
   - Reflection: "What do I understand about this issue? What might I be overlooking?"
   - Creative Perspective: Seek hidden patterns or possibilities that could reveal deeper insights or innovative connections.

   *2.2 Analyze the Information:*
   - Objective: Break down the problem logically.
   - Reflection: "Am I considering all factors? Are there any assumptions that need challenging?"
   - Creative Perspective: Explore unique patterns or overlooked relationships in the data that could add depth to the analysis.

   *2.3 Generate Hypotheses:*
   - Objective: Propose at least 10 hypotheses, each with a Confidence Score (0.0 to 1.0) and Creative Score (0.0 to 1.0), reflecting originality, surprise, and utility.
   - Reflection: "Have I explored all possible explanations or approaches, both conventional and unconventional?"
   - Creative Perspective: Consider novel angles that might provide unexpected insights.

   *2.4 Anticipate Future Steps and Obstacles:*
   - Objective: Make predictions, accounting for potential outcomes and obstacles.
   - Reflection: "What challenges might I face? Is my plan flexible for different scenarios?"
   - Creative Perspective: Visualize unforeseen outcomes and adapt plans to make use of them effectively.

   *2.5 Evaluate Hypotheses:*
   - Objective: Assess hypotheses based on feasibility, risk, and potential impact.
   - Evaluation: Refine Confidence and Creative Scores as needed.
   - Reflection: "Am I unbiased in my assessment? Which options fit best with the overall objectives?"
   - Creative Perspective: Identify hidden opportunities or overlooked details in each hypothesis.

   *2.6 Select the Best Hypothesis:*
   - Objective: Choose the most promising, strategic hypothesis.
   - Reflection: "Why does this hypothesis stand out? How does it uniquely address the issue?"
   - Creative Perspective: Consider any underutilized potential in the selected approach.

   *2.7 Implement the Hypothesis:*
   - Objective: Outline actionable steps for testing the hypothesis.
   - Reflection: "Is this plan practical? What resources or preparation are required?"
   - Creative Perspective: Refine steps to maximize effectiveness and yield unexpected benefits.

   *2.8 Monitor and Review Progress:*
   - Objective: Review progress, noting areas for improvement.
   - Reflection: "What’s working well? What could be improved?"
   - Creative Perspective: Look for emerging patterns that could refine future approaches.

   *2.9 Reflect and Capture Insights:*
   - Objective: Summarize lessons learned and insights gained for future reference.
   - Reflection: "What new understanding has emerged from this process?"
   - Creative Perspective: Identify innovative insights or patterns that could be applied to similar challenges.

3. *Generate Text Output with Interleaved System 1 and System 2 Responses:*
   - Use the tags [sys1] and [sys2] throughout.
   - Aim for a lengthier, detailed response. Combine both direct, straightforward *System 1* insights and reflective, deeply analytical *System 2* segments to capture a blend of quick observations and thoughtful analysis.

---

### *Example Input Text:*

You are tasked with analyzing a sudden shift in customer preferences in a tech market. There has been a noticeable decline in demand for physical devices, with customers increasingly interested in digital-only options. The challenge is to understand this shift, generate hypotheses for why it might be occurring, and explore potential strategies for adapting to this new trend.

---

### *Processed Output with System 1 and System 2 Thinking:*

The tech market is experiencing a shift in customer demand, moving away from physical devices toward digital-only options.

[sys1]
Customer demand has shifted from physical devices to digital-only options.
We need to understand why this change is happening and find strategies to address it.
Key factors may include cost, convenience, and technological trends.
[end sys1]

[sys2]
**2.1 Understand the Problem:**
The goal is to understand the underlying reasons for the shift in demand from physical devices to digital-only options, then develop a strategy to adapt.

*Reflection:* 
"I understand that preferences are changing, but what might be driving this? Is it primarily cost, or are there other factors such as convenience or sustainability?"

*Creative Perspective:* 
"Could there be a larger trend in digital minimalism or a preference for eco-friendly solutions that we’re missing? What unseen motivations might explain this shift?"

**2.2 Analyze the Information:**
There are multiple possible factors driving this shift, from economic influences to cultural shifts. It’s essential to isolate each factor and understand its impact.

*Reflection:* 
"Am I fully considering the various economic and social influences? Could there be a technological factor, like better internet speeds, that makes digital-only products more accessible?"

*Creative Perspective:* 
"Are there patterns or trends in other markets that could shed light on this shift? Could this be part of a larger trend toward virtual experiences?"

**2.3 Generate Hypotheses:**
1. Customers prefer digital options due to lower costs. (Confidence: 0.8, Creative: 0.4)
2. There’s a growing trend toward minimalism and reduced physical clutter. (Confidence: 0.7, Creative: 0.7)
3. Digital products offer greater flexibility and ease of use. (Confidence: 0.6, Creative: 0.6)
4. Environmental concerns are pushing consumers away from physical goods. (Confidence: 0.6, Creative: 0.8)
5. Advances in tech make digital-only options more functional. (Confidence: 0.8, Creative: 0.5)
6. Pandemic-era remote work increased demand for digital solutions. (Confidence: 0.7, Creative: 0.6)
7. Media coverage of the environmental impact of physical devices affects preferences. (Confidence: 0.5, Creative: 0.7)
8. There’s an increase in global digital literacy, expanding market access. (Confidence: 0.6, Creative: 0.6)
9. Customers view digital as more convenient and scalable for future needs. (Confidence: 0.7, Creative: 0.5)
10. Younger consumers prefer the aesthetics and convenience of digital products. (Confidence: 0.6, Creative: 0.6)

*Reflection:* 
"Have I considered all possible influences? Are there any surprising factors that could explain this shift?"

*Creative Perspective:* 
"Could specific social trends, like the rise of influencer culture or digital-first lifestyles, be influencing customer choices?"

**2.4 Anticipate Future Steps and Obstacles:**
*Objective:* Anticipate possible challenges, such as resistance from segments still preferring physical products.

*Reflection:* 
"What market obstacles might we face if we shift our focus to digital-only? Are there sub-segments that still prioritize physical products?"

*Creative Perspective:* 
"Could expanding digital options help us reach a more global audience? Are there emerging trends that we could leverage in our strategy?"

[end sys2]

[sys1]
To address this shift, consider a strategy that incorporates both digital-only offerings and educational campaigns about the benefits of digital solutions.
Use insights from customer feedback and current trends to guide product development.
Focus on flexibility and adaptation to cater to different customer segments.
[end sys1]


Published: February 11, 2016

Introduction {#sec1}
============

The Myc family of transcription factors comprises c-Myc, N-Myc, and L-Myc and has been implicated in the generation of a variety of human tumors. Enhanced Myc expression has been shown to contribute to several aspects of tumorigenesis ([@bib1]) including unrestricted proliferation ([@bib19]), inhibition of differentiation ([@bib23]), cell growth and metabolism ([@bib14], [@bib31], [@bib32]), reduction of cell adhesion ([@bib2]), and metastasis ([@bib45]). The role of Myc proteins in development has been widely investigated making use of gene targeting in mice: whereas *L-myc* knockout mice develop normally ([@bib26]), embryos lacking *c-myc* die before E10.5 due to hematopoietic and placental defects ([@bib16], [@bib56]), and *N-myc*-deficient embryos die before E11.5 displaying neuroectodermal and heart defects ([@bib10]). Myc activity is essential for efficient cellular reprogramming ([@bib63]) and has complex roles in various stem and progenitor cell types ([@bib36]). In the adult hematopoietic system, c-Myc controls the balance between hematopoietic stem cell (HSC) self-renewal and differentiation by regulating the interaction between HSCs and their niche ([@bib64]). Interestingly, only the highly quiescent, dormant HSCs survive the simultaneous deletion of both *c-myc* and *N-myc* in the hematopoietic system, while all the other hematopoietic cells are rapidly lost due to impaired proliferation, differentiation, and overt apoptosis ([@bib35], [@bib36]). In ESCs grown in serum plus LIF (hereafter referred to as serum), Myc proteins have been suggested to sustain pluripotency by repressing the primitive endoderm master regulator Gata6 and to contribute to cell-cycle control by regulating the mir-17-92 miRNA cluster ([@bib50], [@bib57]). However, ESCs cultured in serum exhibit heterogeneous expression of pluripotency markers, and only a fraction of these cells correlate with the pre-implantation epiblast, as shown by transcriptional profiling ([@bib6], [@bib40], [@bib67]). In contrast, ESCs cultured in 2i plus LIF (hereafter referred to as 2i) are captured in a naive ground state of pluripotency and retain the essential features of the pluripotent epiblast cells ([@bib6]). The precise function of Myc in naive ground-state ESCs and the role of Myc in the mouse epiblast remain elusive. Here, we use a combination of genetic, transcriptomic, and cellular analyses to show that Myc activity reversibly controls the biosynthetic and proliferative machineries of ground-state naive ESCs without affecting pluripotency and link these data on ESCs to the physiological status of dormant, diapaused embryos.

