Tissue damage induces a conserved stress response that initiates quiescent muscle stem cell activation

•Capturing in vivo the early cellular responses to tissue injury•Identification of a common stress response signature following tissue damage•Transcriptional changes by cell dissociation impair the accuracy of single-cell atlases•Cooperating MAPK and Notch signaling control quiescence exit of MuSCs Tissue damage dramatically alters how cells interact with their microenvironment. These changes in turn dictate cellular responses, such as stem cell activation, yet early cellular responses in vivo remain ill defined. We generated single-cell and nucleus atlases from intact, dissociated, and injured muscle and liver and identified a common stress response signature shared by multiple cell types across these organs. This prevalent stress response was detected in published datasets across a range of tissues, demonstrating high conservation but also a significant degree of data distortion in single-cell reference atlases. Using quiescent muscle stem cells as a paradigm of cell activation following injury, we captured early cell activation following muscle injury and found that an essential ERK1/2 primary proliferation signal precedes initiation of the Notch-regulated myogenic program. This study defines initial events in response to tissue perturbation and identifies a broadly conserved transcriptional stress response that acts in parallel with cell-specific adaptive alterations. Tissue damage dramatically alters how cells interact with their microenvironment. These changes in turn dictate cellular responses, such as stem cell activation, yet early cellular responses in vivo remain ill defined. We generated single-cell and nucleus atlases from intact, dissociated, and injured muscle and liver and identified a common stress response signature shared by multiple cell types across these organs. This prevalent stress response was detected in published datasets across a range of tissues, demonstrating high conservation but also a significant degree of data distortion in single-cell reference atlases. Using quiescent muscle stem cells as a paradigm of cell activation following injury, we captured early cell activation following muscle injury and found that an essential ERK1/2 primary proliferation signal precedes initiation of the Notch-regulated myogenic program. This study defines initial events in response to tissue perturbation and identifies a broadly conserved transcriptional stress response that acts in parallel with cell-specific adaptive alterations. Cell plasticity is a fundamental property in response to injury in a variety of vertebrate organs. Tissue damage triggers activation and proliferation of stem/progenitor cells in order to restore homeostasis, as seen in the bone, skin, and skeletal muscle, among others (Fuchs and Blau, 2020Fuchs E. Blau H.M. Tissue stem cells: architects of their niches.Cell Stem Cell. 2020; 27: 532-556Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar; Jessen et al., 2015Jessen K.R. Mirsky R. Arthur-Farraj P. The role of cell plasticity in tissue repair: adaptive cellular reprogramming.Dev. Cell. 2015; 34: 613-620Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Differentiated cells too can get activated and eventually proliferate following de-differentiation, as is the case for hepatocytes after hepatectomy (Riehle et al., 2011Riehle K.J. Dan Y.Y. Campbell J.S. Fausto N. New concepts in liver regeneration.J. Gastroenterol. Hepatol. 2011; 26: 203-212Crossref PubMed Scopus (176) Google Scholar) or neonatal mouse cardiomyocytes (Porrello et al., 2011Porrello E.R. Mahmoud A.I. Simpson E. Hill J.A. Richardson J.A. Olson E.N. Sadek H.A. Transient regenerative potential of the neonatal mouse heart.Science. 2011; 331: 1078-1080Crossref PubMed Scopus (1536) Google Scholar). Cell death pathways, however, can also be instigated when cell injury is irreversible (Rathore et al., 2017Rathore R. McCallum J.E. Varghese E. Florea A.M. Büsselberg D. Overcoming chemotherapy drug resistance by targeting inhibitors of apoptosis proteins (IAPs).Apoptosis. 