Perla Le ´o Machado

Jordi Camps, ..., Pascal Geara

Frederic Maire
email: frederic.relaix@inserm.fr

Philippos Relaix
email: philippos.mourikis@inserm.fr

Mourikis

"Enthusiasm is common. Endurance is rare." -Angela Duckworth

Acknowledgments

First, I would like to respectfully thank the jury members for their time and presence. I wish to thank Dr. Colin Crist and Dr. Athanassia Sotiropoulos for reviewing my manuscript and Pr.

Piotr Topilko and Dr. Barbara Geyraud-Morel for accepting to be a part of my thesis jury.

I wish to thank Pr. Frédéric Relaix for granting me the opportunity to kickstart my scientific journey in his lab. You gave me all the tools and techniques I needed to learn and grow as a researcher. Your positivity, good heartedness, and kindness are inspiring.

I would also like to give my outmost gratitude to my supervisor Dr Philippos Mourikis. You're not only a great scientist but a great person as well. I am extremely lucky to have had the opportunity to start my career with you as my mentor and to learn from you. You shaped my scientific mind, "What would Philippos do" is the mindset I followed to make decisions that led to the work presented in this manuscript, and that I will always use. Your calmness, compassion, and good humor were of great help to keep me grounded and focused during the challenging times of this journey. I hope this work and all my future accomplishments make you proud.

A big thank you goes to the Mourikis team. A very special thanks to Stemi for sharing the journey with me under the Mourikis supervision from day one until the very last day. I will miss our complaining and gossiping sessions as well as our conferences adventures. To Léo for taking me under his wing, imparting his unconventional wisdom, and helping me get rid of my leftover food. Eleni, thank you for coming, and thank you and Erica for always being the positive energy in the group. I wish to thank all the BNMS team. I started in the lab when I was only 22 years old, I had the opportunity to meet people from all over the world and to learn a little something from each and every one of them, and that of course has helped me mature as the person I am today.

Maria, Matt, Joana, Alessio, Cyrielle, thank you for your great friendship and the amazing time we got to spend together. And a special thank you for Despoina for choosing me to work with you on your final research project. Thank you for sharing your amazing company, pleasant sense of humor, and incredible food. I'm still hoping to learn your exceptional organizational skills.

I wish to thank my colleague, compatriot, desk neighbor, actual neighbor at some point, and great friend Reem. Thank you for rescuing my cells on the weekends when I couldn't make it to the lab, for "trying" to help me prepare food, granting me access to your secret stocks (oops?), and for being an amazing confinement buddy. My time in the lab and in MDL wouldn't have been the same without you there.

I wish to thank my uncle Michel and his family for taking me in ever since I arrived to France. I would also like to thank all my friends whether here in France, Lebanon, or Poland for their unwavering support.

And finally, my deepest gratitude goes to my family. My parents, you were the biggest examples for hard work and resilience. You taught me to never be limited by people, places, or circumstances and to always go wherever my ambitions take me. Everything I have managed to accomplish so far, I owe it to you, and is dedicated to you. My goal is to always make you proud; I can only hope I managed to do that so far. A tremendous thank you and the biggest hug for my sisters and best friends. I am forever grateful for the unconditional love and support you have always given me; I wish I will be able to give back a small fraction of it.

Abstract

Quiescence is a reversible cell state in which the cells have exited the cell-cycle but can reenter it upon external or intrinsic queues. Even though this cell state is considered as dormant, it has been shown to be regulated by a very dynamic and complex gene network. Many adult organs contain quiescent stem cells that are maintained during the lifespan of an organism, and fine-tuning quiescence and activation is essential for tissue homeostasis and regeneration. Dissecting the mechanisms that regulate the quiescence-to-activation transition of skeletal muscle stem cells (MuSCs) has been the main focus of my Thesis.

Studying adult stem cells requires tissue dissociation and stem cells isolation, methods that we have recently shown to lead to major transcriptomic changes and introduce an activation signature to the cells. In fact, using specialized methods, including a fixation-based protocol developed in the host laboratory and rapid nuclear extraction from whole tissues, have allowed us to capture the transcriptome of MuSCs in their in vivo quiescent state. Comparison to activated MuSCs allowed the identification of an early stress response that is elicited upon tissue damage. In order to compare the transcriptional response to niche disruption in diverse cell types, we performed single nucleus RNA-seq on intact and damaged skeletal muscle and liver, two highly regenerating tissues. By doing this, we were able to identify a conserved, core transcriptional stress response induced in essentially all cell types during tissue damage. This core response included immediate early genes, targets of the oncogenic AP-1 and ERK1/2 MAPK pathways, genes involved in transcription and epigenetic regulation pathways, cytoskeletal organization pathways and the polyamine synthesis pathway among many others.

Moreover, we investigated the functional importance of this stress core response in the early activation of quiescent stem cells. We used a combination of genetic and pharmacological tools to target selected pathways during MuSCs quiescence exit. These included the polyamine synthesis and ERK1/2 signaling pathways that we blocked with specific inhibitors to study their role in MuSCs early activation. We also combined ERK1/2 pathway inhibitors with mice overexpressing activated Notch, a pathway known to be required for MuSCs quiescence, and we were able to generate quiescent-like MuSCs in culture conditions. Furthermore, we have investigated the role of the Krüppel-like transcription factor Klf4, also identified in the stress core, in the early activation of MuSCs, as well as the role of the Serum Response Factor Srf, a master regulator of immediate early genes. Finally, I will present our approaches towards developing culture conditions to maintain but also induce quiescence, with the aim to enhance the regenerative potential of ex vivo amplified MuSCs.

Keywords: Muscle stem cells, Quiescence, Early activation, Stress response, MAPK, Polyamine synthesis, Notch signalling, Klf4, Srf.