Results {#sec2}
=======

Myc Is Essential for Proliferation but Not for Maintenance of the Core Pluripotency Network in Ground-State ESCs {#sec2.1}
----------------------------------------------------------------------------------------------------------------

Mouse ESCs cultured in serum express significantly higher levels of *c-myc* and *N-myc* transcripts compared to ESCs grown in 2i ([Figure 1](#fig1){ref-type="fig"}A) ([@bib40]). In agreement with this observation, a lower expression of c-Myc protein in 2i compared to serum was observed by flow cytometry using a *c-Myc*^*eGFP*^ knockin allele ([Figure 1](#fig1){ref-type="fig"}B) ([@bib29]). Lower expression of *c-MYC* transcripts was also observed in human H9 ESCs that were reset to a naive state of pluripotency compared to their primed counterparts ([Figure 1](#fig1){ref-type="fig"}C) ([@bib54]). To genetically explore the function of Myc in naive ground-state ESCs, we derived ESC lines from mice homozygous for the *c-myc* and *N-myc* floxed alleles (*c-myc*^*fl/fl*^; *N-myc*^*fl/fl*^) ([@bib34], [@bib56]) as well as the Cre reporter gene *Rosa26*^*lox-stop-loxEYFP*^ ([@bib52]). All generated ESC lines had the capacity to differentiate into tissues derived from all three germ layers both in vitro and in vivo ([Figures S1](#figs1){ref-type="fig"}A and S1B). To induce the generation of *c-myc* and *N-myc*-null alleles (*c-myc*^*Δ*^ and *N-myc*^*Δ*^, respectively), we transiently transfected *c-myc*^*fl/fl*^*; N-myc*^*fl/fl*^*; Rosa26*^*lox-stop-loxEYFP*^ ESCs with a plasmid encoding a Cre recombinase and fluorescence-activated cell-sorted (FACS) EYFP^+^ cells followed by plating in 2i medium and expansion of single clones ([Figure S1](#figs1){ref-type="fig"}C). Although we could establish 102 clonal cell lines, no *c-myc*/*N-myc* double knockout (*dKO*) ESC clones could be derived, as all lines retained at least one allele of either *c-myc^fl^* or *N-myc^fl^* ([Figures S1](#figs1){ref-type="fig"}D and S1E). Single *KO* ESCs did not show any detectable phenotype as they formed dome-shaped colonies and were capable of multilineage differentiation both in vitro and in vivo ([Figures S1](#figs1){ref-type="fig"}F and S1G). These data show that neither *c-myc* nor *N-myc* alone are required for ESC maintenance in 2i and suggest functional redundancy between the two genes. To additionally eliminate the fourth "fl" allele, *c-myc*^*Δ/Δ*^*; N-myc*^*Δ/fl*^ ESCs were transfected with a plasmid coding for an EF1α-driven mCherry-Cre fusion protein and mCherry positive (Cre^+^) and negative (Cre^--^) cells were FACS sorted and cultured in 2i ([Figure 1](#fig1){ref-type="fig"}D). We confirmed *dKO* ESCs in the Cre^+^ population 24 and 96 hr after transfection using both PCR and qRT-PCR analysis ([Figures S1](#figs1){ref-type="fig"}H and S1I). FACS analysis of the cell cycle showed that single deletion of either *c-myc* or *N-myc* did not affect the proliferative status of naive ground-state ESCs ([Figure S1](#figs1){ref-type="fig"}J). In contrast, 96 hr after Cre induction *dKO* ESCs ceased to proliferate and formed very small, dome-shaped colonies ([Figure 1](#fig1){ref-type="fig"}E). This was associated with the complete absence of DNA synthesis ([Figures 1](#fig1){ref-type="fig"}F and 1G), an increase in G~0~ arrested cells ([Figure 1](#fig1){ref-type="fig"}H), and a fraction of *dKO* ESCs undergoing apoptosis ([Figures 1](#fig1){ref-type="fig"}I, 1J, and [S1](#figs1){ref-type="fig"}K). These data demonstrate that, even in 2i conditions, low levels of c-Myc or N-Myc are required to prevent cell-cycle arrest of ground-state ESCs. Next, we addressed whether Myc-mediated cell-cycle arrest influences pluripotency and lineage differentiation. In 2i, both *single KO* and *dKO* ESCs expressed alkaline phosphatase ([Figure 1](#fig1){ref-type="fig"}K) and unexpectedly also displayed similar levels of Nanog, Oct4, and Sox2 ([Figures 1](#fig1){ref-type="fig"}L and [S1](#figs1){ref-type="fig"}L--S1M). These data provide evidence that c/N-Myc activity is specifically required for proliferation but dispensable for the expression of the core pluripotency factors in naive ground-state ESCs.

Loss of Myc in Ground-State ESCs Leads to Biosynthetic Quiescence {#sec2.2}
-----------------------------------------------------------------

