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• W4377020153 abstract "•Optogenetic control of ERK and AKT activity in mouse embryonic stem cells•Pluripotency exit is due to cumulative ERK activity, not duration, amplitude, or shape•ESCs retain memory of ERK pulses with memory duration dependent on pulse duration•Lower AKT activity increases pluripotency exit ERK and AKT signaling control pluripotent cell self-renewal versus differentiation. ERK pathway activity over time (i.e., dynamics) is heterogeneous between individual pluripotent cells, even in response to the same stimuli. To analyze potential functions of ERK and AKT dynamics in controlling mouse embryonic stem cell (ESC) fates, we developed ESC lines and experimental pipelines for the simultaneous long-term manipulation and quantification of ERK or AKT dynamics and cell fates. We show that ERK activity duration or amplitude or the type of ERK dynamics (e.g., transient, sustained, or oscillatory) alone does not influence exit from pluripotency, but the sum of activity over time does. Interestingly, cells retain memory of previous ERK pulses, with duration of memory retention dependent on duration of previous pulse length. FGF receptor/AKT dynamics counteract ERK-induced pluripotency exit. These findings improve our understanding of how cells integrate dynamics from multiple signaling pathways and translate them into cell fate cues. ERK and AKT signaling control pluripotent cell self-renewal versus differentiation. ERK pathway activity over time (i.e., dynamics) is heterogeneous between individual pluripotent cells, even in response to the same stimuli. To analyze potential functions of ERK and AKT dynamics in controlling mouse embryonic stem cell (ESC) fates, we developed ESC lines and experimental pipelines for the simultaneous long-term manipulation and quantification of ERK or AKT dynamics and cell fates. We show that ERK activity duration or amplitude or the type of ERK dynamics (e.g., transient, sustained, or oscillatory) alone does not influence exit from pluripotency, but the sum of activity over time does. Interestingly, cells retain memory of previous ERK pulses, with duration of memory retention dependent on duration of previous pulse length. FGF receptor/AKT dynamics counteract ERK-induced pluripotency exit. These findings improve our understanding of how cells integrate dynamics from multiple signaling pathways and translate them into cell fate cues. Different signaling pathways play a central role in controlling pluripotent cell fates. The fibroblast growth factor (FGF)-mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) signaling pathway controls differentiation and self-renewal of pluripotent stem cells. Mouse development requires FGF receptors (FGFRs) and ERK2.1Saba-El-Leil M.K. Vella F.D.J. Vernay B. Voisin L. Chen L. Labrecque N. Ang S.L. Meloche S. An essential function of the mitogen-activated protein kinase Erk2 in mouse trophoblast development.EMBO Rep. 2003; 4: 964-968https://doi.org/10.1038/sj.embor.embor939Crossref PubMed Scopus (308) Google Scholar,2Molotkov A. Mazot P. Brewer J.R. Cinalli R.M. Soriano P. Distinct requirements for FGFR1 and FGFR2 in primitive endoderm development and exit from pluripotency.Dev. Cell. 2017; 41: 511-526.e4https://doi.org/10.1016/j.devcel.2017.05.004Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar Embryonic stem cells (ESCs) are kept pluripotent by inhibiting ERK signaling.3Ying Q.L. Wray J. Nichols J. Batlle-Morera L. Doble B. Woodgett J. Cohen P. Smith A. The ground state of embryonic stem cell self-renewal.Nature. 2008; 453: 519-523https://doi.org/10.1038/nature06968Crossref PubMed Scopus (2574) Google Scholar AKT signaling is implicated in pluripotency control downstream of FGFRs, and overexpressing active AKT can keep cells pluripotent,4Watanabe S. Umehara H. Murayama K. Okabe M. Kimura T. Nakano T. Activation of Akt signaling is sufficient to maintain pluripotency in mouse and primate embryonic stem cells.Oncogene. 