Résumé

La quiescence est un état cellulaire réversible dans lequel les cellules sont sorties du cycle cellulaire mais peuvent le réintégrer en fonction des stimuli externes ou intrinsèques. Bien que cet état cellulaire soit considéré comme un état de dormance, il a été démontré qu'il est régulé par un réseau de gènes très dynamique et complexe. De nombreux organes adultes contiennent des cellules souches quiescentes qui sont maintenues pendant toute la durée de vie d'un organisme, et la régulation de la quiescence et de l'activation est essentiel pour l'homéostasie et la régénération des tissus. La dissection des mécanismes qui régulent la transition entre la quiescence et l'activation des cellules souches du muscle squelettique (MuSCs) a été l'objectif principal de ma thèse.

L'étude des cellules souches adultes nécessite la dissociation des tissus et l'isolement des cellules souches, méthodes dont nous avons récemment montré qu'elles entraînent des modifications transcriptomiques majeures et introduisent une signature d'activation artificielle des cellules. En fait, un protocole basé sur la fixation a été développé dans notre laboratoire qui permet de capturer des cellules souches du muscle squelettique (MuSCs) dans leur état quiescent in vivo. La comparaison de ces MuSCs avec des MuSCs activées nous a permis d'identifier une réponse précoce au stress qui est déclenchée lors de la lésion tissulaire. Afin de comparer la réponse transcriptionnelle de divers types de cellules à la perturbation du microenvironnement, nous avons réalisé une analyse snRNA-seq sur du muscle squelettique et du foie intacts et dissociés, deux tissus à forte régénération. Nous avons pu identifier une réponse de stress commune induite dans la majorité des types de cellules lors de la dissociation. Cette réponse centrale comprend des gènes précoces immédiats, des cibles des voies oncogènes AP-1 et ERK1/2 MAPK, des gènes impliqués dans les voies de transcription et de régulation épigénétique, les voies d'organisation du cytosquelette et la voie de synthèse des polyamines, entre autres.

De plus, nous avons étudié l'importance fonctionnelle de cette réponse au stress dans l'activation précoce des cellules souches quiescentes. Nous avons utilisé une combinaison d'outils génétiques et pharmacologiques pour cibler ces voies identifiées pendant la sortie de la quiescence des cellules souches. Nous avons notamment utilisé des inhibiteurs contre des gènes de la voie de synthèse des polyamines et la voie de signalisation ERK1/2 pour étudier leur rôle dans l'activation précoce des MuSCs. Nous avons également combiné l'inhibition de la voie ERK1/2 avec des souris surexprimant la voie Notch, une voie connue pour son rôle dans la maintenance de quiescence des MuSCs ce qui nous a permis de simuler des MuSCs quasi-quiescentes dans des conditions de culture. De plus, nous avons étudié le rôle du facteur de transcription Krüppel-like Klf4 également identifié dans la réponse au stress, dans l'activation précoce des MuSCs, ainsi que le rôle du Serum Response Factor Srf, un régulateur des gènes précoces immédiats. Enfin, je présenterai nos approches pour développer des conditions de culture permettant de maintenir mais aussi d'induire la quiescence, dans le but d'améliorer le potentiel régénératif des MusCs amplifiées ex vivo.

Mots clés : MuSCs, Quiescence, activation précoce, MAPK, synthèse de polyamines, Notch, Klf4, Srf. Cellular quiescence is a reversible cell cycle arrest state. Unlike senescent or terminally differentiated cells, quiescent cells retain the capacity of re-entering the cell cycle in response to external or internal queues [START_REF] Coller | A new description of cellular quiescence[END_REF]. After the 4 stages of the cell cycle were first described in 1953 [START_REF] Howard | Synthesis of Desoxyribonucleic Acid in Normal and Irradiated Cells and Its Relation to Chromosome Breakage[END_REF], the concept of the existence of a G0 phase that is distinct from the G1 phase became a matter of controversy, as some denied the existence of the G0 and considered it to be a long G1 phase [START_REF] Baserga | BIOCHEMISTRY OF THE CELL CYCLE: A REVIEW[END_REF].

Table of contents

Later, it was observed that quiescence can be induced by several methods, such as serum withdrawal, contact inhibition, or loss of adhesion. Furthermore, it was shown that using different quiescence induction methods in primary fibroblasts resulted in some unique gene expression profiles for each method while retaining some common features [START_REF] Coller | A new description of cellular quiescence[END_REF], demonstrating that cellular quiescence is a complex and broad cellular state.

In fact, for a long time the G0 state was very poorly understood, as we knew more about what these cells are not able to do rather than what they can do. It was believed that quiescent cells, being in a dormant state, have minimal activity. However, it was later shown that they are actively maintained in this state by several signaling pathways to ensure the maintenance of quiescence. These cells reside in specific anatomical locations in tissues and are maintained, thanks to their microenvironment, throughout the organism to ensure tissue regeneration, homeostasis, immunity and reproduction [START_REF] Marescal | Cellular Mechanisms and Regulation of Quiescence[END_REF].

Quiescence in adult stem cells

Adult quiescent stem cells are undifferentiated cells that have the unique ability to activate, following extrinsic or intrinsic stimuli, and give rise to progenitor cells that will eventually differentiate, while at the same time keep their capacity to self-renew to replenish the stem cell pool (Figure 1) [START_REF] Urbán | Stem cell quiescence: the challenging path to activation[END_REF]. Several adult stem cells are maintained in a quiescent state during homeostatic conditions and activate for tissue turnover such as the blood [START_REF] Pietras | Cell cycle regulation in hematopoietic stem cells[END_REF] [START_REF] Sun | Clonal dynamics of native haematopoiesis[END_REF], or in the case of injury such as the muscle [START_REF] Yin | Satellite Cells and the Muscle Stem Cell Niche[END_REF]. [START_REF] Urbán | Stem cell quiescence: the challenging path to activation[END_REF].