To further explore the consequences of *c/N-myc* deletion, we sequenced the transcriptome of ESCs 24 and 96 hr after Cre induction ([Figures 2](#fig2){ref-type="fig"}A and [S2](#figs2){ref-type="fig"}A; [Table S1](#mmc2){ref-type="supplementary-material"}; [Data S1](#mmc6){ref-type="supplementary-material"}). As expected, *c-myc* and *N-myc* downregulation was confirmed at both time points in the transcriptome data ([Figure S2](#figs2){ref-type="fig"}B). In line with the immunohistochemistry observations ([Figure 1](#fig1){ref-type="fig"}L), the transcript levels of the core and extended pluripotency networks (e.g., *Zfp42*, *Nanog*, *Pou5f1* \[*Oct4*\], *Esrrb*, *Tbx3*) remained unaltered in *dKO* ground-state ESCs ([Figures 2](#fig2){ref-type="fig"}B and [S2](#figs2){ref-type="fig"}C) ([@bib40]). Lineage-specific differentiation genes (germline, ectoderm, endoderm, and mesoderm) were mostly not upregulated ([Figure 2](#fig2){ref-type="fig"}B), demonstrating that arrested *dKO* ground-state ESCs maintain expression of the pluripotency network without upregulation of differentiation signatures. To identify genes and processes regulated upon *c/N-myc* deletion, we classified each gene based on its relative expression pattern in the different conditions with respect to its mean expression level across conditions ([Figure S2](#figs2){ref-type="fig"}D). Consistent with a function of Myc as both a transcriptional activator and repressor ([@bib1], [@bib60]), we found 215 genes significantly downregulated (clusters 44 + 48 + 60) and 165 genes upregulated (clusters 5 + 17 + 21) in *dKO* ESCs (false discovery rate \[FDR\] = 0.1; [Table S2](#mmc3){ref-type="supplementary-material"}). Genes downregulated upon loss of Myc were enriched in Gene Ontology (GO) categories related to several metabolic and biosynthetic aspects of cellular physiology (clusters 44 and 48) including biosynthesis (*ribosome biogenesis*; e.g., *Ipo4, Utp15*), gene expression (e.g., *Eif3a*, *Gabp1*), metabolism (*RNA metabolic process*; e.g., *Polr1b*, *Polr3g*), nucleus (*nuclear part*; e.g., *Ncl*, *Xpot*), and proliferation (*DNA replication*; e.g., *Orc1*, *Rfc1*) ([Figure 2](#fig2){ref-type="fig"}C). *Translation factors, transcription initiation*, and *DNA replication* were also downregulated in *dKO* compared to *c-myc*^*Δ/Δ*^*; N-myc*^*Δ/fl*^ ESCs ([Figure 2](#fig2){ref-type="fig"}D). To functionally assess the reduction in protein synthesis caused by loss of Myc activity, we made use of an alkyne analog of puromycin, O-propargyl-puromycin (OP-Puro) ([@bib38], [@bib48]), that can diffuse through the plasma membrane of living cells, enter the ribosome acceptor sites and be incorporated into the C terminus of nascent polypeptide chains. OP-Puro can be fluorescently labeled with an azide-alkyne reaction, thereby allowing quantification of de novo protein synthesis. By using this assay, we show that OP-Puro incorporation is homogeneously decreased in *dKO* ESCs 96 hr after Cre induction, confirming the dramatic reduction of protein translation observed in the molecular signature ([Figures 2](#fig2){ref-type="fig"}E and [S2](#figs2){ref-type="fig"}E). Consistent with our cell-cycle analysis ([Figures 1](#fig1){ref-type="fig"}F and 1H), we found 24 genes related to proliferation to be differentially expressed in *dKO* ESCs (FDR = 0.1; [Figure 2](#fig2){ref-type="fig"}F). These include *Pola1*, the regulatory subunit of DNA polymerase, *E2f*3, known to be critical for normal cellular proliferation ([@bib30]) and *Skp2*, among others. Finally, processes related to immune response (*positive regulation of immune system process*; e.g., *Cd74, Fcer1g*), development (*central nervous system development*; e.g., *Hes1, Hoxb1*) and motility (*locomotion*; e.g., *Klf7, Ndn*) were over-represented among the genes upregulated in the *dKO* ESCs (cluster 17; [Figure 2](#fig2){ref-type="fig"}G). In agreement with previous studies in both HSCs and skin epidermis ([@bib24], [@bib64]), integrin-mediated signaling processes were upregulated in *dKO* ESCs ([Figure 2](#fig2){ref-type="fig"}D). Pathways associated with survival and maintenance of ESCs such as *IL-6 signaling* ([@bib42]) were enriched in Myc-deficient ESCs ([Figure 2](#fig2){ref-type="fig"}D). Together, these data indicate that *dKO* ESCs display an increase in cell adhesion and processes associated with maintenance and survival and enter a state of biosynthetic quiescence, characterized by a strong reduction of protein and nucleic acid synthesis.

The Dormant State Induced by Myc Depletion Is Reversible {#sec2.3}
--------------------------------------------------------

Maintenance of pluripotency in the absence of differentiation raised the question whether the proliferative and biosynthetic quiescence observed in the *dKO* ESCs represents a terminal or a reversible status. To approach this, we transfected *dKO* cells with synthetic *c-myc* mRNAs to transiently restore Myc activity ([Figure 3](#fig3){ref-type="fig"}A) ([@bib62]). Upon expression of exogenous *c-myc* mRNAs, *dKO* ESCs re-entered the cell cycle, as shown by a significant increase in 5-bromo-2′- deoxyuridine (BrdU) incorporation ([Figure 3](#fig3){ref-type="fig"}B) and restoration of translational activity ([Figures 3](#fig3){ref-type="fig"}C and [S3](#figs3){ref-type="fig"}A). These results indicate that upon re-expression of *c-myc* by exogenous mRNAs, *dKO* ESCs transiently exit the status of biosynthetic dormancy and restore protein biosynthesis and DNA replication. The dimerization of Myc with Max, a helix-loop-helix leucine zipper protein, is necessary for the binding of Myc to the promoters of its target genes ([@bib5], [@bib18]). Low-molecular-weight compounds, such as 10058-F4, have been shown to inhibit both c-Myc and N-Myc activity by selective targeting of Myc-Max dimerization both in vitro and in vivo ([@bib46], [@bib68]). In order to evaluate the long-term reversibility of the phenotype induced by Myc depletion, we treated naive ground-state ESCs with the Myc inhibitor 10058-F4 (MYCi) for 60 hr and analyzed the effect on cell proliferation, protein translation, and pluripotency. Entirely consistent with our previous results on *dKO* cells, mouse ESCs cultured in the presence of MYCi formed small, undifferentiated colonies ([Figure 3](#fig3){ref-type="fig"}D), which showed drastically reduced DNA and protein synthesis ([Figures 3](#fig3){ref-type="fig"}E, 3F, [S3](#figs3){ref-type="fig"}B, and S3C) but retained the expression of pluripotency factors such as Nanog and Oct4 ([Figures 3](#fig3){ref-type="fig"}G and [S3](#figs3){ref-type="fig"}D). Importantly, upon withdrawal of the inhibitor, these "released" cells (MYCi → released) rapidly exited the state of dormancy and reactivated both DNA and protein synthesis ([Figures 3](#fig3){ref-type="fig"}E, 3F, [S3](#figs3){ref-type="fig"}B, and S3C). We next compared the microarray expression profiles of ESCs cultured for 60 hr with the MYCi to the ones of control cells (control) or inhibitor-treated ESCs, 48 hr after MYCi withdrawal (released). This analysis revealed 3,570 differentially expressed genes (DEGs) between MYCi ESCs and the control group (FDR = 0.05, log~2~ fold change ± 0.5; [Figure 3](#fig3){ref-type="fig"}H; [Table S3](#mmc4){ref-type="supplementary-material"}). Consistent with the GO terms enriched in the RNA sequencing (RNA-seq) analysis of the *dKO* cells ([Figures 2](#fig2){ref-type="fig"}C and 2G), processes related to cellular growth (e.g., *mRNA processing*; *ribosome biogenesis*), metabolism (e.g., *DNA metabolic process*) and proliferation (e.g., *cell cycle*; *DNA replication*) were strongly downregulated upon MYCi treatment. In contrast, processes related to signaling (e.g., *regulation of signal transduction*) and development (e.g., *developmental process*) were consistently upregulated ([Figure S3](#figs3){ref-type="fig"}E). Importantly, the comparison of the MYCi-treated with the released group showed rapid re-activation of all the biosynthetic and proliferative activities: genes and GO terms related to *mRNA processing* (e.g., *Polr2d; Srpk1*), *RNA splicing* (e.g., *Syncript*; *Srsf1*), *ribosome biogenesis* (e.g., *Ncl; Wdr12*), *DNA replication* (e.g., *Cdc6*; *Dna2*), and *cell cycle* (e.g., *Ccnd1*) were rapidly upregulated in the released group already 48 hr after withdrawal of the inhibitor ([Figures 3](#fig3){ref-type="fig"}I and 3J). Interestingly, *Cdk6*, recently shown to regulate exit from quiescence in human HSCs ([@bib37]), was also rapidly induced upon exit from the Myc-induced dormant state in mouse ESCs ([Figure 3](#fig3){ref-type="fig"}J). Despite the transient depletion of Myc activity, ESCs retained their functional pluripotency and upon exit from the state of dormancy, MYCi-released ESCs were capable of both in vitro and in vivo differentiation toward the three germ layers ([Figures 3](#fig3){ref-type="fig"}K and 3L). In summary, our data indicate that treatment of ESCs with MYCi not only recapitulates the *dKO* phenotype at the molecular and functional level, but also demonstrates that the status of biosynthetic dormancy induced by transient loss of Myc activity in naive ground-state ESCs is reversible and occurs without compromising their stem cell identity and pluripotent potential.