2006; 25: 2697-2707https://doi.org/10.1038/sj.onc.1209307Crossref PubMed Scopus (279) Google Scholar,5Brewer J.R. Molotkov A. Mazot P. Hoch R.V. Soriano P. FGFR1 regulates development through the combinatorial use of signaling proteins.Genes Dev. 2015; 29: 1863-1874https://doi.org/10.1101/gad.264994.115Crossref PubMed Scopus (0) Google Scholar but the role of its dynamics or interaction with ERK dynamics in controlling ESC fate is unknown. Recently, it was recognized that relevant information is encoded not only by simple on/off signaling activity but also by pathway activity over time (i.e., dynamics), which causes different molecular target programs and cell fate choices. Live visualization of signaling pathway activity over time revealed that individual cells can display heterogeneous dynamics, including sustained, transient, and oscillatory activity.6Purvis J.E. Karhohs K.W. Mock C. Batchelor E. Loewer A. Lahav G. P53 dynamics control cell fate.Science. 2012; 336: 1440-1444https://doi.org/10.1126/science.1218351Crossref PubMed Scopus (550) Google Scholar,7Lane K. Van Valen D. DeFelice M.M. Macklin D.N. Kudo T. Jaimovich A. Carr A. Meyer T. Pe’er D. Boutet S.C. et al.Measuring signaling and RNA-seq in the same cell links gene expression to dynamic patterns of NF-κB activation.Cell Syst. 2017; 4: 458-469.e5https://doi.org/10.1016/j.cels.2017.03.010Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar,8Wang W. Zhang Y. Dettinger P. Reimann A. Kull T. Loeffler D. Manz M.G. Lengerke C. Schroeder T. Cytokine combinations for human blood stem cell expansion induce cell-type- and cytokine-specific signaling dynamics.Blood. 2021; 138: 847-857https://doi.org/10.1182/BLOOD.2020008386Crossref PubMed Scopus (0) Google Scholar,9Nandagopal N. Santat L.A. LeBon L. Sprinzak D. Bronner M.E. Elowitz M.B. Dynamic ligand discrimination in the Notch signaling pathway.Cell. 2018; 172: 869-880.e19https://doi.org/10.1016/J.CELL.2018.01.002Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar,10Marshall C.J. Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation.Cell. 1995; 80: 179-185https://doi.org/10.1016/0092-8674(95)90401-8Abstract Full Text PDF PubMed Scopus (4249) Google Scholar In PC-12 cells, sustained versus transient ERK activity leads to differentiation versus proliferation.10Marshall C.J. Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation.Cell. 1995; 80: 179-185https://doi.org/10.1016/0092-8674(95)90401-8Abstract Full Text PDF PubMed Scopus (4249) Google Scholar ERK dynamics can dictate lineage choice between endoderm and ectoderm in Drosophila gastrulation.11Johnson H.E. Toettcher J.E. Signaling dynamics control cell fate in the early Drosophila embryo.Dev. Cell. 2019; 48: 361-370.e3https://doi.org/10.1016/j.devcel.2019.01.009Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar Different p53 signaling dynamics regulate cell survival by activating different gene sets.6Purvis J.E. Karhohs K.W. Mock C. Batchelor E. Loewer A. Lahav G. P53 dynamics control cell fate.Science. 2012; 336: 1440-1444https://doi.org/10.1126/science.1218351Crossref PubMed Scopus (550) Google Scholar In PC-12 cells, certain frequencies of ERK oscillations are better for differentiation.12Ryu H. Chung M. Dobrzyński M. Fey D. Blum Y. Lee S.S. Peter M. Kholodenko B.N. Jeon N.L. Pertz O. Frequency modulation of ERK activation dynamics rewires cell fate.Mol. Syst. Biol. 2015; 11: 838https://doi.org/10.15252/msb.20156458Crossref PubMed Scopus (124) Google Scholar ERK dynamics are also heterogeneous with variable ERK amplitudes, transient activation, and sometimes oscillations in individual mouse blastocyst cells13Simon C.S. Rahman S. Raina D. Schröter C. Hadjantonakis A.K. Live visualization of ERK activity in the mouse blastocyst reveals lineage-specific signaling dynamics.Dev. Cell. 2020; 55: 341-353.e5https://doi.org/10.1016/j.devcel.2020.09.030Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar and in differentiating14Deathridge J. Antolović V. Parsons M. Chubb J.R. Live imaging of erk signalling dynamics in differentiating mouse embryonic stem cells.Development. 2019; 146https://doi.org/10.1242/dev.172940Crossref PubMed Scopus (14) Google Scholar and pluripotent15Raina D. Fabris F. Morelli L.G. Schröter C. Intermittent ERK oscillations downstream of FGF in mouse embryonic stem cells.Development. 