Due to their low numbers in the tissue, adult quiescent stem cells are complicated to identify. Several methods have been however optimized to identify these cells. Quiescent cells are known by the absence of cell cycle markers such as Ki67, PCNA and the phospho-histone H3 [START_REF] Gerdes | Immunobiochemical and molecular biologic characterization of the cell proliferation-associated nuclear antigen that is defined by monoclonal antibody Ki-67[END_REF]. Quiescent cells are also characterized by their low capacity of label uptake. Pulses with nuclear analogs such as tritiated thymidine , 5-bromo 20deoxyuridine (BrdU), or 5-ethynyl-20 -deoxyuridine (EdU), will only label actively proliferating cells [START_REF] Rodriguez | Identifying Quiescent Stem Cells in Hair Follicles[END_REF], and spare quiescent cells. Furthermore, quiescent cells are known for their unreplicated genomes and low RNA content due to their lower transcriptional activity compared to activated cells. The cells being in the G0 phase, have unreplicated genome and therefore one copy of their DNA. This is how, coupling DNA and RNA measurement for detecting cells with both low RNA and DNA content is a method to identify quiescent stem cells [START_REF] Gothot | Functional heterogeneity of human CD34(+) cells isolated in subcompartments of the G0 /G1 phase of the cell cycle[END_REF].

In addition to their low RNA content compared to activated cells, quiescent stem cells across tissues present several common features such as higher translational repression and therefore lower protein synthesis [START_REF] Signer | Haematopoietic stem cells require a highly regulated protein synthesis rate[END_REF] [START_REF] Llorens-Bobadilla | Single-Cell Transcriptomics Reveals a Population of Dormant Neural Stem Cells that Become Activated upon Brain Injury[END_REF] [START_REF] Zismanov | Phosphorylation of eIF2α Is a Translational Control Mechanism Regulating Muscle Stem Cell Quiescence and Self-Renewal[END_REF]. Quiescent stem cells are also known to rely on different metabolic pathways compared to their activated counterparts. For example, neural and hematopoietic stem cells use glycolysis and fatty acid oxidation during quiescence and switch to oxidative phosphorylation upon activation [START_REF] Shin | Single-Cell RNA-Seq with Waterfall Reveals Molecular Cascades underlying Adult Neurogenesis[END_REF][START_REF] Simsek | The Distinct Metabolic Profile of Hematopoietic Stem Cells Reflects Their Location in a Hypoxic Niche[END_REF], MuSCs on the other hand switch from fatty acid oxidation to glycolysis when activated [START_REF] Ryall | The NAD+-Dependent SIRT1 Deacetylase Translates a Metabolic Switch into Regulatory Epigenetics in Skeletal Muscle Stem Cells[END_REF]. In addition, it has been show that quiescent stem cells rely on autophagy to eliminate unwanted cellular components and prevent their accumulation [START_REF] Guan | Autophagy in stem cells[END_REF]. Finally, even though quiescent stem cells can reside in this state for a long time, they are equipped to be rapidly activated when stimulated to do so, as these cells are epigenetically poised, and their epigenome can be rapidly modified to allow the transcription of genes required for their activation and differentiation [START_REF] Lee | Signalling couples hair follicle stem cell quiescence with reduced histone H3 K4/K9/K27me3 for proper tissue homeostasis[END_REF].

Adult stem cells usually reside in a very specific microenvironment or niche. The interaction between stem cells and the different components of their microenvironment plays an essential role in the quiescence maintenance of these cells. This niche is mainly composed of the stem cell itself, neighboring cells that interact with the stem cell, and soluble factors coming from the blood or the extracellular matrix (ECM). These factors can also provide mechanical signals to the cells [START_REF] Maul | Mechanical stimuli differentially control stem cell behavior: morphology, proliferation, and differentiation[END_REF].

As previously stated, quiescence is an active and complex state, which brings forward the question of why cells go a long way to stay quiescent. Several studies have been published

to answer this dilemma. Studies on quiescence performed in yeast and cellular cell lines proved its role as a protective cellular mechanism to endure stress events and prolong the lifespan of a cell. Similarly, adult stem cells are maintained in a quiescent state as a protective mechanism against stress for their long-term maintenance. As a matter of fact, it has been shown that quiescence maintenance is essential for tissue homeostasis and proper regeneration throughout the lifespan of the organism. For example, loss of hematopoietic stem cells (HSCs) quiescence leads to an overexpansion of progenitor cells, an impairment in the adaptive immune response as well as myeloproliferative diseases [START_REF] Orford | Deconstructing stem cell self-renewal: genetic insights into cell-cycle regulation[END_REF]. Moreover, loss of quiescence of adult muscle stem cells (MuSCs) results in impaired muscle regeneration following injury and the loss of the stem cell pool [START_REF] Abou-Khalil | Autocrine and paracrine angiopoietin 1/Tie-2 signaling promotes muscle satellite cell self-renewal[END_REF] (Mourikis et al., 2012) [START_REF] Rozo | Targeting β1-integrin signaling enhances regeneration in aged and dystrophic muscle in mice[END_REF] (Zismanov et al., 2016, p. 2) [START_REF] Goel | Niche Cadherins Control the Quiescence-to-Activation Transition in Muscle Stem Cells[END_REF] (Baghdadi et al., 2018a) (Baghdadi et al., 2018b) [START_REF] Low | Delta-Like 4 Activates Notch 3 to Regulate Self-Renewal in Skeletal Muscle Stem Cells[END_REF] (Eliazer et al., 2019, p. 4). In the light of these findings, the importance of quiescence maintenance and the proper activation of adult stem cells at the right time with the right stimuli has become very evident.