2017; 22: 898-919Crossref PubMed Scopus (149) Google Scholar). Uncovering the early transcriptional roadmap of the cells as they exit homeostasis is critical for understanding their properties and developing tools for experimental manipulation. Despite extensive research, however, the degree, kinetics, and nature of the early, cell type-specific or conserved responses to tissue damage as they occur in vivo remain poorly defined. A major limitation in capturing the early responses in vivo is that the process of cell isolation induces major transcriptional modifications that mask the actual injury-induced changes. Early studies that uncovered the global transcriptional changes in standard cell dissociation methods were performed on quiescent skeletal muscle stem cells (MuSCs) (van den Brink et al., 2017van den Brink S.C. Sage F. Vértesy Á. Spanjaard B. Peterson-Maduro J. Baron C.S. Robin C. van Oudenaarden A. Single-cell sequencing reveals dissociation-induced gene expression in tissue subpopulations.Nat. Methods. 2017; 14: 935-936Crossref PubMed Scopus (350) Google Scholar; Machado et al., 2017Machado L. Esteves de Lima J. Fabre O. Proux C. Legendre R. Szegedi A. Varet H. Ingerslev L.R. Barrès R. Relaix F. et al.In situ fixation redefines quiescence and early activation of skeletal muscle stem cells.Cell Rep. 2017; 21: 1982-1993Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar; van Velthoven et al., 2017van Velthoven C.T.J. de Morree A. Egner I.M. Brett J.O. Rando T.A. Transcriptional profiling of quiescent muscle stem cells in vivo.Cell Rep. 2017; 21: 1994-2004Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Yet it was perceived that these changes are a particular feature of quiescent stem cells, and MuSCs in particular, as they have evolved to become activated following disruptions in their niche homeostasis. Several studies, however, have reported transcriptional changes in diverse cellular systems during ex vivo cell isolation, challenging the notion that this is unique to MuSCs (Adam et al., 2017Adam M. Potter A.S. Potter S.S. Psychrophilic proteases dramatically reduce single cell RNA-seq artifacts: a molecular atlas of kidney development.Development. 2017; 144: 3625-3632Crossref PubMed Scopus (138) Google Scholar; Bakken et al., 2018Bakken T.E. Hodge R.D. Miller J.A. Yao Z. Nguyen T.N. Aevermann B. Barkan E. Bertagnolli D. Casper T. Dee N. et al.Single-nucleus and single-cell transcriptomes compared in matched cortical cell types.PLoS ONE. 2018; 13: e0209648Crossref PubMed Scopus (146) Google Scholar; Lacar et al., 2016Lacar B. Linker S.B. Jaeger B.N. Krishnaswami S.R. Barron J.J. Kelder M.J.E. Parylak S.L. Paquola A.C.M. Venepally P. Novotny M. et al.Nuclear RNA-seq of single neurons reveals molecular signatures of activation.Nat. Commun. 2016; 7: 11022Crossref PubMed Scopus (194) Google Scholar; O’Flanagan et al., 2019O’Flanagan C.H. Campbell K.R. Zhang A.W. Kabeer F. Lim J.L.P. Biele J. Eirew P. Lai D. McPherson A. Kong E. et al.CRUK IMAXT Grand Challenge TeamDissociation of solid tumor tissues with cold active protease for single-cell RNA-seq minimizes conserved collagenase-associated stress responses.Genome Biol. 2019; 20: 210Crossref PubMed Scopus (63) Google Scholar; Wu et al., 2017Wu Y.E. Pan L. Zuo Y. Li X. Hong W. Detecting activated cell populations using single-cell RNA-seq.Neuron. 2017; 96: 313-329.e6Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, Wu et al., 2019Wu H. Kirita Y. Donnelly E.L. Humphreys B.D. Advantages of single-nucleus over single-cell RNA sequencing of adult kidney: rare cell types and novel cell states revealed in fibrosis.J. Am. Soc. Nephrol. 30. 2019; : 23-32Crossref Scopus (180) Google Scholar). Considering the ever growing importance of transcriptional studies and reference atlases, understanding whether the changes are technical artifacts linked to cell isolation and RNA recovery methods or if they are caused by loss of niche signals is critical. Here, we have developed specialized protocols to identify the kinetics and nature of the stress response to tissue damage in diverse tissues and cell types. We identify a broadly conserved stress response across organs and cell types and uncover a functional role of this response, using MuSCs as a model of quiescent stem cell activation. In order to test the impact of cell dissociation across cell types and tissues, we