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 damage•Transcriptional changes by cell dissociation impair accuracy single-cell atlases•Cooperating MAPK and Notch signaling control quiescence exit MuSCs Tissue damage dramatically alters how cells interact with their microenvironment. These turn dictate responses, such as stem activation, yet remain ill defined. We generated nucleus atlases from intact, dissociated, injured muscle liver identified shared multiple types across these organs. This prevalent was detected published datasets range tissues, demonstrating high conservation but also significant degree data distortion reference atlases. Using quiescent paradigm activation injury, we captured injury found that an essential ERK1/2 primary proliferation signal precedes initiation Notch-regulated myogenic program. study defines initial events perturbation identifies broadly conserved transcriptional acts parallel cell-specific adaptive alterations. Cell plasticity is fundamental property variety vertebrate triggers stem/progenitor order restore homeostasis, seen bone, skin, skeletal muscle, among others (Fuchs Blau, 2020Fuchs E. Blau H.M. cells: architects niches.Cell Stem Cell. 2020; 27: 532-556Abstract Full Text PDF PubMed Scopus (44) Google Scholar; Jessen et al., 2015Jessen K.R. Mirsky R. Arthur-Farraj P. The role repair: reprogramming.Dev. 2015; 34: 613-620Abstract (70) Scholar). Differentiated too can get activated eventually proliferate de-differentiation, case for hepatocytes after hepatectomy (Riehle 2011Riehle K.J. Dan Y.Y. Campbell J.S. Fausto N. New concepts regeneration.J. Gastroenterol. Hepatol. 2011; 26: 203-212Crossref (176) Scholar) or neonatal mouse cardiomyocytes (Porrello 2011Porrello E.R. Mahmoud A.I. Simpson Hill J.A. Richardson Olson E.N. Sadek H.A. Transient regenerative potential heart.Science. 331: 1078-1080Crossref (1536) death pathways, however, be instigated when irreversible (Rathore 2017Rathore McCallum J.E. Varghese Florea A.M. Büsselberg D. Overcoming chemotherapy drug resistance targeting inhibitors apoptosis proteins (IAPs).Apoptosis. 2017; 22: 898-919Crossref (149) Uncovering roadmap they homeostasis critical understanding properties developing tools experimental manipulation. Despite extensive research, degree, kinetics, nature early, type-specific occur poorly A major limitation capturing process isolation induces modifications mask actual injury-induced changes. Early studies uncovered global standard methods were performed on (MuSCs) (van den Brink 2017van S.C. Sage F. Vértesy Á. Spanjaard B. Peterson-Maduro J. Baron C.S. 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Nephrol. 30. : 23-32Crossref (180) Considering ever growing importance atlases, whether technical artifacts linked recovery if caused loss signals critical. Here, developed specialized protocols identify kinetics types. organs uncover functional response, model activation. In test impact (snRNA-seq) intact dissociated murine droplet-based platform (Figure 1A) 26,074 (Figures S1A–S1D), distributed ten main seven 1B, S1E, S1F). When all nuclei merged, noticed tended cluster type, there clear separation between indicating strong differences 1C). Remarkably, nearly showed induction Jun, prototypical stress-response S1G) (Bohmann 1987Bohmann Bos T.J. Admon Nishimura Vogt P.K. Tjian Human proto-oncogene c-jun encodes DNA binding protein structural transcription factor AP-1.Science. 1987; 238: 1386-1392Crossref (945) Differential analysis indicated variable dissociation, average 1,226 differentially expressed genes (DEGs) studied S1H). Notably, MuSCs, DEGs highly similar previous bulk (Machado Scholar), validating approach S1I S1J). Analysis upregulated enrichment biological processes, described Figures S1K–S1N (Ishikawa 2012Ishikawa Factor V.M. Marquardt J.U. Raggi Seo Kitade Conner E.A. Thorgeirsson Hepatocyte growth factor/c-met required cell-mediated regeneration mice.Hepatology. 