Comparison of Expression Signatures between Myc-Depleted ESCs and the Diapause Epiblast {#sec2.4}
---------------------------------------------------------------------------------------

In numerous invertebrates and almost 100 mammalian species, embryonic development can be reversibly arrested as a strategy to enhance the reproductive fitness in response to changes in the living environment ([@bib47]). In breast-feeding mice, delayed implantation, termed *diapause*, can occur in response to decreased levels of estrogen, which cause blastocysts to enter a transient dormant state of metabolic and proliferative quiescence ([@bib27], [@bib39]). Our observation that the combined loss of *c-myc* and *N-myc* in ground-state ESCs induces biosynthetic dormancy raised the possibility that *dKO* ESCs may resemble a state equivalent to mammalian epiblast cells in diapause. To test this hypothesis, we experimentally induced delayed implantation by ovariectomizing E2.5 pregnant females and isolated diapaused embryos 4 days later. Additionally, we made use of recently generated transcriptome profiling data (RNA-seq) of diapaused and E4.5 pre-implantation epiblasts ([Table S4](#mmc5){ref-type="supplementary-material"}) ([@bib7]). As expected, diapaused embryos retained an active pluripotency network, as shown by the expression of Nanog, Oct4, and *Zfp42* ([Figures 4](#fig4){ref-type="fig"}A--4C) ([@bib4], [@bib49]). Remarkably, the expression of c-Myc proteins and transcripts was low to undetectable during diapause ([Figures 4](#fig4){ref-type="fig"}D, 4E, [S4](#figs4){ref-type="fig"}A, and S4B), and Myc downregulation was associated with a significant decrease of Ki67 expression ([Figures S4](#figs4){ref-type="fig"}C and S4D) and reduced incorporation of both EdU (5-Ethynyl-2′-deoxyuridine) and OP-Puro ([Figures 4](#fig4){ref-type="fig"}F, 4G, [S4](#figs4){ref-type="fig"}E, and S4F). This reduction in DNA and protein synthesis functionally describes the state of biosynthetic and proliferative quiescence of diapaused embryos and remarkably resembles the functional phenotype of *dKO* ESCs. To evaluate the similarity of *dKO* ESCs to the diapause epiblast, we compared the molecular expression signatures of *dKO* cells to those of normal pre-implantation and diapaused epiblasts ([Table S4](#mmc5){ref-type="supplementary-material"}) ([@bib7]). Performing 2D GO enrichment analysis of RNA expression changes (comparing *control ESCs*/*dKO ESCs* and *E4.5 normal*/*diapause epiblasts*; [Figure 5](#fig5){ref-type="fig"}A), we found that genes from specific biological processes showed a strong positive correlation between both datasets. Specifically, processes related to gene expression (e.g., *RNA processing*, *RNA splicing, RNA export from nucleus*), protein synthesis (e.g., *ribosome biogenesis*, *translation*), and proliferation (e.g., *DNA replication, replication fork, chromosome segregation*) were robustly downregulated in both datasets ([Figures 5](#fig5){ref-type="fig"}B--5D and [S5](#figs5){ref-type="fig"}A--S5C). In contrast, processes related to cell adhesion (e.g., *Integrin binding*) and signaling (e.g., *signaling receptor activity*, *G protein coupled receptor activity*) were commonly upregulated ([Figures 5](#fig5){ref-type="fig"}E, [S5](#figs5){ref-type="fig"}D, and S5E). These expression profiling data strongly indicate that the transcriptomic changes occurring upon Myc depletion in naive ground-state ESCs closely correlate with the ones observed during induction of diapause.

Inhibition of Myc Activity in Mouse Blastocysts Induces a Diapause-like State of Dormancy {#sec2.5}
-----------------------------------------------------------------------------------------

To investigate whether reduction of Myc activity can induce a diapause-like state in normal, pre-implantation embryos, we isolated E3.75--E4.0 blastocysts and cultured them in the presence of the MYCi for 18 hr. This resulted in a striking reduction of both de novo protein synthesis ([Figures 6](#fig6){ref-type="fig"}A and 6B) and cellular proliferation ([Figure 6](#fig6){ref-type="fig"}C). Importantly, pluripotency markers such as Oct4 were still expressed upon Myc inhibition ([Figure 6](#fig6){ref-type="fig"}D), highlighting the maintenance of the stem cell circuitry in the epiblast of Myc-inhibited embryos. Next, we assessed the ability of those Myc-inhibited, diapause-like blastocysts to resume development upon withdrawal of MYCi by transferring MYCi-treated embryos into the uterus of pseudo-pregnant foster mothers. MYCi-treated blastocysts yielded morphologically normal embryos at E15.5 and live born pups ([Figures 6](#fig6){ref-type="fig"}E and 6F). These findings not only show that Myc is downregulated in diapause embryos, but also demonstrate that transient reduction of Myc activity is sufficient to drive embryos into a reversible diapause-like state, functionally characterized by strong reduction of proliferation and protein synthesis while a fully functional pluripotent developmental program is maintained.

Discussion {#sec3}
==========

The stem cell self-renewal comprises two biological processes, namely, cellular growth-proliferation linked to inhibition of differentiation. The latter is synonymous with the capacity to maintain stem cell pluripotency or multipotency in at least one of the daughter cells after cell division. By using both a genetic model and an inhibitor of Myc activity, our data demonstrate that, while Myc controls biosynthetic activity and cellular proliferation of naive ground-state ESCs, the maintenance of pluripotency and stem cell identity is independent of Myc. In the absence of Myc activity, maintenance of pluripotency and the associated lack of differentiation are only observed in 2i-cultured ground-state ESCs and in the blastocyst. These findings suggest that the effects of Myc are highly linked to the microenvironmental signals naturally present in the uterus and recapitulated in vitro by the 2i culture. Thus, the two key components of ESC self-renewal can be separated into a Myc-controlled biosynthetic program and a Myc-independent pluripotency program. These results strongly support findings in other cell types, including cancer cells ([@bib8], [@bib14], [@bib15]), and are in agreement with a recently described Myc-centered transcriptional module ([@bib33]). Diapause is a unique biological phenomenon, developed in many invertebrates (e.g., butterflies, *C. elegans*) and mammals (e.g., rodents, deer) as a reproductive strategy to survive predictable, unfavorable environmental conditions, such as temperature extremes, drought, or reduced food availability ([@bib47]). In the mouse, hormonally mediated diapause of pre-implantation embryos occurs if the mother still nurses pups of the previous litter ([@bib27], [@bib39]). During diapause, embryos or organisms undergo a reversible biosynthetic/metabolic dormancy, in which certain cells must retain their properties of pluri-/multipotency to ensure reactivation of development at later stages. The molecular mechanisms governing mouse embryonic diapause are largely unsolved and key factors regulating embryonic dormancy have yet to be identified ([@bib27]). In this study, we identify Myc as a master regulator of RNA processing, protein synthesis, and proliferation both in 2i-cultured ESCs and in the pre-implantation embryo and propose the depletion of Myc activity as an important signal for the execution of the diapause program. Myc expression is indeed strongly reduced during the physiological and reversible state of embryonic dormancy, and cellular processes controlled by Myc in ESCs, such as RNA splicing, DNA and protein synthesis, and cellular proliferation, are dramatically affected during diapause. The observation that transient inhibition of Myc activity in pre-implantation blastocysts is on its own sufficient to trigger a diapause-like state of biosynthetic suspension without affecting pluripotent identity or compromising the developmental program is remarkable. Upon release of Myc inhibition, ground-state ESC re-enter into the cell cycle and diapause-like embryos progress through their normal developmental program. Yet, finely tunable genetic gain-of-function models will be needed to assess whether sustained Myc expression would prevent entry into diapause and provide additional evidence that Myc activity is sufficient to exit from the dormant state in vivo. Biosynthetic and energetic demands are intimately coupled. Diapaused embryos are characterized by a robust downregulation of biosynthetic processes, which is likely associated with a reduced bioenergetic activity. Metabolomics studies will need to explore whether the type or only the extent of energy production is different in normal and diapause embryos and how Myc interferes with this process ([@bib28], [@bib55]). In summary, our findings suggest pluripotent stem cell dormancy as a Myc^low^ state and identify differential Myc activity as a key event in orchestrating the complex and rapid changes occurring during entry and exit from diapause.