performed single-nucleus RNA sequencing (snRNA-seq) on intact or dissociated murine skeletal muscle and liver using a droplet-based platform (Figure 1A) and identified 26,074 single-nucleus transcriptomes (Figures S1A–S1D), distributed in ten main populations in the muscle and seven in the liver (Figures 1B, S1E, and S1F). When all nuclei of a tissue were merged, we noticed that cells tended to cluster by type, yet there was a clear separation between intact and dissociated cells, indicating strong transcriptional differences (Figure 1C). Remarkably, nearly all dissociated cells showed high induction of Jun, a prototypical stress-response gene (Figure S1G) (Bohmann et al., 1987Bohmann D. Bos T.J. Admon A. Nishimura T. Vogt P.K. Tjian R. Human proto-oncogene c-jun encodes a DNA binding protein with structural and functional properties of transcription factor AP-1.Science. 1987; 238: 1386-1392Crossref PubMed Scopus (945) Google Scholar). Differential gene expression analysis indicated variable yet strong modifications in all cell types during dissociation, with an average of 1,226 differentially expressed genes (DEGs) in the ten main cell types studied (Figure S1H). Notably, for MuSCs, the detected DEGs were highly similar to previous dissociation-induced differences found by bulk RNA-seq (Machado et al., 2017Machado L. Esteves de Lima J. Fabre O. Proux C. Legendre R. Szegedi A. Varet H. Ingerslev L.R. Barrès R. Relaix F. et al.In situ fixation redefines quiescence and early activation of skeletal muscle stem cells.Cell Rep. 2017; 21: 1982-1993Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), validating the single-nucleus approach (Figures S1I and S1J). Analysis of the upregulated genes showed enrichment for cell-specific biological processes, as described in Figures S1K–S1N (Ishikawa et al., 2012Ishikawa T. Factor V.M. Marquardt J.U. Raggi C. Seo D. Kitade M. Conner E.A. Thorgeirsson S.S. Hepatocyte growth factor/c-met signaling is required for stem cell-mediated liver regeneration in mice.Hepatology. 2012; 55: 1215-1226Crossref PubMed Scopus (137) Google Scholar; van Meeteren and ten Dijke, 2012van Meeteren L.A. ten Dijke P. Regulation of endothelial cell plasticity by TGF-β.Cell Tissue Res. 2012; 347: 177-186Crossref PubMed Scopus (226) Google Scholar; Contreras et al., 2019Contreras O. Cruz-Soca M. Theret M. Soliman H. Tung L.W. Groppa E. Rossi F.M. Brandan E. Cross-talk between TGF-β and PDGFRα signaling pathways regulates the fate of stromal fibro-adipogenic progenitors.J. Cell Sci. 2019; 132: jcs232157Crossref PubMed Scopus (33) Google Scholar). We then examined the extent to which the dissociation-induced modifications were shared among cell types. Approximately half of the upregulated genes were cell type specific, while the rest were induced in at least two populations (Figure 1D). A similar pattern was observed for the downregulated genes (Figure S2A). Importantly, we uncovered a conserved transcriptional core composed of 98 genes that was induced in at least seven of the ten main cell types (Figures 1E and S2B; Table S1). The average expression of this gene set was designated as the stress index and was validated in our snRNA-seq dataset (Figure S2C). Gene Ontology analysis of the stress core was enriched for terms linked to MAPK signaling, response to growth factors and cytokines, and initiation of transcription (Figure 1F). Consistently, phospho-ERK1/2 showed rapid induction of MAPK activation at 15 min of muscle digestion (Figure S2D). On the basis of the broad modifications we observed in muscle and liver cells during dissociation, we extended our analysis to a multi-organ murine single-cell atlas (Tabula Muris; Schaum et al., 2018Schaum N. Karkanias J. Neff N.F. May A.P. Quake S.R. Wyss-Coray T. Darmanis S. Batson J. Botvinnik O. Chen M.B. et al.Tabula Muris ConsortiumOverall coordinationLogistical coordinationOrgan collection and processingLibrary preparation and sequencingComputational data analysisCell type annotationWriting groupSupplemental text writing groupPrincipal investigatorsSingle-cell transcriptomics of 20 mouse organs creates a Tabula Muris.Nature. 