2022; 149https://doi.org/10.1242/dev.199710Crossref PubMed Scopus (3) Google Scholar ESCs. However, technical challenges of quantifying signaling over time in asynchronous cells lead to noisy data and make it difficult to parse how individual cells interpret these dynamics and their potential functional relevance for fate control. Here, we therefore use optogenetics to force specific ERK or AKT dynamics in mouse ESCs, while simultaneously quantifying single-cell signaling dynamics and future fate choices. We show a causal link between ERK dynamics and ESC exit from pluripotency, determine relevant ERK dynamics properties, and quantify the combinatorial effects of ERK and AKT signaling. To force different ERK dynamics in ESCs, we used the previously published optoFGFR1 optogenetic system.16Kim N. Kim J.M. Lee M. Kim C.Y. Chang K.Y. Do Heo W.D. Spatiotemporal control of fibroblast growth factor receptor signals by blue light.Chem. Biol. 2014; 21: 903-912https://doi.org/10.1016/j.chembiol.2014.05.013Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar Of the four FGFRs, FGF receptor 1 (FGFR1) has been identified as the most critical in early blastocyst and ESC differentiation.2Molotkov A. Mazot P. Brewer J.R. Cinalli R.M. Soriano P. Distinct requirements for FGFR1 and FGFR2 in primitive endoderm development and exit from pluripotency.Dev. Cell. 2017; 41: 511-526.e4https://doi.org/10.1016/j.devcel.2017.05.004Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar,17Kang M. Garg V. Hadjantonakis A.K. Lineage establishment and progression within the inner cell mass of the mouse blastocyst requires FGFR1 and FGFR2.Dev. Cell. 2017; 41: 496-510.e5https://doi.org/10.1016/j.devcel.2017.05.003Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar In the presence of extracellular ligands, FGFR1 dimerizes and activates downstream signaling pathways. In optoFGFR1, the extracellular portion is removed, and the intracellular portion is fused with the Cry2 blue light-responsive protein (Figure 1A). In the presence of blue light, Cry2 and thus FGFR1 reversibly dimerizes, triggering downstream signaling. In the absence of blue light, optoFGFR1 returns to the monomeric state (Figure 1A) with a reported dissociation time of 5 min.16Kim N. Kim J.M. Lee M. Kim C.Y. Chang K.Y. Do Heo W.D. Spatiotemporal control of fibroblast growth factor receptor signals by blue light.Chem. Biol. 2014; 21: 903-912https://doi.org/10.1016/j.chembiol.2014.05.013Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar Thus, optoFGFR1 mimics wild-type (WT) FGFR1 activation with better temporal control than growth factor stimulation.16Kim N. Kim J.M. Lee M. Kim C.Y. Chang K.Y. Do Heo W.D. Spatiotemporal control of fibroblast growth factor receptor signals by blue light.Chem. Biol. 2014; 21: 903-912https://doi.org/10.1016/j.chembiol.2014.05.013Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar Activation of several pathways including ERK and AKT downstream of FGFR1 has been reported.5Brewer J.R. Molotkov A. Mazot P. Hoch R.V. Soriano P. FGFR1 regulates development through the combinatorial use of signaling proteins.Genes Dev. 2015; 29: 1863-1874https://doi.org/10.1101/gad.264994.115Crossref PubMed Scopus (0) Google Scholar Due to fluorescence spectral limitations we could quantify only one of them per cell. In ESCs, ERK has been implicated in differentiation1Saba-El-Leil M.K. Vella F.D.J. Vernay B. Voisin L. Chen L. Labrecque N. Ang S.L. Meloche S. An essential function of the mitogen-activated protein kinase Erk2 in mouse trophoblast development.EMBO Rep. 2003; 4: 964-968https://doi.org/10.1038/sj.embor.embor939Crossref PubMed Scopus (308) Google Scholar,18Kunath T. Saba-El-Leil M.K. Almousailleakh M. Wray J. Meloche S. Smith A. FGF stimulation of the ERK1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment.Development. 2007; 134: 2895-2902https://doi.org/10.1242/dev.02880Crossref PubMed Scopus (602) Google Scholar while AKT has been implicated in pluripotency maintenance.4Watanabe S. Umehara H. Murayama K. Okabe M. Kimura T. Nakano T. Activation of Akt signaling is sufficient to maintain pluripotency in mouse and primate embryonic stem cells.Oncogene. 