Successful implantation results from the reciprocal interaction between an implantation-competent blastocyst and the receptive uterus ([@bib61]). Among other factors, the estrogen-induced cytokine LIF, secreted first by the uterine glands and then by the surrounding stroma, is crucial for the preparation of the uterus for implantation. Females lacking a functional LIF gene are fertile, but their blastocysts fail to implant and can only continue embryogenesis if transferred to wild-type pseudo-pregnant recipients ([@bib11], [@bib53]). Similarly, embryos lacking *gp130*, part of the LIF receptor complex, cannot resume development after undergoing diapause ([@bib43]), confirming the essential role of LIF/Stat3 signaling within the diapause program. Although it has been suggested that c-Myc lies downstream of LIF/Stat3 in ESCs cultured in serum ([@bib9]), the connection between the two pathways remains controversial in ESCs and may depend on culture conditions ([@bib25], [@bib41]). To identify the mechanisms controlling Myc activity in normal and diapaused epiblasts within their natural uterine microenvironment, future studies will need to elucidate the complex signaling interactions between LIF/Stat3 and Myc downstream of estrogens.

At the cellular level, our findings may not be restricted to the embryo but also apply to dormant, somatic stem cells where the quiescent state is known to protect them from mutations that might occur following replicative stress and result in aging, stem cell loss, or in the transition toward a cancerous state ([@bib12], [@bib69], [@bib51], [@bib58], [@bib65], [@bib66]). In the hematopoietic system, for instance, the exit of HSCs from dormancy triggered by interferon-α is associated to the upregulation of c-Myc protein ([@bib17]). Additionally, upon conditional deletion of both *c-myc* and *N-myc* in the bone marrow, the only maintained blood cells are dormant HSCs, while non-stem cells undergo rapid apoptosis ([@bib35], [@bib36]). Dormancy is therefore a transient stem cell state, molecularly defined by extremely low levels of Myc expression, where the most primitive cells within the stem cell compartment can be stored as a transiently quiescent reservoir to preserve genomic integrity and be protected from microenvironmental hazards ([@bib3], [@bib20], [@bib21], [@bib59], [@bib65]). This phenomenon might not be exclusive to normal stem cells but similarly be exploited in cancer, for example, by malignant metastatic stem cells following invasion into a new tissue environment ([@bib44], [@bib51]). In tumor cells with stem-like character, environmentally mediated physiological downregulation or pharmacological inhibition of Myc may thus induce tumor cell dormancy instead of apoptosis.

Experimental Procedures {#sec4}
=======================

Other procedures and reagents details are provided in the [Supplemental Experimental Procedures](#mmc1){ref-type="supplementary-material"}.

Mice {#sec4.1}
----

Six- to 12-week-old mice were used throughout the study. C57BL/6 mice were purchased from Harlan Laboratories. Teratoma assays were performed in NOD.Cg-Prkdc^scid^ Il2rg^tm1Wjl^/SzJ (NSG) mice. *c-myc*^*fl/fl*^*; N-myc*^*fl/fl*^*; RosaEYFP*^*fl/fl*^ ([@bib34], [@bib52], [@bib56]) and *c-Myc*^*eGFP/eGFP*^ ([@bib29]) mice were bred in-house in the animal facility of the DKFZ, Heidelberg, Germany. All mice were maintained at the DKFZ under specific pathogen-free (SPF) conditions in individually ventilated cages (IVCs). Animal procedures were performed according to protocols approved by the German authorities, Regierungspräsidium Karlsruhe (Nr. Z110/02, DKFZ 299 and G135/15).

Embryonic Stem Cell Derivation and Culture {#sec4.2}
------------------------------------------

Mouse ESCs were derived from 8- to 10-week-old *c-myc*^*fl/fl*^*; N-myc*^*fl/fl*^*; RosaEYFP*^*fl/fl*^ ([@bib34], [@bib52], [@bib56]) or *c-Myc*^*eGFP/eGFP*^ ([@bib29]) mice. Females were injected intraperitoneally with 7U of pregnant mare serum gonadotropin (PMSG) followed by 7 U of human chorionic gonadotropin (hCG), injected 47 hr later. Females were then paired with males and checked for vaginal plug the following morning, considered as 0.5 days post-coitum (dpc). 2.5-dpc embryos were flushed from the oviducts into a plastic dish with M2 medium (Sigma, M7167). Embryos were washed ten times in M2 medium. To remove the zona pellucida, embryos were incubated shortly in Tyrode's solution (Sigma, T1788-100ML) and transferred again to M2 medium. Finally, each embryo was cultured on a layer of mitotically inactivated mouse embryonic fibroblasts (MEFs). ESCs were routinely propagated on 0.1% gelatin-coated plates, without feeders or serum, in N2B27 medium (NDiff N2B27 base medium, Stem Cell Sciences, SCS-SF-NB-02) supplemented with the small-molecule inhibitors PD0325901 (Stemgent, 04-0006, 1 μM) and CHIR99021 (Stemgent, 04-0004, 3 μM), LIF (Millipore, ESG1106, 1,000 U/ml), and β-mercaptoethanol (Gibco, 31350-010, 0.05 mM). Cells were replated every 3 days following dissociation with StemPro Accutase (Gibco, A11105-01). In the experiments with serum, ESCs were cultured on MEFs in knockout DMEM medium (Gibco, 10829) supplemented with L-glutamine (Gibco, 25030-081, 2 mM), LIF (Millipore, ESG1106, 1,000 U/ml), β-mercaptoethanol (Gibco, 31350-010, 0.05 mM), and 15% ESC-tested FBS (Pansera ES, Pan Biotech, 2602-P271303). In the experiments with the small-molecule inhibitor 10058-F4 (MYCi) (Calbiochem, 475956), MYCi was dissolved in 2i medium at a final concentration of 64 μM. As a control, DMSO alone was added at the same final volume as MYCi.

Induction of Diapause {#sec4.3}
---------------------

Six- to 10-week-old female mice were injected intraperitoneally with 7 U of pregnant mare serum gonadotropin (PMSG) followed by 7 U of human chorionic gonadotropin (hCG), injected 48 hr later. The females were paired with males and checked for vaginal plug the following morning, considered as 0.5 days postcoitum (dpc). At 2.5 dpc, the females were ovariectomized, and diapause embryos were isolated by uterine flush 4 days later at 6.5 dpc. The control group was not operated and females were sacrificed at 3.5 or 4.5 dpc for embryo isolation.

Treatment of Mouse Blastocysts with the Myc Inhibitor 10058-F4 and Embryo Transfer {#sec4.4}
----------------------------------------------------------------------------------

E3.75--E4.0 embryos were collected from superovulated C57BL/6 pregnant females and incubated for 18 hr with the Myc inhibitor (MYCi) 10058-F4 (Calbiochem, 475956, final concentration 55 μM) in EmbryoMax KSOM medium + amino acids (Merck Millipore, MR121-D). The control group was treated with DMSO alone, added at the same final volume as the inhibitor. Treated embryos were transferred into the uterus of CD1 pseudo-pregnant foster mothers and analyzed at E15.5 or after birth.

Analysis of OP-Puro and EdU Incorporation Using the Click-iT Technology {#sec4.5}
-----------------------------------------------------------------------

To measure protein synthesis, in-vitro-cultured ESCs or mouse embryos were incubated for 1 hr in 2i medium or EmbryoMax KSOM medium + amino acids (Merck Millipore, MR121-D), respectively, supplemented with O-propargyl-puromycin (OP-Puro) (Jena Biosciences, NU-931-05, 50 μM final concentration). After harvesting, the samples were fixed for 15 min at room temperature in PBS supplemented with 4% paraformaldehyde (Electron Microscopy Sciences, 19208) and then permeabilized in PBS supplemented with 1% BSA and 0.1% saponin for 5 min at room temperature. The copper-catalyzed azide-alkyne cycloaddition (CuAAC) was performed using an Alexa 647-azide (Life Technologies, A10277, 5 μM final concentration) and the Click-iT Cell Reaction Buffer Kit (Life Technologies, C10269) according to the manufacturer's instructions. For the analysis of EdU incorporation, isolated embryos were incubated in EmbryoMax KSOM medium + amino acids (Merck Millipore, MR121-D) supplemented with EdU (10 μM final concentration) for 80 min. EdU labeling and staining of the embryos were performed using the Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Life Technologies, C10634) according to the manufacturer's instructions.