2018; 562: 367-372Crossref PubMed Scopus (787) Google Scholar), (Figures 1G and S2E). Stress index was high for most cells of the muscle but only a fraction of the liver atlas (Figure S2F), in contrast to our data on dissociated liver (Figure S2C). We postulated that these variations were linked to the time of the cell dissociation procedure. To corroborate this hypothesis, we inferred the digestion time for every cell from the Tabula Muris dataset (Table S2) and found a strong correlation between dissociation time and stress index (Figures 1G, 1H, S2G, and S2H). This correlation held across organs, with those dissociated for more than 60 min (such as the bladder, the tongue, the trachea, the limb muscle, and the mammary gland) presenting with a high stress index and tissues dissociated for less than 20 min (such as the liver, the marrow, the spleen, the pancreas, and the brain) presenting with a low stress index (Figures S2I and S2J). In order to test for bias due to cell type composition, we calculated the median stress index per cell type across all tissues and, consistently, observed the same positive correlation with median dissociation time (Figures 1I and S2K). The gene expression modifications we found were identified in cells during experimental cell dissociation. However, the early transcriptional response to tissue damage in vivo remained unknown. To address this, we performed single-nucleus transcriptomic analysis shortly after chemically induced injury of the liver and the skeletal muscle (Figure 2A). Following screening for the stress-response kinetics (Figure S3A), we generated a snRNA-seq atlas from injured livers 4 h post-carbon tetrachloride (CCl4) injection (Teixeira-Clerc et al., 2010Teixeira-Clerc F. Belot M.P. Manin S. Deveaux V. Cadoudal T. Chobert M.N. Louvet A. Zimmer A. Tordjmann T. Mallat A. Lotersztajn S. Beneficial paracrine effects of cannabinoid receptor 2 on liver injury and regeneration.Hepatology. 2010; 52: 1046-1059Crossref PubMed Scopus (91) Google Scholar) and compared with to uninjured liver (Figures 2B and S3B–S3D). Strong transcriptional changes were detected in all identified populations, as shown by the clear separation of the uninjured from the injured clusters (Figure 2C) with a high stress index detected specifically in the injured liver-derived hepatocytes (Figure 2D). We also performed injury of the tibialis anterior (TA) hindlimb muscle by intramuscular injection of barium chloride (BaCl2) (Casar et al., 2004Casar J.C. McKechnie B.A. Fallon J.R. Young M.F. Brandan E. Transient up-regulation of biglycan during skeletal muscle regeneration: delayed fiber growth along with decorin increase in biglycan-deficient mice.Dev. Biol. 2004; 268: 358-371Crossref PubMed Scopus (83) Google Scholar). We first determined the stress-response kinetics (Figure S3E) and generated snRNA-seq atlases from muscles collected 4 h post-BaCl2 injection (Figures 2B, S3C, S3F, and S3G). Analysis showed that the activated populations clustered separately from their corresponding populations in intact muscles (Figure 2C) and a high stress index (Figure 2D). To expand our analysis on the kinetics of a specialized stem cell population in vivo, we performed single-cell RNA-seq (scRNA-seq) on MuSCs at different time points post-injury, by adapting the SMARTseq2 scRNA-seq for in situ fixation (iSiFi)-treated samples. MuSCs were isolated using fluorescence-activated cell sorting (FACS) at 2 and 4 h post-BaCl2 injury (T2 and T4) and compared with uninjured muscle (T0) (Figure 2E). Following quality controls, we analyzed 280 MuSCs (Figures S4A–S4F). Principal-component analysis (PCA) grouped the cells according to their experimental condition and showed transcriptional consistency within clusters yet marked differences among time points (Figure 2F). Of note, quiescent T0 MuSCs formed a unique, compact cluster indicating a relatively homogeneous cell population, whereas the activated cells appeared more scattered while remaining distinct (Figure 2F and S4G). To precisely order cells along the activation path, we performed pseudotime analysis (Street et al., 2018Street K. Risso D. Fletcher R.B. Das D. Ngai J. Yosef N. Purdom E. Dudoit S. Slingshot: cell lineage and pseudotime inference for single-cell transcriptomics.BMC Genomics. 