2006; 25: 2697-2707https://doi.org/10.1038/sj.onc.1209307Crossref PubMed Scopus (279) Google Scholar Inhibiting ERK is a key feature of defined media arresting ESCs in pluripotency.3Ying Q.L. Wray J. Nichols J. Batlle-Morera L. Doble B. Woodgett J. Cohen P. Smith A. The ground state of embryonic stem cell self-renewal.Nature. 2008; 453: 519-523https://doi.org/10.1038/nature06968Crossref PubMed Scopus (2574) Google Scholar AKT is involved in essential ESC functions such as proliferation and apoptosis19Yu J.S.L. Cui W. Proliferation, survival and metabolism: the role of PI3K/AKT/ mTOR signalling in pluripotency and cell fate determination.Development. 2016; 143: 3050-3060https://doi.org/10.1242/dev.137075Crossref PubMed Scopus (642) Google Scholar,20Wang L. Huang D. Jiang Z. Luo Y. Norris C. Zhang M. Tian X. Tang Y. Akt3 is responsible for the survival and proliferation of embryonic stem cells.Biol. Open. 2017; 6: 850-861https://doi.org/10.1242/bio.024505Crossref PubMed Scopus (24) Google Scholar and active in pluripotency promoting media.3Ying Q.L. Wray J. Nichols J. Batlle-Morera L. Doble B. Woodgett J. Cohen P. Smith A. The ground state of embryonic stem cell self-renewal.Nature. 2008; 453: 519-523https://doi.org/10.1038/nature06968Crossref PubMed Scopus (2574) Google Scholar Thus, based on prior literature, we considered the ERK pathway activity to be the main catalyst to ESC exit from pluripotency with pathways such as AKT playing a potentially synergistic or antagonistic role only in combination with ERK. Therefore, we focused initially on optoFGFR1-induced ERK dynamics and their effect on ESC fate. To simultaneously quantify ERK dynamics in response to optoFGFR1 activation in individual cells, we used the fluorescent ERKKTR biosensor (Figure 1A).21Regot S. Hughey J.J. Bajar B.T. Carrasco S. Covert M.W. High-sensitivity measurements of multiple kinase activities in live single cells.Cell. 2014; 157: 1724-1734https://doi.org/10.1016/j.cell.2014.04.039Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar When ERK is inactive, ERKKTR is located in the nucleus, and upon ERK activation, ERKKTR translocates to the cytoplasm (Figure 1A).13Simon C.S. Rahman S. Raina D. Schröter C. Hadjantonakis A.K. Live visualization of ERK activity in the mouse blastocyst reveals lineage-specific signaling dynamics.Dev. Cell. 2020; 55: 341-353.e5https://doi.org/10.1016/j.devcel.2020.09.030Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar,21Regot S. Hughey J.J. Bajar B.T. Carrasco S. Covert M.W. High-sensitivity measurements of multiple kinase activities in live single cells.Cell. 2014; 157: 1724-1734https://doi.org/10.1016/j.cell.2014.04.039Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar To generate the required optoFGFR1/ERKKTR ESC line, we transfected ESCs with one plasmid comprising ERKKTRmRuby28Wang W. Zhang Y. Dettinger P. Reimann A. Kull T. Loeffler D. Manz M.G. Lengerke C. Schroeder T. Cytokine combinations for human blood stem cell expansion induce cell-type- and cytokine-specific signaling dynamics.Blood. 2021; 138: 847-857https://doi.org/10.1182/BLOOD.2020008386Crossref PubMed Scopus (0) Google Scholar and iRFPnucmem joined by P2A linker under the constitutive CAG promoter (Figure 1B). iRFPnucmem constitutively labels the nucleus for computational segmentation and tracking.22Filipczyk A. Marr C. Hastreiter S. Feigelman J. Schwarzfischer M. Hoppe P.S. Loeffler D. Kokkaliaris K.D. Endele M. Schauberger B. et al.Network plasticity of pluripotency transcription factors in embryonic stem cells.Nat. Cell Biol. 2015; 17: 1235-1246https://doi.org/10.1038/ncb3237Crossref PubMed Scopus (94) Google Scholar Stably transfected ERKKTR cells were isolated by fluorescence-activated cell sorting (FACS) and stably transfected with one plasmid comprising optoFGFR1mCitrine16Kim N. Kim J.M. Lee M. Kim C.Y. Chang K.Y. Do Heo W.D. Spatiotemporal control of fibroblast growth factor receptor signals by blue light.Chem. Biol. 2014; 21: 903-912https://doi.org/10.1016/j.chembiol.2014.05.013Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar and a truncated form of CD4 under the CAG promoter (Figure 1B). To avoid light exposure to optoFGFR1 transfected cells, they were MACS sorted for CD4 in the dark to obtain optoFGFR1/ERKKTR ESCs (Figure 1B). Of these cells, 98% co-expressed ERKKTR and optoFGFR1 (Figures S1A and S1B), with optoFGFR1 localized to plasma membranes as expected (Figure S1C). They also expressed the pluripotency markers NANOG and KLF4 (Figure S2A)23Guo G. Yang J. Nichols J. Hall J.S. Eyres I. Mansfield W. Smith A. Klf4 reverts developmentally programmed restriction of ground state pluripotency.Development. 