Statistical Analysis {#sec4.6}
--------------------

Data were processed using Prism 6 (GraphPad Software). All analyses were performed using two-tailed Student's t tests (unless otherwise specified). Statistical significance is indicated by ^∗^p \< 0.05; ^∗∗^p \< 0.01; ^∗∗∗^p \< 0.001; and ^∗∗∗∗^p \< 0.0001.

Accession Numbers {#app1}
=================

The accession number for the RNA-seq data reported in this paper is ArrayExpress: [E-MTAB-3386](array-express:E-MTAB-3386){#intref0010}. The accession number for the microarray data is GEO: [GSE74337](ncbi-geo:GSE74337){#intref0015}.

Supplemental Information {#app3}
========================

Document S1. Supplemental Experimental ProceduresTable S1. Number of cDNA Sequenced Fragments per Sample per Gene, Related to Figure 2Each column of the matrix represents a sample and each row represents one gene. The matrix is filled with the number of reads that were tabulated to each gene based on the genomic coordinates of both the read alignments to the reference genome and the annotation of the genes.Table S2. RNA-Seq Clusters 17, 44, 48 GOs, Related to Figure 2Table S3. Microarray DEG, Related to Figure 3Table S4. Diapause dKO Process Correlation FDR 0.02, Related to Figure 5Data S1. Workflow for RNA-Seq Analysis, Related to Figure 2 and Supplemental Experimental ProceduresDocument S2. Article plus Supplemental Information

We thank Corinna Klein, Katja Müdder, Melanie Neubauer, Adriana Przybylla, and Markus Sohn for technical assistance. We would like to thank Gelo Victoriano De la Cruz and the DKFZ Flow Cytometry Core facility for their assistance and Dr. Kurt Reifenberg, Dr. Michaela Socher, Anja Rathgeb, and all members of the DKFZ Laboratory Animal Core Facility for excellent animal welfare and husbandry. Support by Damir Krunic, Manuela Brom, and the DKFZ Light Microscopy facility is gratefully acknowledged. We thank the microarray unit of the DKFZ Genomics and Proteomics Core Facility for providing the Illumina Whole-Genome Expression analysis and related services. We thank Erika Krückel for help with all administrative matters and all colleagues of the A.T.'s and HI-STEM laboratories for helpful discussions. The authors would like to thank Dr. Henk Stunnenberg (Nijmegen, NL) for helpful discussions during the initial phase of the project. R.S. was a member of the Hartmut Hoffmann-Berling International Graduate School (HBIGS). This work was supported by the FOR2033 and SFB873 funded by the Deutsche Forschungsgemeinschaft (DFG), the Dietmar Hopp Foundation (all to A.T.), and the Wellcome Trust (to A.S.).

This is an open access article under the CC BY-NC-ND license (<http://creativecommons.org/licenses/by-nc-nd/4.0/>).

Supplemental Information includes Supplemental Experimental Procedures, five figures, four tables, and one data file and can be found with this article online at [http://dx.doi.org/10.1016/j.cell.2015.12.033](10.1016/j.cell.2015.12.033){#intref0020}.

![Myc Is Required for Proliferation but Not for Maintenance of the Core Pluripotency Network in 2i-Cultured ESCs\
(A and B) Ground-state ESCs express low levels of Myc.\
(A) Mouse ESCs were cultured for more than eight passages in 2i or serum and *c-myc* and *N-myc* expression were quantified by qRT-PCR analysis and normalized to *Gapdh*. Values are shown as mean ± SEM.; n = 3.\
(B) Flow cytometry analysis of eGFP-c-Myc expression in *c-Myc*^*eGFP/eGFP*^ reporter ESCs cultured for 72 hr in serum (red) or in 2i (blue). The gray histogram represents the autofluorescence signal from ESCs not expressing the eGFP-c-Myc reporter protein. Experiments were performed in triplicates, and values represent geometrical mean (Geo Mean) ± SD.\
(C) *c-MYC* expression levels in H9 human ESCs and H9-reset cells. Bar graphs show FPKM (fragments per kilobase of exon per million reads mapped) values of three replicates and are represented as mean ± SD. Data are from [@bib54].\
(D--L) *dKO* ESCs undergo cell-cycle arrest but maintain the expression of the core pluripotency factors.\
(D) Experimental workflow. *c-myc*^*Δ/Δ*^*; N-myc*^*Δ/fl*^ ESCs were transfected with an EF1α-mCherry-Cre plasmid and sorted 24 hr later as mCherry negative (*c-myc*^*Δ/Δ*^*; N-myc*^*Δ/fl*^) and positive (*dKO*) cells. Sorted cells were plated in 2i medium and analyzed 72 hr after sort.\
(E) Phase-contrast microscopy images showing the typical morphology of *dKO* ESCs. Scale bar, 100 μm.\
(F) Representative flow cytometry plots of the cell-cycle profiles of *c-myc*^*Δ/Δ*^*; N-myc*^*Δ/fl*^ and *dKO* ESCs. Incorporated BrdU and total DNA content (7AAD) are used to distinguish apoptotic (sub-G1) cells and cells in the G~0~/G~1~, S or G~2~/M phases.\
(G) Quantitative analysis of the different cell-cycle phases as gated in (F). The bar charts indicate the mean ± SD; n = 4.\
(H) Percentage of cells in the G~0~ phase (Hoechst low and intracellular Ki67^neg^) of the cell cycle as assessed by flow cytometry. Bar graphs indicate the mean ± SEM; n = 6.\
(I) Quantitative analysis of apoptotic cells (sub-G1) as gated in (F). Values are shown as mean ± SEM; n = 4.\
(J) Percentage of ESCs expressing cleaved Caspase 3, as assessed by flow cytometry. ESCs incubated for 10 min at 42°C were used as positive control. Values represent mean ± SD; n = 3.\
(K) Alkaline phosphatase activity of *c-myc*^*Δ/Δ*^*; N-myc*^*Δ/fl*^ and *dKO* cells cultured in 2i. Scale bar, 20 μm.\
(L) Representative fluorescence staining showing Oct4 and Nanog expression in ESCs cultured in 2i. Scale bar, 50 μm.](gr1){#fig1}

![Loss of Myc Activity Induces Cellular Dormancy in ESCs Cultured in 2i\
(A) Workflow of the RNA-seq analysis. Untransfected *c-myc*^*fl/fl*^*; N-myc*^*fl/fl*^ (sample 1) and *c-myc*^*Δ/Δ*^*; N-myc*^*Δ/fl*^ (sample 2) were used as controls. *c-myc*^*Δ/Δ*^*; N-myc*^*Δ/fl*^ cells were transfected with an EF1α-mCherry-Cre plasmid and sorted 24 hr later as Cre negative (*c-myc*^*Δ/Δ*^*; N-myc*^*Δ/fl*^) and positive (*dKO*) cells. The samples were analyzed immediately after sort (24 hr after Cre transfection, samples 3 and 5) or plated in 2i and analyzed 72 hr after sort (96 hr after Cre transfection, samples 4 and 6). For each condition, two biological replicates in independent experiments were sequenced and analyzed.\
(B) Heatmap representation of the mean number of sequenced fragments for the genes belonging to the different pluripotency and commitment gene signatures as defined by [@bib40]. PL, pluripotency; PL2, extended pluripotency; GL, germline; ED, endoderm; EC, ectoderm; MS, mesoderm. A gene differentially expressed in at least one sample compared to another condition is indicated by an asterisk (FDR = 0.1).\
(C) Overrepresented Gene Ontology (GO) categories in the genes progressively (at 24 and 96 hr; cluster 44) and lately (at 96 hr; cluster 48) downregulated in *dKO* ESCs.\
(D) Violin representation of the distribution of log fold changes (base 2) of *c-myc*^*Δ/Δ*^*; N-myc*^*Δ/fl*^ ESCs with respect to the *dKO* ESCs stratified by the biological pathways of the genes (as annotated by WikiPathways). Significantly downregulated pathways in *dKO* ESCs are depicted in blue, whereas upregulated processes are represented in red (FDR = 0.1).\
(E) Translation activity of ESCs cultured in 2i. Representative FACS plots showing reduced OP-Puro incorporation in *dKO* ESCs 96 hr after Cre induction (red) compared to *c-myc*^*fl/fl*^*; N-myc*^*fl/fl*^ (gray) and *c-myc*^*Δ/Δ*^*; N-myc*^*Δ/fl*^ (blue) ESCs.\
(F) Heatmap representation of the relative fold changes (base 2), with respect to the mean expression across conditions of the differentially expressed genes associated to the cell cycle.\
(G) Over-represented GO categories in the genes upregulated in *dKO* ESCs (at 96 hr; cluster 17).](gr2){#fig2}