To further highlight the complexity of quiescence in adult stem cells, the notion of quiescence depth has emerged in many tissues. For example, in muscle stem cells, it has been recently shown that fasting can induce a deep quiescent state of the cells with a slower ability to activate [START_REF] Benjamin | Fasting induces a highly resilient deep quiescent state in muscle stem cells via ketone body signaling[END_REF]. Moreover, this concept was already described for the hematopoietic system, as two distinct populations of HSCs have been identified, the long term LT-HSCs, known for their deep quiescent state, their very high regeneration potential but low turnover, and the short term ST-HSCs that are more primed for activation and are the main source of blood turnover throughout the lifetime of an organism [START_REF] Wilson | Hematopoietic Stem Cells Reversibly Switch from Dormancy to Self-Renewal during Homeostasis and Repair[END_REF].

Quiescent cells upon activation are characterized by a longer G1 phase compared to cells that are already cycling. A lot of studies have tried to understand why these cells take so long to activate. One of the hypotheses is that this long G1 phase is due to the absence of proteins responsible for creating the origins for DNA replication from the chromatin of quiescent cells. This theory is extensively explained in this review [START_REF] Coller | What's taking so long? S-phase entry from quiescence versus proliferation[END_REF]. One can speculate that quiescent stem cells, other than the need to prepare for DNA replication and the subsequent cell division, also need to prepare to make important fate decisions for commitment, differentiation, and self-renewal, which might also explain their need to go into this long G1 phase.

Early response to stress

Cells have acquired the ability to respond in several ways upon stress stimuli, depending on the type of stress, its amplitude, as well as its duration. This response ranges from a protective reaction to a destructive one that will eventually lead to cell death. Ultimately, pathways that play important roles in cell fate decision are highly conserved in cellular response to stress and determine the outcome of the induced stress [START_REF] Fulda | Cellular Stress Responses: Cell Survival and Cell Death[END_REF].

In response to a wide range of cellular stimuli, a handful of genes (about a hundred), called immediate early genes (IEGs), are induced within minutes. The IEGs can be transcribed very rapidly even in the absence of protein synthesis, suggesting that proteins required for their activation already exist in the cells [START_REF] Herschman | Primary response genes induced by growth factors and tumor promoters[END_REF]. These genes, encoding transcription factors, were first identified in cells as a response to mitogen exposure and are known for their role in cell cycle regulation [START_REF] Greenberg | Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene[END_REF]. These transcription factors, will in turn, induce the expression of a wider range of genes known as secondary response genes (SRGs) [START_REF] Herschman | Primary response genes induced by growth factors and tumor promoters[END_REF]. Examples of IEGs include Fos and Jun, also known as the AP-1 complex, which are one of the most studied and well characterized IEGs. Other IEGs include Egr1, cMyc, and Ier5 [START_REF] Lee | Activation and Repression of Cellular Immediate Early Genes by Serum Response Factor Cofactors[END_REF].

The kinetics of IEGs have been studied in response to growth factor stimulation.

Interestingly, these genes are upregulated in the first 20 minutes of growth factors stimulation and are highly dynamic in the first 4 hours following stimulation [START_REF] Amit | A module of negative feedback regulators defines growth factor signaling[END_REF]. Fos, Jun, and Egr1 are one of the very first responders with a characteristic bell-shaped expression profile, as they are rapidly downregulated, likely by negative feedback mechanisms. In contrast, other genes, such as Sox13, are upregulated later, after 2 hours of growth factor stimulation (Figure 2). In fact, Fos and Jun were found to be induced in the first 20 min following growth factors stimulation and return to their initial levels after 90 min [START_REF] Greenberg | Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene[END_REF]. It was also shown that the ERK1/2 MAPK pathway is a robustly induced and dynamically regulated pathway in these first hours. Remarkably, diving deeper into the genes identified, it seems that the ERK pathway constitutes a node of feedback regulation upon mitogen stimulation [START_REF] Amit | A module of negative feedback regulators defines growth factor signaling[END_REF]. [START_REF] Amit | A module of negative feedback regulators defines growth factor signaling[END_REF]. The genes were arranged in order of peak expression time. Color-coding of genes is based on their primary role in signaling networks (green, transcription activators; red, transcription repressors).

The main pathways that are activated upon stress or mitogen stimuli include the Rho-Actin, ERK MAPK, P38 MAPK and PI3K pathways (Figure 3) [START_REF] Bahrami | Gene regulation in the immediate-early response process[END_REF]) [START_REF] Kann | An injury-responsive Rac-to-Rho GTPase switch drives activation of muscle stem cells through rapid cytoskeletal remodeling[END_REF]. The Rho-Actin and MAPK pathways are known for activating coregulators of the Serum Response Factor (SRF) already bound to the Serum Response Elements (SRE) of IEGs promoters [START_REF] Treisman | Identification and purification of a polypeptide that binds to the c-fos serum response element[END_REF]. In the MAPK pathway, the kinase cascade phosphorylates Ternary Complex Factors (TCF) such as ELK1 [START_REF] Janknecht | Activation of ternary complex factor Elk-1 by MAP kinases[END_REF]. Upon activation, ELK1 binds to the SRE domains along with SRF and p300 to facilitate transcriptional activation [START_REF] Li | MAP kinase phosphorylation-dependent activation of Elk-1 leads to activation of the coactivator p300[END_REF]. In the RhoA-GTPase pathway, other SRF cofactors are induced, the MKL proteins, MKL1 (MRTF-A, MAL and BSAC) and MKL2 (MRTF-B) [START_REF] Lee | Activation and Repression of Cellular Immediate Early Genes by Serum Response Factor Cofactors[END_REF]. The activation of these cofactors through the Rho-actin pathway causes their translocation to the nucleus where they will form a complex with SRF, resulting in transcriptional activation [START_REF] Fowler | Regulation of Primary Response Genes[END_REF]. The ERK1/2 MAPK pathway ERK1 and ERK2 are a part of the mitogen activated protein kinases (MAPK). This signaling pathway depends majorly on a series of kinases down to ERK1/2, which will in turn phosphorylate context and cell dependent downstream targets [START_REF] Cargnello | Activation and Function of the MAPKs and Their Substrates, the MAPK-Activated Protein Kinases[END_REF].