2018; 19: 477Crossref PubMed Scopus (454) Google Scholar) and observed a continuous trajectory, suggesting that all cells transitioned evenly from T0 to T2 to T4 state but with variable kinetics (Figure 2G). Furthermore, to cross-correlate in vivo activation and the stress response core genes, we visualized the stress index of each cell according to its pseudotime value. We noticed that MuSCs activated in vivo exhibited a strong and transient induction of the stress core genes (Figure 2H), indicating that the transcription of the early response genes is transient and dynamic. Finally, implementation of the iSiFi protocol during early activation enabled us to provide an explanation for some puzzling features in high-quality single-cell datasets on quiescent MuSCs (Camps et al., 2020Camps J. Breuls N. Sifrim A. Giarratana N. Corvelyn M. Danti L. Grosemans H. Vanuytven S. Thiry I. Belicchi M. et al.Interstitial cell remodeling promotes aberrant adipogenesis in dystrophic muscles.Cell Rep. 2020; 31: 107597Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar; Dell’Orso et al., 2019Dell’Orso S. Juan A.H. Ko K.-D. Naz F. Gutierrez-Cruz G. Feng X. Sartorelli V. Single-cell analysis of adult skeletal muscle stem cells in homeostatic and regenerative conditions.Development. 2019; 146: dev174177Crossref PubMed Scopus (67) Google Scholar; Giordani et al., 2019Giordani L. He G.J. Negroni E. Sakai H. Law J.Y.C. Siu M.M. Wan R. Corneau A. Tajbakhsh S. Cheung T.H. Le Grand F. High-dimensional single-cell cartography reveals novel skeletal muscle-resident cell populations.Mol. Cell. 2019; 74: 609-621.e6Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar; Schaum et al., 2018Schaum N. Karkanias J. Neff N.F. May A.P. Quake S.R. Wyss-Coray T. Darmanis S. Batson J. Botvinnik O. Chen M.B. et al.Tabula Muris ConsortiumOverall coordinationLogistical coordinationOrgan collection and processingLibrary preparation and sequencingComputational data analysisCell type annotationWriting groupSupplemental text writing groupPrincipal investigatorsSingle-cell transcriptomics of 20 mouse organs creates a Tabula Muris.Nature. 2018; 562: 367-372Crossref PubMed Scopus (787) Google Scholar), namely, the low detection of the MuSCs hallmark gene Pax7 and, paradoxically, the high abundance of several oncogene transcripts, such as Egr1, Jun, and Fos (Figures S4H–S4J), which have been shown to be expressed in activated MuSCs (Wang et al., 2018aWang G. Zhu H. Situ C. Han L. Yu Y. Cheung T.H. Liu K. Wu Z. p110α of PI3K is necessary and sufficient for quiescence exit in adult muscle satellite cells.EMBO J. 2018; 37: e98239Crossref PubMed Scopus (15) Google Scholar). To further define the kinetics of MuSC quiescence exit, we generated a comprehensive time course transcriptional map on iSiFi-processed MuSCs. In situ fixed MuSCs were isolated using FACS every 30 min for 2 h during the standard dissociation procedure and RNA-sequenced in bulk. A time point of 15 min (T15) was introduced to detect earlier transcriptional events (Figure 3A). PCA on the transcriptomes clustered samples by time points and formed a clear activation trajectory along the three main principal components (Figure 3B). After 15 min, 42 genes were upregulated (fold change [FC] > 2, p < 0.05), and most differential expression events took place at T60, reaching a maximum at T120 (>3,000 DEGs, FC > 2; Figure 3C). The identity of the DEGs at T120 was highly similar to that observed in our previous study (Machado et al., 2017Machado L. Esteves de Lima J. Fabre O. Proux C. Legendre R. Szegedi A. Varet H. Ingerslev L.R. Barrès R. Relaix F. et al.In situ fixation redefines quiescence and early activation of skeletal muscle stem cells.Cell Rep. 2017; 21: 1982-1993Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) (Figure S4K). Using hierarchical clustering, transcripts with related kinetics were grouped in five clusters (Figures 3D–3F). Cluster 1 contained 36 of the 42 upregulated genes following 15 min of dissociation, including immediate and early response genes (Ier2, Ier3, Egr1, and Egr2) and members of the AP-1 family of oncogenes and ERK1/2 targets (Jun, Junb, Jund, Fos, and Fosb). Of note, the cluster 1 pattern of expression was characterized by a peak at T60 that rapidly reverted to close to T0 levels after 2 h of digestion (Figure 3F), a stereotypical expression pattern of growth factor- or stress-driven responses (Barton et al., 1996Barton K. Muthusamy N. Chanyangam M. Fischer C. Clendenin C. Leiden J.M. Defective thymocyte proliferation and IL-2 production in transgenic mice expressing a dominant-negative form of CREB.Nature. 