2009; 136: 1063-1069https://doi.org/10.1242/dev.030957Crossref PubMed Scopus (604) Google Scholar,24Pan G. Thomson J.A. Nanog and transcriptional networks in embryonic stem cell pluripotency.Cell Res. 2007; 17: 42-49https://doi.org/10.1038/sj.cr.7310125Crossref PubMed Scopus (441) Google Scholar and formed colonies with typical ESC morphology (Figure S2B). Their doubling time of ∼15 h was not significantly different from WT cells (∼16 h) (Figure S2C) and is in agreement with previously published results.25Tamm C. Pijuan Galitó S. Annerén C. A comparative study of protocols for mouse embryonic stem cell culturing.PLoS One. 2013; 8e81156https://doi.org/10.1371/journal.pone.0081156Crossref Scopus (63) Google Scholar They could differentiate into all three germ layers, as detected by Sox1 (neuroectoderm marker) or Sox17 (endoderm marker) expression (Figures S2D and S2E), or development of contracting cardiomyocytes (originating from mesoderm progenitors) after undirected differentiation into 3D embryoid body aggregates. Taken together, this validated the used optoFGFR1/ERKKTR line as an ESC line. We then subjected cells to light pulses to quantify how ERK responds to FGFR1 activation in different ESC media. Cells were switched from “SerumLIF” pluripotency medium (used for routine maintenance) to either chemically defined pluripotency media 2iLIF (“2iL”)3Ying Q.L. Wray J. Nichols J. Batlle-Morera L. Doble B. Woodgett J. Cohen P. Smith A. The ground state of embryonic stem cell self-renewal.Nature. 2008; 453: 519-523https://doi.org/10.1038/nature06968Crossref PubMed Scopus (2574) Google Scholar or aiLIF (“aiL”)26Shimizu T. Ueda J. Ho J.C. Iwasaki K. Poellinger L. Harada I. Sawada Y. Dual inhibition of Src and GSK3 maintains mouse embryonic stem cells, whose differentiation is mechanically regulated by Src signaling.Stem Cells. 2012; 30: 1394-1404https://doi.org/10.1002/stem.1119Crossref PubMed Scopus (43) Google Scholar prior to light stimulation. The basal medium for both aiL and 2iL is N2B27. In 2iL, ERK activity is abrogated by the PD0325901 inhibitor directly inhibiting Mek and thus preventing ERK activation by FGFR1 and by optoFGFR1 (Figures S3A–S3C). In aiL, the Src kinase inhibitor CGP77675 (Figures S3B and S3C) abrogates ESC exit from pluripotency, which is usually induced by baseline ERK activity due to autocrine FGF signaling.27Lanner F. Rossant J. The role of FGF/Erk signaling in pluripotent cells.Development. 2010; 137: 3351-3360https://doi.org/10.1242/dev.050146Crossref PubMed Scopus (294) Google Scholar However, it still allows ERK activation by (opto)FGFR1 (Figure 1C). Cells in aiL are transcriptionally similar to cells in 2iL.28Kolodziejczyk A.A. Kim J.K. Tsang J.C.H. Ilicic T. Henriksson J. Natarajan K.N. Tuck A.C. Gao X. Bühler M. Liu P. et al.Single cell RNA-sequencing of pluripotent states unlocks modular transcriptional variation.Cell Stem Cell. 2015; 17: 471-485https://doi.org/10.1016/j.stem.2015.09.011Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar However, while cells in 2iL exhibit loss of DNA methylation and impaired development, cells in aiL maintain epigenetic markers and show no development impairment.29Yagi M. Yamanaka S. Yamada Y. Epigenetic foundations of pluripotent stem cells that recapitulate in vivo pluripotency.Lab. Invest. 2017; 97: 1133-1141https://doi.org/10.1038/labinvest.2017.87Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar,30Choi J. Huebner A.J. Clement K. Walsh R.M. Savol A. Lin K. Gu H. Di Stefano B. Brumbaugh J. Kim S.Y. et al.Prolonged Mek1/2 suppression impairs the developmental potential of embryonic stem cells.Nature. 