Generally, the ERK1/2 MAPK pathway is activated by mitogen or growth factor stimulated cell surface receptors [START_REF] Boulton | An Insulin-Stimulated Protein Kinase Similar to Yeast Kinases Involved in Cell Cycle Control[END_REF]. Signaling is transduced by Ras GTPases which will trigger the activation of a series of kinases including ERK1/2. Eventually, ERK1/2 can relocate to diverse compartments of the cell in order to phosphorylate its targets, leading to a cell specific response. Depending on the activated targets, these responses include survival, proliferation, differentiation, migration, metabolism, and growth (Figure 4) [START_REF] Lavoie | ERK signalling: a master regulator of cell behaviour, life and fate[END_REF]. The ERK1/2 MAPK signaling pathway plays a key role in a variety of cellular processes. As a result, it has been the subject of extensive research for decades. For instance, ERK signaling is known for its important role in cell cycle regulation of proliferating cells, as it promotes the G1 -S phase transition [START_REF] Voisin | Genetic Demonstration of a Redundant Role of Extracellular Signal-Regulated Kinase 1 (ERK1) and ERK2 Mitogen-Activated Protein Kinases in Promoting Fibroblast Proliferation[END_REF]. As already mentioned, ERK1/2 also indirectly regulates SRF activation in order to induce IEGs expression [START_REF] Hollenhorst | Genomic and Biochemical Insights into the Specificity of ETS Transcription Factors[END_REF]. ERK1/2 can also directly phosphorylate several IEGs such as FOS, JUN and cMYC leading to their stabilization and the inhibition of their degradation [START_REF] Sears | Multiple Rasdependent phosphorylation pathways regulate Myc protein stability[END_REF].

Furthermore, there is a direct regulation of the G1 -S transition by ERK1/2 as it can activate the SRG Cyclin D1 (CCND1), which has several IEGs binding sites on its promoter [START_REF] Lavoie | Cyclin D1 Expression Is Regulated Positively by the p42/p44MAPK and Negatively by the p38/HOGMAPK Pathway[END_REF] [START_REF] Klein | Transcriptional regulation of the cyclin D1 gene at a glance[END_REF]. Upon Cyclin D1 induction, the cyclin-D-cyclindependent kinase 4 and 6 (CDK4 and CDK6) complexes will phosphorylate the retinoblastoma associated protein (RB) which will in turn depress E2F transcription factors resulting in Cyclin E induction. Finally, the Cyclin E-CDK2 complex will further phosphorylate RB, thus further depressing E2F resulting in an irreversible entry into the S phase [START_REF] Bertoli | Control of cell cycle transcription during G1 and S phases[END_REF].

Furthermore, cells transitioning from the G1 to the S phase are subject to a cell size checkpoint.

During the G1 phase, cells are increasing in size, which reflects an increase in biogenesis such as an increase in RNA and protein production. ERK1/2 is a known regulator of biogenesis, by inducing the transcriptional and translational machinery through several pathways [START_REF] Lavoie | ERK signalling: a master regulator of cell behaviour, life and fate[END_REF].

Another role of ERK1/2 is promoting cell survival, as it acts as a pro-survival factor in most cells by counteracting apoptosis [START_REF] Berra | The Activation of p38 and Apoptosis by the Inhibition of Erk Is Antagonized by the Phosphoinositide 3-Kinase/Akt Pathway *[END_REF]. ERK1/2 has been identified as an inhibitor of BIM, a potent proapoptotic protein [START_REF] Hübner | Multisite Phosphorylation Regulates Bim Stability and Apoptotic Activity[END_REF]. ERK1/2 can also induce antiapoptotic factors such as MCL1, BCL2 and BCL1-2L to promote cell survival [START_REF] Lavoie | ERK signalling: a master regulator of cell behaviour, life and fate[END_REF].

The MAPK pathway can also affect cellular metabolism by promoting glycolysis.

Although most studies on the role of ERK1/2 in metabolism have been performed on cancer cells with an ERK1/2 hyperactivity, we can clearly appreciate a role of the MAPK in inducing glycolysis even in highly proliferating cells [START_REF] Falck Miniotis | MEK1/2 Inhibition Decreases Lactate in BRAF-Driven Human Cancer Cells[END_REF]. Notwithstanding that cancer cells have developed different cellular responses compared to stem cells; we can assume that the induction of ERK signaling in stem cells during activation could be driving the observed metabolic switch towards glycolysis.

Furthermore, ERK1/2 activation can have a great impact on cellular motility by affecting the actin cytoskeleton. As a matter of fact, ERK1/2 can directly phosphorylate the myocardin related transcription factors MRTFA and MRTFB, co-factors of SRF, resulting in their translocation to the nucleus and the activation of SRF, which will in turn induce a transcriptional program involved in actin cytoskeleton homeostasis (Panayiotou et al., n.d.). In parallel, ERK1/2 can regulated cell migration and proliferation by mediating the transduction of mechanical stimuli [START_REF] Haroon | Myofiber stretch induces tensile and shear deformation of muscle stem cells in their native niche[END_REF].

Finally, the MAPK pathway is very well studied for its role in differentiation, primarily in ESCs results in the translocation of the former into the cytoplasm where it will be degraded [START_REF] Dhaliwal | KLF4 Nuclear Export Requires ERK Activation and Initiates Exit from Naive Pluripotency[END_REF]). An even earlier role for ERK1/2 in ESCs differentiation has been identified, through which ERK1/2 drives epigenetic modifications through the transcriptional coactivator MED24 resulting in a more primed epigenetic landscape to promote ESCs differentiation [START_REF] Hamilton | Dynamic lineage priming is driven via direct enhancer regulation by ERK[END_REF].