1996; 379: 81-85Crossref PubMed Scopus (214) Google Scholar). Transcriptional events directly linked to the myogenic program were found well into the second hour of digestion, such as the induction of Myod (cluster 3, 11.9 upregulated at T120 compared to T0) or the downregulation of the MuSC quiescence markers Pax7 (Seale et al., 2000Seale P. Sabourin L.A. Girgis-Gabardo A. Mansouri A. Gruss P. Rudnicki M.A. Pax7 is required for the specification of myogenic satellite cells.Cell. 2000; 102: 777-786Abstract Full Text Full Text PDF PubMed Scopus (1577) Google Scholar), Spry1 (Chakkalakal et al., 2012Chakkalakal J.V. Jones K.M. Basson M.A. Brack A.S. The aged niche disrupts muscle stem cell quiescence.Nature. 2012; 490: 355-360Crossref PubMed Scopus (502) Google Scholar), and Calcr (Yamaguchi et al., 2015Yamaguchi M. Watanabe Y. Ohtani T. Uezumi A. Mikami N. Nakamura M. Sato T. Ikawa M. Hoshino M. Tsuchida K. et al.Calcitonin receptor signaling inhibits muscle stem cells from escaping the quiescent state and the niche.Cell Rep. 2015; 13: 302-314Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) (cluster 4, FCs of 3.5, 3.2, and 1.7, respectively, at T0/T120). Similarly, we scored attenuation of Notch signaling at the latest time points, with pathway canonical target genes Hey1, Hes1, and HeyL being downregulated at T60, T90, and T120, respectively, compared with T0. Counterintuitively, but consistent with its role as an adaptor protein of kinase complexes, the cell cycle inhibitor encoding gene Cdkn1a (p21) was upregulated by the dissociation procedure (cluster 2, FC of 29.9 at T120/T0). By ontology analysis, we propose that the five clusters did not differ merely on the gene expression patterns but also exhibited distinct functional identities (Figure S4L). To investigate the similarities in MuSC activation between in vivo muscle injury and ex vivo muscle dissociation, we integrated the scRNA-seq from the injured TA with the bulk RNA-seq time course (Figures 2E and 3A, respectively). First, we projected the dissociated MuSCs onto the PCA space of in vivo activated MuSCs. Principal component 1 (PC1) and principal component 2 (PC2) accurately positioned the ex vivo dissociated samples according to their digestion time, from T0 to T120 (Figure 3G). Next, we investigated the behavior of the five genetic clusters identified ex vivo into the injury-activated MuSC and found a striking similarity in the dynamic behavior of genes across all clusters (Figure 3H). Finally, we confirmed statistically this observation using an analysis of covariance (ANCOVA) and found no significant differences in the genetic behavior of four of the five clusters investigated (Figure S4M). Of note, cluster 1 genes differed between experimental models, possibly because of the highly dynamic nature of genes in this cluster. Having established that a broadly conserved stress response occurs with cell activation, we investigated its functional role on MuSC activity. As part of the early stress response, we identified Odc1 and Azin1 (Figures 1E and 3E), whose products form a complex for the synthesis of the polyamine spermidine that is important in cell proliferation, growth, translation initiation and elongation (Mandal et al., 2013Mandal S. Mandal A. Johansson H.E. Orjalo A.V. Park M.H. Depletion of cellular polyamines, spermidine and spermine, causes a total arrest in translation and growth in mammalian cells.Proc. Natl. Acad. Sci. U S A. 2013; 110: 2169-2174Crossref PubMed Scopus (161) Google Scholar). In turn, spermidine is the source of the amino acid hypusine, a unique post-translational addition to the eukaryotic translation initiation factor eIF5A (Schuller et al., 2017Schuller A.P. Wu C.C.