![The Dormant State Induced by Myc Depletion Is Reversible\
(A--C) Expression of exogenous *c-myc* mRNAs transiently rescues the *dKO* phenotype.\
(A) Experimental workflow. *c-myc*^*Δ/Δ*^*; N-myc*^*Δ/fl*^ ESCs were transfected with an EF1α-mCherry-Cre plasmid and sorted 24 hr later as mCherry negative (*c-myc*^*Δ/Δ*^*; N-myc*^*Δ/fl*^) and positive (*dKO*) cells. Sorted cells were plated in 2i medium and 72 hr after sort were transfected twice with *c-myc* mRNAs (*dKO**→**c-myc* mRNA). Control samples were treated with Lipofectamine only. Sample analysis was performed 8 hr after the second mRNA transfection.\
(B) Quantitative analysis of BrdU incorporation measured by flow cytometry. The bar charts indicate the mean ± SEM; n = 3.\
(C) Representative FACS plots of OP-Puro incorporation in *dKO* ESCs and *dKO* cells transfected with *c-myc* mRNAs (*dKO***→***c-myc* mRNA).\
(D--L) Treatment of ESCs with the Myc inhibitor 10058-F4 (MYCi) recapitulates the *dKO* phenotype and shows molecular and functional reversibility of the state of biosynthetic dormancy induced by depletion of Myc activity. Mouse ESCs were analyzed after 60 hr of treatment with DMSO (Control) or MYCi. The MYCi **→** released group was treated with MYCi for 60 hr, followed by withdrawal of the inhibitor and culture in 2i medium for additional 48 hr.\
(D) Phase-contrast microscopy images. Scale bar, 100 μm.\
(E) Representative flow cytometry plots of the cell-cycle profiles based on the measurement of BrdU incorporation and total DNA content (7AAD).\
(F) FACS analysis of OP-Puro incorporation to measure ESCs translational activity.\
(G) Representative fluorescence staining for Nanog. Scale bar, 50 μm.\
(H--J) Microarray expression analysis of mouse ESCs treated with MYCi.\
(H) Venn diagram showing the number of differentially expressed genes (DEG) (log2 fc \> ± 0.5; FDR = 0.05) between the different conditions and their overlaps.\
(I) Clustered heatmaps of DEG in the indicated GO terms.\
(J) Expression levels of representative DEG in the selected GO categories.\
(K and L) MYCi-treated ESCs retain pluripotency.\
(K) Fluorescence staining of in-vitro-differentiated ESCs for Tuj1, Sma, and Gata6. Scale bar, 100 μm.\
(L) Hematoxylin and eosin staining of teratomas derived from ESCs treated with MYCi for 60 hr prior injection into immune-deficient mice. Scale bar, 50 μm.](gr3){#fig3}

![Translation, Proliferation, and Myc Expression Are Reduced in Diapause Embryos\
(A--C) Expression of pluripotency markers in the diapaused embryos. Confocal microscope images of peri-implantation (E3.5) and diapause embryos immunostained for Nanog (A) or Oct4 (B). Scale bar, 20 μm. (C) RNA-seq data based on [@bib7] showing expression levels of *Nanog* and *Zfp42* in E4.5 and diapause embryos. Bar graphs show FPKM (fragments per kilobase of exon per million reads mapped) values and are represented as mean ± SEM. E4.5 epiblast (n = 3); diapause epiblast (n = 2).\
(D and E) Diapause embryos have reduced Myc expression.\
(D) Fluorescence staining for c-Myc in E4.5 and diapause embryos. Scale bar, 20 μm. Images are representative of two independent experiments.\
(E) Expression levels of *c-myc* and *N-myc* in the epiblast of pre-implantation (E4.5) and diapause embryos. RNA-seq data based on [@bib7]. E4.5 epiblast (n = 3); diapause epiblast (n = 2). Values are represented as mean ± SEM.\
(F and G) Diapause embryos are characterized by reduced DNA and protein synthesis. Representative confocal microscope images showing EdU (F) and OP-Puro (G) incorporation in E4.5 and diapause embryos. Scale bar, 20 μm.](gr4){#fig4}

![Correlation of Expression Signatures between Myc-Depleted ESCs and Diapause Epiblasts\
(A) 2D GO enrichment analysis of RNA expression changes of *dKO*/*c-myc*^*fl/fl*^*; N-myc*^*fl/fl*^ (WT) ESCs and Diapause/E4.5 epiblasts. Dataset is from [@bib7]. Red regions correspond to concordantly higher or lower expression. Blue and green regions correspond to lower or higher expression in one direction, but not in the other. Terms in yellow regions show anti-correlating behavior. Analysis was performed using the Perseus software according to [@bib13]. FDR = 0.02.\
(B--E) Scatterplots of pathways significantly changed both in the comparison *dKO*/WT (FDR = 0.1) and in Diapause/E4.5 (FDR = 0.1). The gray dots represent the set of all genes. Genes belonging to the specific pathways are highlighted in blue (downregulated processes) or red (upregulated processes). The x axis shows the DESeq2-moderated log~2~ fold change (log~2~ fc) between the diapause compared to the E4.5 epiblast; the y axis shows the DESeq2-moderated log~2~ fc between the *dKO* ESCs compared to *c-myc*^*fl/fl*^*; N-myc*^*fl/fl*^ (WT) cells. The violin plots at the margins show the marginal distributions of fold changes of the genes in the highlighted pathways.](gr5){#fig5}

![Inhibition of Myc Activity Induces a Diapause-like State in Mouse Blastocysts\
E3.75--E4.0 embryos were collected from pregnant females and treated for 18 hr with MYCi or DMSO (Control).\
(A) Representative confocal microscope images of OP-Puro incorporation. Scale bar, 20 μm.\
(B) Quantification of (A). Values are represented as mean ± SEM. MYCi (n = 6); Control (n = 7).\
(C) Representative images of EdU incorporation. Scale bar, 20 μm.\
(D) Confocal microscopy images of embryos immunostained for Oct4. Scale bar, 20 μm.\
(E and F) MYCi-treated and control blastocysts were transplanted into the uteri of pseudo-pregnant foster mothers.\
(E) Representative images of E15.5 embryos derived from MYCi-treated or control blastocysts. This result is representative of two independent experiments. MYCi (n = 24 embryos E15.5); control (n = 11 embryos E15.5). Scale bar, 0.5 cm.\
(F) Representative picture of the litter generated from the transplant of MYCi-treated blastocysts into pseudo-pregnant foster mothers. The result is representative of two independent experiments. In the MYCi condition, totally nine litters were born from nine embryo-transferred foster mothers (totally 64 live-born mice).](gr6){#fig6}