The polyamine synthesis pathway

Another mitogenic and stress induced pathway is the polyamine synthesis pathway [START_REF] Bachrach | Polyamines: New Cues in Cellular Signal Transduction[END_REF]) [START_REF] Kucharzewska | The polyamines regulate endothelial cell survival during hypoxic stress through PI3K/AKT and MCL-1[END_REF] [START_REF] Zahedi | Activation of endoplasmic reticulum stress response by enhanced polyamine catabolism is important in the mediation of cisplatin-induced acute kidney injury[END_REF]. Interestingly, this pathway has also been shown to be activated upon dissociation in MuSCs (Machado et al., 2017). Polyamines are polycationic alkylamines, commonly found in cells, the most common ones being putrescine, spermine and spermidine. These positively charged molecules interact with negatively charged macromolecules such as DNA, RNA and proteins, acquiring them important roles in cell growth, proliferation, differentiation, migration, gene expression, and biosynthesis. Polyamine regulation is tightly regulated by a feedback mechanism at the transcriptional, translational and post-translational level and their presence is ubiquitous and vital in all living cells [START_REF] Li | Polyamines and related signaling pathways in cancer[END_REF].

During the urea cycle, arginine is catalyzed by arginase to produce ornithine, the substrate for polyamine synthesis. The polyamine synthesis pathway is then triggered by the decarboxylation of ornithine by ornithine decarboxylase (ODC1) to generate putrescine [START_REF] Pegg | Regulation of Ornithine Decarboxylase *[END_REF], whose levels are adjusted by the ornithine decarboxylase Antizyme Inhibitor 1, AZIN1 (Qiu et al., 2017). Following that, putrescine is converted to spermidine, and then to spermine by spermidine synthase (SPDSY) and spermine synthase (SPMSY), respectively [START_REF] Ikeguchi | Aminopropyltransferases: Function, Structure and Genetics[END_REF].

Interestingly, studies have shown the induction of Odc1 by the ERK1/2 MAPK pathway as well as several IEGs, such as the AP-1 complex (FOS and JUN) and MYC [START_REF] Shantz | Transcriptional and translational control of ornithine decarboxylase during Ras transformation[END_REF]) [START_REF] Chaturvedi | Arginine and polyamines in Helicobacter pylori-induced immune dysregulation and gastric carcinogenesis[END_REF][START_REF] Bello-Fernandez | The ornithine decarboxylase gene is a transcriptional target of c-Myc[END_REF]. Furthermore, Odc1 can be induced by mTORC and AKT signaling pathways, all of which are known to be induced upon stress stimuli (Ye et al., 2019, p. 1) [START_REF] Ray | Interaction of polyamines and mTOR signaling in the synthesis of antizyme (AZ)[END_REF].

A unique molecule that is synthesized by this pathway is hypusine. Hypusine is generated in 2 steps: the first one is catalyzed by deoxyhypusine synthase (DHPS) that cleaves the 4-aminobuthyl of spermidine and transfers it to a very specific lysine residue of the eIF5a precursor protein to form deoxyhypusine, which is then hydroxylated by deoxyhypusine hydroxylase (DOHH) to form hypusine in eIF5a. Hypusination is a unique posttranslational modification of eIF5A that is required for its activity (Figure 5) [START_REF] Park | Hypusine, a polyamine-derived amino acid critical for eukaryotic translation[END_REF].

The function of eIF5a is not for translation initiation as its name indicates, but to promote translation elongation by relieving ribosome stalling, facilitating translation termination, and the formation of stress granules and processing bodies. Both these structures are formed in the cytosol, containing proteins and mRNA that appear when the cells are exposed to stress, acting as safeguards for these macromolecules from this stress [START_REF] Park | Post-translational formation of hypusine in eIF5A: implications in human neurodevelopment[END_REF]. Interestingly, eIF5a knock out leads to embryonic lethality in mice [START_REF] Abbruzzese | Deoxyhypusine hydroxylase from rat testis. Partial purification and characterization[END_REF] and the heterozygous eIF5a germline causes disorders in humans [START_REF] Faundes | Impaired eIF5A function causes a Mendelian disorder that is partially rescued in model systems by spermidine[END_REF] demonstrating its important role in development and disease.

Uncovering a new activation state of quiescent stem cells

Due to the increasing interest in stem cells in general, and more specifically in quiescent stem cells, several isolation techniques from various tissues have been optimized to isolate those cells in order to study them in their in vivo quiescent state. However, applying these techniques requires removing the stem cells from their niche. Furthermore, all these protocols involve mechanical and/or enzymatic steps, sometimes, for a significant amount of time, methods that have been shown to stress the cells and lead to their activation and the loss of their quiescence (Figure 6) (Machado et al., 2017) (Lacar et al., 2016) (van Velthoven et al., 2017) (van den Brink et al., 2017) (Adam et al., 2017) [START_REF] Marsh | Dissection of artifactual and confounding glial signatures by single-cell sequencing of mouse and human brain[END_REF]. Interestingly, this response to dissociation that was identified in several tissues, included the induction of several pathways, like ERK1/2 and p38 MAPK, as well as genes involved in cytoskeletal organization, the polyamine synthesis pathway among many others (Machado et al., 2017). However, whether this response was an artefact of the dissociation method or if it was important for cellular activation was not addressed. ( [START_REF] Marsh | Dissection of artifactual and confounding glial signatures by single-cell sequencing of mouse and human brain[END_REF].

To capture the in vivo molecular profile of cells, several protocols have been optimized

to circumvent dissociation induced changes, all of which have their advantages and weaknesses (Machado et al., 2021).