2012; 55: 1215-1226Crossref (137) Meeteren Dijke, 2012van L.A. Dijke Regulation endothelial TGF-?.Cell Res. 347: 177-186Crossref (226) Contreras 2019Contreras Cruz-Soca Theret Soliman Tung L.W. Groppa Rossi F.M. Brandan Cross-talk TGF-? PDGFR? pathways regulates fate stromal fibro-adipogenic progenitors.J. Sci. 132: jcs232157Crossref (33) then examined extent which Approximately half type specific, while rest induced at least two 1D). pattern observed downregulated S2A). Importantly, core composed 98 1E S2B; Table S1). set designated index validated our snRNA-seq dataset S2C). Gene Ontology enriched terms signaling, factors cytokines, 1F). Consistently, phospho-ERK1/2 rapid 15 min digestion S2D). On basis broad extended multi-organ (Tabula Muris; Schaum 2018Schaum Karkanias Neff N.F. May A.P. Quake Wyss-Coray Darmanis S. Batson Botvinnik Chen M.B. al.Tabula Muris ConsortiumOverall coordinationLogistical coordinationOrgan collection processingLibrary preparation sequencingComputational analysisCell annotationWriting groupSupplemental text writing groupPrincipal investigatorsSingle-cell transcriptomics 20 creates Tabula Muris.Nature. 562: 367-372Crossref (787) 1G S2E). Stress most only fraction S2F), contrast postulated variations time procedure. To corroborate hypothesis, inferred every (Table S2) correlation 1G, 1H, S2G, S2H). held organs, those more than 60 (such bladder, tongue, trachea, limb mammary gland) presenting less liver, marrow, spleen, pancreas, brain) low S2I S2J). bias due composition, calculated median per and, consistently, same positive 1I S2K). dissociation. However, remained unknown. address this, transcriptomic shortly chemically 2A). Following screening S3A), livers 4 h post-carbon tetrachloride (CCl4) injection (Teixeira-Clerc 2010Teixeira-Clerc Belot M.P. Manin Deveaux V. Cadoudal Chobert M.N. Louvet Zimmer Tordjmann Mallat Lotersztajn Beneficial paracrine effects cannabinoid receptor 2 regeneration.Hepatology. 2010; 52: 1046-1059Crossref (91) uninjured 2B S3B–S3D). Strong populations, shown clusters 2C) specifically liver-derived 2D). tibialis anterior (TA) hindlimb intramuscular barium chloride (BaCl2) (Casar 2004Casar J.C. McKechnie B.A. Fallon J.R. Young M.F. up-regulation biglycan regeneration: delayed fiber along decorin increase biglycan-deficient mice.Dev. 2004; 268: 358-371Crossref (83) first determined S3E) muscles collected post-BaCl2 2B, S3C, S3F, S3G). clustered separately corresponding expand population vivo, (scRNA-seq) different points post-injury, adapting SMARTseq2 scRNA-seq (iSiFi)-treated samples. isolated fluorescence-activated sorting (FACS) (T2 T4) (T0) 2E). quality controls, analyzed 280 S4A–S4F). Principal-component (PCA) grouped according condition consistency within marked 2F). Of note, T0 formed unique, compact relatively homogeneous population, whereas appeared scattered remaining distinct 2F S4G). precisely path, pseudotime (Street 2018Street K. Risso Fletcher R.B. Das Ngai Yosef Purdom Dudoit Slingshot: lineage inference transcriptomics.BMC Genomics. 19: 477Crossref (454) continuous trajectory, suggesting transitioned evenly T2 T4 state 2G). Furthermore, cross-correlate genes, visualized each its value. exhibited transient 2H), dynamic. Finally, implementation iSiFi protocol enabled us provide explanation some puzzling features high-quality (Camps 2020Camps Breuls Sifrim Giarratana Corvelyn Danti Grosemans Vanuytven Thiry I. Belicchi al.Interstitial remodeling promotes aberrant adipogenesis dystrophic muscles.Cell 31: 107597Abstract (27) Dell’Orso 2019Dell’Orso Juan A.H. Ko K.-D. Naz Gutierrez-Cruz G. Feng Sartorelli homeostatic conditions.Development. 