2017; 548: 219-223https://doi.org/10.1038/nature23274Crossref PubMed Scopus (148) Google Scholar aiL is thus well suited for our analysis of the role of ERK dynamics in ESC fate control. Illuminating cells with light in aiL induced fast ERK activation, peaking at 6 min post light pulse and returning to baseline by 12 min, indicating a lack of positive feedback (Figure 1C). This activation was repeatable, with cells responding to a second dose of light at 12 min, again with peak activation by additional 6 min (Figure 1C). We confirmed that ERKKTR is an accurate readout for ERK activity by staining for phosphorylated ERK (pERK). Without light, ERK activity is off (or very low), as indicated by nuclear ERKKTR localization and the absence of pERK (Figure 1D). Light stimulation activates ERK, as indicated by pERK presence and ERKKTR translocating to cytoplasm (Figure 1D). As expected, we observed no sensor translocation upon light stimulation in 2iL where ERK is inhibited via Meki3Ying Q.L. Wray J. Nichols J. Batlle-Morera L. Doble B. Woodgett J. Cohen P. Smith A. The ground state of embryonic stem cell self-renewal.Nature. 2008; 453: 519-523https://doi.org/10.1038/nature06968Crossref PubMed Scopus (2574) Google Scholar (Figure S3A). Importantly, even long-term light stimulation to induce sustained ERK activation (250 mson750 msoff) for 24 h was not toxic to the cells (Figure S3D). Having validated the ERK activation and quantification system, we tested the degree of control over ERK by optogenetic manipulation (Figures 1E and 1F). Different light patterns induced sustained or oscillatory ERK dynamics, highly synchronized between individual cells (Figures 1E and 1F; Videos S1 and S2). The oscillation frequency could be tuned from as fast as ∼10 min to 1 h by changing the frequency of light pulses (Figure 1E). For example, continuous ERK oscillations with a ∼10-min period and without an extended ERK OFF period could be induced by 2 minon10 minoff light stimulation. Changing the light intensity changed the amplitude of ERK activation wi1thout changing the dynamics pattern (Figure 1F). Thus, the optoFGFR1/ERKKTR ESC line enables a high degree of control over induced ERK dynamics. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJiMDJmNTI0MGM5YTk3ZmQxNzc2ODY4OGZkYjY3M2MwMyIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjg4MzQ2NjA5fQ.At26Umygqe8olRMmrZW9LDcblRaZskzlKfwWopZd6fbtP3lqtj471-2_gOJFCqAPbRrNC55CTMZGVesoAcC3suBeNyT4YM7n2kijbWBmpERUoXRV-R34owPXHHNF1kFHWTwxJmEt69vgx849UsFQsxShsOy_Cp0xq_z87S_63HSBFAZ6_d6xy0xnKVHjHW23NN4Ff04i2eGa5y13FNDYnNpzcIj53mglBuriiUcosiBdQ0_QWQOhBUAbuW6f2Sek1f7ThKQ31bL2jDD0j1rxkuTzjFplbkmDD1EBLea1VUwUTDd5DURbUbbNGLWZzTBmxtYJC1_fyoDh1bLQ2lCSRA Download .mp4 (1.25 MB) Help with .mp4 files Video S1. Constant light stimulation leads to sustained ERK activation in a synchronous manner, related to Figure 1Time-lapse microscopy of R1 optoFGFR1/ERKKTR ESCs subjected to sustained light at high intensity. ERK activation indicated by ERKKTR translocation from nucleus to cytoplasm. Movie duration 140 min, frame interval 2 min. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI4ODExZmRkYTA1N2JiZDJjZWZhNjE5Mzk0YzIxMWY3ZSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjg4MzQ2NjA5fQ.ZS9F6pqrvzgGZzmCJ0IGYWFRRmAk60jpoMKUbotfYetOKalnAUVRRRt9XoxK4oMKGExAEwTjamZ5Jr1UblfXMvW1wwl2lHs_t-VIOlKSGMhuvhKV-ddYLEM2Ba_mmhx743iafVOt-eG968f89o5vYYJ468WnrE1GeK5M74UOhF3tuy2UQWjnwMearD6wK7ZblNXcQibvYy1qcpxmmnnOvUcPIvMWv_ERF1sYfzsZtqLhW8SRCk6nhxza9CXyyj0sEA0ib3wFQQDON-OA9qQWw4waJHxRD5wcrGmCRw86G5kZaCnn0G7qSwXfV9DwsKH8PaBtOtRH9b6VzjiqPefesQ Download .mp4 (1.76 MB) Help with .mp4 files Video S2. Pulsed light stimulation leads to oscillatory ERK activation in a synchronous manner, related to Figure 1Time-lapse microscopy of R1 optoFGFR1/ERKKTR cells subjected to light pulses every 10 min at high intensity. ERK activation indicated by ERKKTR translocation from nucleus to cytoplasm. Movie duration 200 min, frame interval 2 min. We quantified if ERK activation dynamics affect ESC self-renewal. We cultured ESCs in aiL, induced different ERK dynamics for 24 h, and then quantified pluripotency marker expression (Figure 2A). Sustained ERK activation at highest light intensity for 24 h downregulated pluripotency markers NANOG and KLF4 both in aiL and N2B27 media (Figure 2B) and upregulated OTX2 (Figures S3F and S3G), a transcription factor involved in transition out of naive pluripotency.31Kalkan T. Olova N. Roode M. Mulas C. Lee H.J. Nett I. Marks H. Walker R. Stunnenberg H.G. Lilley K.S. et al.Tracking the embryonic stem cell transition from ground state pluripotency.Development. 