Chapter 2: Skeletal muscle stem cells during quiescence and regeneration

The skeletal muscle tissue

The skeletal muscle is one of the most multifunctional tissues of the body and contributes to several functions such as movement, posture, basal metabolism, and maintenance of core temperature. Each muscle is surrounded by connective tissue called epimysium. The muscle fibers are arranged in bundles and each of these bundles is surrounded by another connective tissue called the perimysium. Within these bundles, the muscle fibers are separated by the endomysium, or the basal lamina. These muscle fibers are post mitotic multinucleated cells that originate from the fusion of several muscle progenitors (Figure 7) [START_REF] Frontera | Skeletal Muscle: A Brief Review of Structure and Function[END_REF]. Interestingly, muscle fibers exist in various types, that have different biochemical, mechanical, and metabolic phenotypes, which also reflects the heterogeneity between the different muscles [START_REF] Schiaffino | Fiber Types in Mammalian Skeletal Muscles[END_REF]. Recent studies also showed heterogeneity between myonuclei in terms of gene expression in adult mice (Dos Santos et al., 2020) [START_REF] Kim | Single-nucleus transcriptomics reveals functional compartmentalization in syncytial skeletal muscle cells[END_REF] [START_REF] Petrany | Single-nucleus RNA-seq identifies transcriptional heterogeneity in multinucleated skeletal myofibers[END_REF].

Even though the muscle fiber is the main cell type of the muscle, several other mononucleated cells exist in the muscle which can be mainly found in the different connective tissues. For example, fibroblasts, mesenchymal cells, immune cells as well as endothelial cells ensuring the vascularization of the fibers can be found in the muscle. Finally, a small population of muscle stem cells, also called satellite cells, is found between the basal lamina and the fiber, which will be extensively described later.

Figure 7:

The skeletal muscle tissue and its diverse structures [START_REF] Tajbakhsh | Skeletal muscle stem cells in developmental versus regenerative myogenesis[END_REF].

The skeletal muscle cell types

The different cell populations of the skeletal muscle tissue have been long identified and characterized over time. Recent studies using high throughput single cell RNA-seq have confirmed the existence of at least 10 populations in the muscle: muscle stem cells, fibroadipogenic progenitors (FAPs), neutrophils, B cells, T cells, macrophages, endothelial cells, glial cells, smooth muscle cells and tenocytes (Figure 10) (Schaum et al., 2018) (Giordani et al., 2019) (Dell'Orso et al., 2019) [START_REF] Oprescu | Temporal Dynamics and Heterogeneity of Cell Populations during Skeletal Muscle Regeneration[END_REF]. However, these studies rely on single cell dissociation methods that filter out the long myofibers. Recent single nucleus RNA-seq (snRNA-seq) studies managed to preserve the myonuclei for analysis and show that they account for more than half of the total nuclei from the muscle tissue (Dos Santos et al., 2020) [START_REF] Machado | Tissue damage induces a conserved stress response that initiates quiescent muscle stem cell activation[END_REF]. Of note, the percentage of all muscle cells changes during muscle regeneration, as recently shown by scRNA-seq analyses on regenerating muscle (Figure 8).

The skeletal Muscle Stem Cells

The skeletal muscle is a stable tissue in homeostatic conditions; however, it has a great capacity to regenerate following injury or pathological conditions. This high regeneration ability is due to the skeletal muscle stem cells (MuSCs), also called the satellite cells, localized between the sarcolemma of the muscle fiber and the underlying basal lamina [START_REF] Relaix | Perspectives on skeletal muscle stem cells[END_REF]. Following their discovery by [START_REF] Katz | The termination of the afferent nerve fibre in the muscle spindle of the frog[END_REF]then later Mauro in 1961 (Katz, 1961) [START_REF] Mauro | Satellite cell of skeletal muscle fibers[END_REF], they were shown to proliferate following injury in vivo in 1967 [START_REF] Mintz | Normal mammalian muscle differentiation and gene control of isocitrate dehydrogenase synthesis[END_REF]. However, the myogenic potential of these cells remained a matter of controversy for years after their discovery. In 1975, isolated muscle fibers studies showed the ability of these cells to proliferate and form myotubes in culture [START_REF] Bischoff | Regeneration of single skeletal muscle fibers in vitro[END_REF] [START_REF] Konigsberg | The regenerative response of single mature muscle fibers isolated in vitro[END_REF].

Fast forward to 2005, transplantation experiments showed the myogenic potential of these cells in vivo [START_REF] Montarras | Direct isolation of satellite cells for skeletal muscle regeneration[END_REF] [START_REF] Collins | Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche[END_REF]. And then later, serial transplantation of MuSCs proved their ability to self-renew [START_REF] Sacco | Self-renewal and expansion of single transplanted muscle stem cells[END_REF]. A few years later, three different studies showed that conditional genetic ablation of these cells lead to a total failure of muscle regeneration following injuries and thus proving that tMuSCs are indispensable for the muscle regeneration and that they are the bona fide stem cell population of the skeletal muscle tissue (Lepper et al., 2009) [START_REF] Sambasivan | Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration[END_REF])(Murphy et al., 2011).

Several markers have been identified for MuSCs, the main one being the paired homeodomain transcription factor Pax7 (Seale et al., 2000). This factor has been shown to be required for the specification of the muscle lineage during development as well as the regeneration potential of MuSCs in adulthood (Seale et al., 2000). Several other surface markers can be used in combination or alone to identify MuSCs, such as CD34, α7-integrin, VCAM1, Calcitonin receptor (CALCR), c-Met, and Syndecan4 [START_REF] Zhou | A Long Journey before Cycling: Regulation of Quiescence Exit in Adult Muscle Satellite Cells[END_REF].