146: dev174177Crossref (67) Giordani 2019Giordani He G.J. Negroni Sakai Law J.Y.C. Siu M.M. Wan Corneau Tajbakhsh Cheung T.H. Le High-dimensional cartography muscle-resident populations.Mol. 74: 609-621.e6Abstract (114) namely, detection hallmark Pax7 paradoxically, abundance several oncogene transcripts, Egr1, Fos S4H–S4J), been (Wang 2018aWang Zhu Situ Han Yu Liu p110? PI3K necessary sufficient satellite cells.EMBO 37: e98239Crossref (15) further define MuSC exit, comprehensive course map iSiFi-processed MuSCs. fixed FACS 30 procedure RNA-sequenced bulk. point (T15) introduced detect earlier 3A). PCA samples trajectory three principal components 3B). After min, 42 (fold change [FC] > 2, p < 0.05), differential took place T60, reaching maximum T120 (>3,000 DEGs, FC 2; Figure 3C). identity S4K). hierarchical clustering, transcripts related five 3D–3F). Cluster 1 contained 36 including immediate (Ier2, Ier3, Egr2) members AP-1 family oncogenes targets (Jun, Junb, Jund, Fos, Fosb). characterized peak T60 rapidly reverted close levels 3F), stereotypical factor- stress-driven (Barton 1996Barton Muthusamy Chanyangam Fischer Clendenin Leiden J.M. Defective thymocyte IL-2 production transgenic mice expressing dominant-negative form CREB.Nature. 1996; 379: 81-85Crossref (214) directly program well into second hour digestion, Myod (cluster 3, 11.9 T0) downregulation markers (Seale 2000Seale Sabourin Girgis-Gabardo Mansouri Gruss Rudnicki M.A. specification cells.Cell. 2000; 102: 777-786Abstract (1577) Spry1 (Chakkalakal 2012Chakkalakal J.V. Jones K.M. Basson Brack aged disrupts quiescence.Nature. 490: 355-360Crossref (502) Calcr (Yamaguchi 2015Yamaguchi Watanabe Ohtani Uezumi Mikami Nakamura Sato Ikawa Hoshino Tsuchida al.Calcitonin inhibits escaping niche.Cell 302-314Abstract (54) 4, FCs 3.5, 3.2, 1.7, respectively, T0/T120). Similarly, scored attenuation latest points, pathway canonical target Hey1, Hes1, HeyL being T90, T120, T0. Counterintuitively, consistent adaptor kinase complexes, cycle inhibitor encoding Cdkn1a (p21) 29.9 T120/T0). By ontology analysis, propose did not differ merely patterns identities S4L). investigate similarities integrated TA 2E 3A, respectively). First, projected onto space Principal component (PC1) (PC2) accurately positioned time, 3G). Next, investigated behavior genetic injury-activated striking similarity dynamic 3H). confirmed statistically observation covariance (ANCOVA) no four S4M). differed models, possibly because cluster. Having established occurs activity. As part Odc1 Azin1 3E), whose products complex synthesis polyamine spermidine important proliferation, growth, translation elongation (Mandal 2013Mandal Mandal Johansson H.E. Orjalo A.V. Park M.H. Depletion polyamines, spermine, causes total arrest mammalian cells.Proc. Natl. Acad. U S 2013; 110: 2169-2174Crossref (161) turn, source amino acid hypusine, post-translational addition eukaryotic eIF5A (Schuller 2017Schuller C.C.-C. Dever Buskirk A.R. Green functions globally termination.Mol. 66: 194-205.e5Abstract (190) transcriptionally biosynthesis G0-to-S transition cultured freshly ODC1 d,l-?-difluoromethylornithine (DFMO) 40 quantified EdU incorporation. DFMO treatment resulted significantly decelerated entry. Interestingly, decreased lead accelerated differentiation, instead retained upstream PAX7+/Myogenin? 4A 4B ). focused Notch, opposite potent damage. strongly immediately induced, gradually entered 3E HeyL; S4D phosphorylation). significance interaction conditionally overexpressing constitutively Notch1 (NICD) (Pax7CreERT2; Rosastop-NICD) without SCH772984 4C). NICD impeded marker MYOD probably consequence, entry phase (Mourikis Tajbakhsh, 2014Mourikis Distinct contextual roles signalling cells.BMC Dev. 2014; 2Crossref Treatment affect antagonistic effect blocked 3 days culture, majority cycled, PAX7 4D). interest, combination ERK inhibition maintained quiescent-like 72 culture (EdU?, Pax7+, Myog?) spindle-shaped nuclei, characteristic Taken together, suggest partially uncoupled, whereby driving force primes progression differentiation process, plated medium 4E, S4N, S4O). impaired capacity enter phase, providing evidence 4F S4P). study, damage, focusing liver. analyzing large number defined appears depend primarily duration stressors’ rather type. serve regeneration, likely operate functions. Indeed, progenitors (FAPs) terminally differentiated myonuclei wasted replaced still manifest response. Moreover, encounter leading remains unclear. involves lysis release mechanical adhesion physical barriers, contact ligand-bearing name few. With complexity damage-induced trigger multifactorial. Our results mitigates activat