2017; 144: 1221-1234https://doi.org/10.1242/dev.142711Crossref PubMed Scopus (160) Google Scholar,32Mulas C. Kalkan T. von Meyenn F. Leitch H.G. Nichols J. Smith A. Defined conditions for propagation and manipulation of mouse embryonic stem cells.Development. 2019; 146dev173146https://doi.org/10.1242/dev.173146Crossref Scopus (45) Google Scholar However, given the narrow dynamic range of OTX2, we focused on NANOG and KLF4 to quantify ESCs exiting pluripotency. NANOG and KLF4 are not only markers for pluripotency but also core members of the pluripotency network. NANOG expression has been used as a measure of pluripotency exit.14Deathridge J. Antolović V. Parsons M. Chubb J.R. Live imaging of erk signalling dynamics in differentiating mouse embryonic stem cells.Development. 2019; 146https://doi.org/10.1242/dev.172940Crossref PubMed Scopus (14) Google Scholar,33Jin K.X. Zuo R. Anastassiadis K. Klungland A. Marr C. Filipczyk A. N6-methyladenosine (m6A) depletion regulates pluripotency exit by activating signaling pathways in embryonic stem cells.Proc. Natl. Acad. Sci. USA. 2021; 118e2105192118https://doi.org/10.1073/pnas.2105192118Crossref Scopus (7) Google Scholar Decrease of nuclear Klf4 has been shown to be the first step that initiates exit from naive pluripotency.34Dhaliwal N.K. Miri K. Davidson S. Tamim El Jarkass H. Mitchell J.A. KLF4 nuclear export requires ERK activation and initiates exit from naive pluripotency.Stem Cell Rep. 2018; 10: 1308-1323https://doi.org/10.1016/j.stemcr.2018.02.007Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar To be conservative, we considered cells to be exiting pluripotency only when both NANOG and KLF4 expression were below their thresholds. Typically, the intersection point of a bimodal distribution33Jin K.X. Zuo R. Anastassiadis K. Klungland A. Marr C. Filipczyk A. N6-methyladenosine (m6A) depletion regulates pluripotency exit by activating signaling pathways in embryonic stem cells.Proc. Natl. Acad. Sci. USA. 2021; 118e2105192118https://doi.org/10.1073/pnas.2105192118Crossref Scopus (7) Google Scholar,35Gunne-Braden A. Sullivan A. Gharibi B. Sheriff R.S.M. Maity A. Wang Y.F. Edwards A. Jiang M. Howell M. Goldstone R. et al.GATA3 mediates a fast, irreversible commitment to BMP4-driven differentiation in human embryonic stem cells.Cell Stem Cell. 2020; 26: 693-706.e9https://doi.org/10.1016/j.stem.2020.03.005Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar,36Hormoz S. Singer Z.S. Linton J.M. Antebi Y.E. Shraiman B.I. Elowitz M.B. Inferring.Cell Syst. 2016; 3: 419-433.e8https://doi.org/10.1016/j.cels.2016.10.015Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar or the tailing ends of a unimodal distribution are used as the threshold.37Chambers I. Silva J. Colby D. Nichols J. Nijmeijer B. Robertson M. Vrana J. Jones K. Grotewold L. Smith A. Nanog safeguards pluripotency and mediates germline development.Nature. 2007; 450: 1230-1234https://doi.org/10.1038/nature06403Crossref PubMed Scopus (1170) Google Scholar,38Abranches E. Guedes A.M.V. Moravec M. Maamar H. Svoboda P. Raj A. Henrique D. Stochastic NANOG fluctuations allow mouse embryonic stem cells to explore pluripotency.Development. 2014; 141: 2770-2779https://doi.org/10.1242/dev.108910Crossref PubMed Scopus (98) Google Scholar The intersection points between our two distributions furthest apart in Figure 2C, “aiL + no light” and “N2B27 + sustained light,” could thus be thresholds, but we decided to be more conservative. We defined NANOG and KLF4 expression thresholds from aiL + no light control self-renewal conditions, where their expression is at its highest, as expected (Figure 2C). Observing the spread of the distributions, we set thresholds to the bottom" @default.