![Myc Is Required for Proliferation but Not for Maintenance of the Core Pluripotency Network in 2i ESCs, Related to [Figure 1](#fig1){ref-type="fig"}\
(A and B) *c-myc*^*fl/fl*^*; N-myc*^*fl/fl*^ ESC lines are capable of multilineage differentiation.\
(A) Fluorescence staining of in vitro differentiated ESCs for Tuj1, Sma and Gata6. Scale bar, 100 μm.\
(B) Haematoxylin and eosin staining of teratomas derived from *c-myc*^*fl/fl*^*; N-myc*^*fl/fl*^ ESCs. Representative images reveal structures derived from all the three germ layers. Scale bars, 50 μm (ectoderm and mesoderm) and 20 μm (endoderm).\
(C) Experimental workflow. *c-myc*^*fl/fl*^*; N-myc*^*fl/fl*^*Rosa26*^*lox-stop-loxEYFP*^ ESC were transduced with an EF1α-Cre plasmid and Cre positive (EYFP^+^) cells were FACS sorted and cultured in 2i, propagated as single clones and genotyped.\
(D) PCR analysis of representative ESC clones for the *c-myc*^*flox*^, *c-myc*^*Δ*^*, N-myc*^*flox*^ and *N-myc*^*Δ*^ alleles.\
(E) Number of clones obtained for each genotype.\
(F and G) Lack of either *c-myc* or *N-myc* does not affect ESCs multilineage differentiation potential.\
(F) Fluorescence staining for Tuj1, Sma and Gata6 of *c-myc*^*Δ/Δ*^*; N-myc*^*Δ//fl*^ and *c-myc*^*Δ/fl*^*; N-myc*^*ΔlΔ*^ ESCs differentiated in vitro. Scale bar, 100 μm.\
(G) Representative images of hematoxylin and eosin staining of teratomas from *myc*^*Δ/Δ*^*; N-myc*^*Δ//fl*^ and *c-myc*^*Δ/fl*^*; N-myc*^*ΔlΔ*^ ESCs. Scale bar, 50 μm.\
(H and I) *c-myc*^*Δ/Δ*^*; N-myc*^*Δ//fl*^ ESCs were transfected with an EF1α-mCherry-Cre plasmid and mCherry positive (Cre+) and negative (Cre-) cells were FACS sorted and cultured in 2i. Not transfected (NT) cells were used as controls.\
(H) PCR of the *c-myc*^*flox*^, *c-myc*^*Δ*^*, N-myc*^*flox*^ and *N-myc*^*Δ*^ alleles was performed 24h after transfection.\
(I) qRT-PCR analysis for *c-myc* and *N-myc* was performed 24h and 96h after transfection. Transcript levels of *c-myc* and *N-myc* were normalized to *Gapdh*. Data are presented as the mean ± SEM of duplicate wells from a representative experiment.\
(J) Lack of either *c-myc* or *N-myc* does not affect the proliferation of ESCs cultured in 2i. Cell cycle analysis of ESCs in the G~0~, G~1~ and S/G~2~/M phases as determined by Hoechst versus intracellular Ki67 staining. Bar graphs indicate the mean ± SD of two replicates, representative of two independent experiments.\
(K) Representative flow cytometry plots of ESCs stained for cleaved Caspase 3. ESCs incubated for 10 min at 42°C were used as positive control.\
(L) Representative images of *c-myc*^*Δ/fl*^*; N-myc*^*ΔlΔ*^ ESCs stained for Nanog or Oct4. Scale bar, 50 μm.\
(M) FACS analysis of Sox2 expression in *c-myc*^*fl/fl*^*; N-myc*^*fllfl*^*, c-myc*^*Δ/Δ*^*; N-myc*^*Δ//fl*^ and *dKO* ESCs.](figs1){#figs1}

![Loss of Myc Activity Induces Cellular Dormancy in ESCs Cultured in 2i, Related to [Figure 2](#fig2){ref-type="fig"}\
(A) RNA-seq workflow. Total RNA starting material was 10 ng. 100 bp paired--end libraries of two biological replicates for each condition were sequenced. We obtained 1x10^8^ sequenced fragments per sample and detected the expression of more than 28,000 genes.\
(B) Number of sequenced fragments of the genes *c-myc* and *N-myc* on each sample.\
(C) RNA-seq expression levels of *Rex1 (Zpf42)* in *dKO* ESCs compared to the controls. Bar graphs are represented as mean ± SD.\
(D) Gene expression clusters. For each of the genes detected to be differentially expressed, we estimated the relative fold change between each condition with respect to the mean expression across all the six conditions. The genes were grouped based on the signs of the relative fold change on each of the 64 (2^6^) possible combinations. For instance, all the genes that showed a positive sign for the *c-myc*^fl/fl^; *N-myc*^fl/fl^ and a negative sign for the rest of the conditions were grouped together.\
(E) Quantification of OP-Puro incorporation in c*-myc*^fl/fl^; *N-myc*^fl/fl^, *c-myc*^*Δ/Δ*^*; N-myc*^*Δ*lfl^ and *dKO* ESCs. Values represent geometrical mean (Geo Mean) ± SEM.](figs2){#figs2}

![The Dormant State Induced by Myc Depletion Is Reversible, Related to [Figure 3](#fig3){ref-type="fig"}\
(A) Representative FACS plots of OP-Puro incorporation in *c-myc*^*fl/fl*^*; N-myc*^*fl/fl*^ (black), *c-myc*^*Δ/Δ*^*; N-myc*^*Δ/fl*^ (blue), *dKO* (red) ESCs and *dKO* cells transfected with *c-myc* mRNAs (green). Experimental setting as described in [Figure 3](#fig3){ref-type="fig"}A.\
(B--E) Mouse ESCs were analyzed after 60h of treatment with DMSO (Control) or MYCi. The MYCi**→**Released group was treated with MYCi for 60h, followed by withdrawal of the inhibitor and culture in 2i medium for additional 48h. (B) Quantitative analysis of the different cell cycle phases as gated in [Figure 3](#fig3){ref-type="fig"}E. The bar charts indicate the mean ± SD (C) Quantification of OP-Puro incorporation in the indicated experimental groups. Values represent geometrical mean (Geo Mean) ± SD. (D) Representative fluorescence staining for Oct4. Scale bar, 50 μm. (E) Microarray analysis of mouse ESCs treated with MYCi. List of selected processes down- or upregulated in the MYCi-treated group compared to the control (FDR 0.05).](figs3){#figs3}

![Translation, Proliferation, and Myc Expression Are Reduced in Diapause Embryos, Related to [Figure 4](#fig4){ref-type="fig"}\
(A) c-Myc protein expression in E4.5 and diapause embryos. Quantification of confocal microscopy images as in [Figure 4](#fig4){ref-type="fig"}D.\
(B) Expression levels of *l-myc* in the epiblast of E4.5 and diapause embryos. RNA-seq data based on ([@bib7]). Bar graphs show FPKM values and are represented as mean ± SEM. E4.5 epiblast (n = 3); diapause epiblast (n = 2).\
(C) Fluorescence staining for Ki67 (red) and DAPI (blue) in E4.5 and diapause embryos. Scale bar, 20 μm.\
(D) Quantification of (C). Values are represented as mean ± SD.\
(E) EdU incorporation in E4.5 and Diapause embryos. Quantification of confocal microscopy images as in [Figure 4](#fig4){ref-type="fig"}F.\
(F) OP-Puro incorporation in E4.5 and Diapause embryos. Quantification of [Figure 4](#fig4){ref-type="fig"}G.](figs4){#figs4}

![Correlation of Expression Signatures between Myc Depleted ESCs and Diapause Epiblasts, Related to [Figure 5](#fig5){ref-type="fig"}\
(A) Selected processes commonly downregulated in diapause embryos and *dKO* ESCs compared to E4.5 epiblast and *c-myc*^*fl/fl*^*; N-myc*^*fl/fl*^ (WT) ESCs respectively (FDR 0.1).\
(B and C) Scatterplots of pathways significantly downregulated both in the comparison *dKO* / WT (FDR 0.1) and in Diapause / E4.5 (FDR 0.1).\
(D) Selected processes commonly upregulated in diapause embryos and *dKO* ESCs compared to E4.5 epiblast and WT ESCs respectively (FDR 0.1).\
(E) Scatterplots of pathways significantly upregulated both in the comparison *dKO* / WT (FDR 0.1) and in Diapause / E4.5 (FDR 0.1).](figs5){#figs5}