-C. Dever T.E. Buskirk A.R. Green R. eIF5A functions globally in translation elongation and termination.Mol. Cell. 2017; 66: 194-205.e5Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar), which was also found to be transcriptionally induced during activation. To test the role of polyamine biosynthesis on the G0-to-S transition of quiescent cells, we cultured freshly isolated MuSCs with the ODC1 inhibitor d,l-α-difluoromethylornithine (DFMO) for 40 h and quantified EdU incorporation. DFMO treatment resulted in significantly decelerated cell cycle entry. Interestingly, decreased proliferation did not lead to accelerated differentiation, but instead DFMO treatment retained a more upstream PAX7+/Myogenin− cell population, compared with control (Figures 4A and 4B ). We then focused on two major signaling pathways, MAPK and Notch, that showed opposite yet potent responses to tissue damage. MAPK was strongly and immediately induced, whereas Notch was gradually downregulated as MuSCs entered the myogenic program (Figure 3E for Hes1, Hey1, and HeyL; Figure S4D for ERK1/2 phosphorylation). To test the significance of these pathways and their potential interaction during early MuSC activation, we generated mice conditionally overexpressing a constitutively active form of Notch1 (NICD) in MuSCs (Pax7CreERT2; Rosastop-NICD) and cultured muscle cells with or without the ERK1/2 inhibitor SCH772984 (Figure 4C). NICD impeded the expression of the activation marker MYOD and, probably as a consequence, delayed entry to S phase (Mourikis and Tajbakhsh, 2014Mourikis P. Tajbakhsh S. Distinct contextual roles for Notch signalling in skeletal muscle stem cells.BMC Dev. Biol. 2014; 14: 2Crossref PubMed Scopus (83) Google Scholar) (Figure 4C). Treatment with SCH772984 did not affect the antagonistic effect of NICD on MYOD but strongly blocked cell cycle entry (Figure 4C). After 3 days in culture, the majority of control NICD cells cycled, and all were positive for PAX7 (Figure 4D). Of interest, the combination of ERK inhibition and Notch activation maintained MuSCs in a quiescent-like state for at least 72 h in culture (EdU−, Pax7+, Myog−) with spindle-shaped nuclei, characteristic of quiescent MuSCs (Machado et al., 2017Machado L. Esteves de Lima J. Fabre O. Proux C. Legendre R. Szegedi A. Varet H. Ingerslev L.R. Barrès R. Relaix F. et al.In situ fixation redefines quiescence and early activation of skeletal muscle stem cells.Cell Rep. 2017; 21: 1982-1993Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) (Figure 4D). Taken together, our data suggest that Notch and ERK pathways are partially uncoupled, whereby an early ERK signal is the driving force of MuSCs proliferation and Notch downregulation primes the progression into the myogenic differentiation program. Next, to test directly the significance of the early stress response to stem cell activation, ERK signaling was blocked during the 2 h of the dissociation process, and then MuSCs were plated in proliferation medium (Figures 4E, S4N, and S4O). Remarkably, this treatment significantly impaired the capacity of these cells to enter S phase, providing strong evidence for the importance of the early stress response on MuSC quiescence exit (Figures 4F and S4P). In this study, we have captured the primary cellular responses to niche damage, focusing on two highly regenerative tissues, the skeletal muscle and the liver. By analyzing a large number of diverse cells, we defined a conserved transcriptional response to tissue damage that appears to depend primarily on the duration of the stressors’ signal rather than the cell type. As different cells serve diverse functions during regeneration, the core stress response is also likely to operate in a variety of specialized functions. Indeed, in the muscle, quiescent MuSCs and fibro-adipogenic progenitors (FAPs) enter a proliferation phase in response to injury, whereas terminally differentiated myonuclei are wasted and replaced yet still manifest the stress core response. Moreover, the nature of the stress cells encounter leading to this response remains unclear. Tissue damage involves cell lysis and release of growth factors and cytokines, mechanical changes such as loss of adhesion and physical barriers, and loss of cell contact with ligand-bearing cells, to name a few. With such complexity of damage-induced processes, it is likely that the trigger of the stress response is multifactorial. Our results that ERK1/2 inhibition mitigates the activat