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• W4377020153 title "Optogenetic manipulation identifies the roles of ERK and AKT dynamics in controlling mouse embryonic stem cell exit from pluripotency" @default.
• W4377020153 cites W1966697925 @default.
• W4377020153 cites W1966758700 @default.
• W4377020153 cites W1966868529 @default.
• W4377020153 cites W1970065245 @default.
• W4377020153 cites W1978018545 @default.
• W4377020153 cites W1990180850 @default.
• W4377020153 cites W1995559307 @default.
• W4377020153 cites W1995819749 @default.
• W4377020153 cites W1998203489 @default.
• W4377020153 cites W2000246164 @default.
• W4377020153 cites W2001038614 @default.
• W4377020153 cites W2002464225 @default.
• W4377020153 cites W2002973634 @default.
• W4377020153 cites W2004046942 @default.
• W4377020153 cites W2005941786 @default.
• W4377020153 cites W2011135937 @default.
• W4377020153 cites W2015852145 @default.
• W4377020153 cites W2018765068 @default.
• W4377020153 cites W2048897339 @default.
• W4377020153 cites W2049345986 @default.
• W4377020153 cites W2058521801 @default.
• W4377020153 cites W2060123496 @default.
• W4377020153 cites W2066218351 @default.
• W4377020153 cites W2074591015 @default.
• W4377020153 cites W2075682647 @default.
• W4377020153 cites W2075805565 @default.
• W4377020153 cites W2101326169 @default.
• W4377020153 cites W2105241566 @default.
• W4377020153 cites W2106008571 @default.
• W4377020153 cites W2111882541 @default.
• W4377020153 cites W2117623697 @default.
• W4377020153 cites W2123335871 @default.
• W4377020153 cites W2129223507 @default.
• W4377020153 cites W2130044807 @default.
• W4377020153 cites W2133351686 @default.
• W4377020153 cites W2139194392 @default.
• W4377020153 cites W2158771952 @default.
• W4377020153 cites W2161300409 @default.
• W4377020153 cites W2168979150 @default.
• W4377020153 cites W2176424950 @default.
• W4377020153 cites W2192104980 @default.
• W4377020153 cites W2193067168 @default.
• W4377020153 cites W2465367340 @default.
• W4377020153 cites W2468991623 @default.
• W4377020153 cites W2510435007 @default.
• W4377020153 cites W2550701680 @default.
• W4377020153 cites W2580093245 @default.
• W4377020153 cites W2592920807 @default.
• W4377020153 cites W2604169508 @default.
• W4377020153 cites W2618900678 @default.
• W4377020153 cites W2619140115 @default.
• W4377020153 cites W2622865236 @default.
• W4377020153 cites W2625834976 @default.
• W4377020153 cites W2738015524 @default.
• W4377020153 cites W2753012972 @default.
• W4377020153 cites W2786741514 @default.
• W4377020153 cites W2793967558 @default.
• W4377020153 cites W2810807384 @default.
• W4377020153 cites W2950402574 @default.
• W4377020153 cites W2950434985 @default.
• W4377020153 cites W2951962060 @default.
• W4377020153 cites W2987479280 @default.
• W4377020153 cites W3016331313 @default.
• W4377020153 cites W3094240904 @default.
• W4377020153 cites W3162242653 @default.
• W4377020153 cites W3169281636 @default.
• W4377020153 cites W4200405938 @default.
• W4377020153 cites W4224317049 @default.
• W4377020153 cites W4226160114 @default.
• W4377020153 cites W4281742107 @default.
• W4377020153 cites W4282549436 @default.
• W4377020153 cites W4285091897 @default.
• W4377020153 cites W4291991127 @default.
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