The myogenic program

In response to several stimuli, MuSCs have the fascinating ability to trigger a set of tightly organized events that allow the proper activation, proliferation, differentiation, and fusion or self-renewal of these cells. All these events are grouped in what is called the myogenic program. As already mentioned, MuSCs are mitotically quiescent during homeostasis. Upon activation, they undergo a very long G1 phase of about 35 hours, then start proliferation at a rate of 10 hours per cell cycle [START_REF] Rocheteau | A subpopulation of adult skeletal muscle stem cells retains all template DNA strands after cell division[END_REF]. Machado et al. managed to identify an early activation step happening during muscle dissociation including the upregulation of stress response signals such as the MAPK pathway among others (Machado et al., 2017). Following quiescence exit, the myogenic program is orchestrated by key transcription factors. Other than PAX7, a family of basic helix-loop-helix (bHLH) factors also called MRFs are responsible for the myogenic commitment of activated MuSCs. Myf5 and MyoD1 are the MRFs expressed in adult muscle stem and progenitor cells [START_REF] Tajbakhsh | Skeletal muscle stem cells in developmental versus regenerative myogenesis[END_REF].

MuSCs express Myf5, but its expression increases upon their activation by the release of the Myf5 mRNA from mRNPs [START_REF] Crist | Muscle satellite cells are primed for myogenesis but maintain quiescence with sequestration of Myf5 mRNA targeted by microRNA-31 in mRNP granules[END_REF]. MyoD1 is also induced upon activation where it can play a context-dependent role in activated myoblasts. Actually, MYOD has been shown to play a dual role in myoblasts, the first by promoting proliferation through the activation of Cdc6 and then later by playing a role in the commitment of cells in the myogenic program [START_REF] Zhang | Activation of Cdc6 by MyoD is associated with the expansion of quiescent myogenic satellite cells[END_REF] [START_REF] Relaix | Perspectives on skeletal muscle stem cells[END_REF]. Following the amplification phase, myoblasts will express the muscle differentiation factor Myogenin and myocytes will exit the cell cycle terminally and fuse to form the myofibers (Figure 9) [START_REF] Schmidt | Adult stem cells at work: regenerating skeletal muscle[END_REF]. The fusion process is orchestrated by different factors such as Myomaker and Minion for cell-cell hemifusion and Myomerger for the creation of fusion pores [START_REF] Millay | Myomaker is a membrane activator of myoblast fusion and muscle formation[END_REF]) [START_REF] Quinn | Myomerger induces fusion of non-fusogenic cells and is required for skeletal muscle development[END_REF]) [START_REF] Leikina | Myomaker and Myomerger Work Independently to Control Distinct Steps of Membrane Remodeling during Myoblast Fusion[END_REF], this is followed by the expression of the Myosin Heavy Chain (MHC) isoforms [START_REF] Schiaffino | Developmental myosins: expression patterns and functional significance[END_REF]. During this process, some of the activated MuSCs will self-renew in order to replenish the stem cells pool. Heavy Chain (MHC) and mature into myofibers. At the same time, a fraction of the activated cells will self-renew in order to replenish the stem cell pool. Modified from [START_REF] Schmidt | Adult stem cells at work: regenerating skeletal muscle[END_REF].

Muscle injury and regeneration

The skeletal muscle has an innate capacity to regenerate following any disruption of the tissue integrity. This disruption can be caused by minor events such as physical activity or minor and local injuries, or by major traumas that can affect the entire tissue. Laumonnier et al describe 3 main phases of muscle regeneration: (1,2) the denervation/inflammation phase during which the myofibers become necrotic, and inflammation occurs. (3) The regeneration phase, in this phase the damaged tissue is phagocytosed, the MuSCs are activated and myofibers are regenerated. [START_REF] Mondor | Service d'histologie, F-94010 Creteil, France * equally contributed to this work[END_REF]5) The remodeling phase, where newly formed myofibers mature and the muscle regains its functional capacity. Fibrosis and tissue scarring are also formed. All these phases, even though distinguishable are overlapping during the regeneration process [START_REF] Laumonier | Muscle injuries and strategies for improving their repair[END_REF] (Figure 10A).

Several injury models are used to study injury. The most common ones are mechanical injury (freezing injury), myotoxins (netotoxin and cardiotoxin), and chemical injury (BaCl2) (Figure 10B). All these models have different intensities leading to a significant heterogeneity in the regeneration process. However, the aforementioned regeneration phases are conserved during the regeneration process but with different kinetics and efficiencies [START_REF] Hardy | Comparative Study of Injury Models for Studying Muscle Regeneration in Mice[END_REF].

Of note, until recently, it remained unclear whether MuSCs exhibit the same transcriptional changes following tissue injury by any of these models compared to their response to enzymatic dissociation ex vivo. during regeneration [START_REF] Bentzinger | Cellular dynamics in the muscle satellite cell niche[END_REF]. B) Different injury models leading to a heterogeneity in the regeneration process (Baghdadi and Tajbakhsh, 2018).

Even though MuSCs are indispensable for the proper regeneration of the muscle tissue, they are not the only contributors. During regeneration, several cell types are recruited to the damage site and assist the stem cells to ensure the return to homeostasis of the skeletal muscle tissue (Figure 11 A-B).

Muscle injury can trigger a very orchestrated inflammatory response in the aim to remove the damaged tissue and ultimately restore its homeostasis. Neutrophils are the first immune cells to be recruited to the damaged area as they appear within 3 hours post injury [START_REF] Heredia | Type 2 innate signals stimulate fibro/adipogenic progenitors to facilitate muscle regeneration[END_REF]. Their role is to clear the damaged area of the necrotic tissue. At 3 days post injury, the proinflammatory M1 macrophages are recruited and secrete several cytokines such as TNFα, INFα, and IL6 that promote myoblasts proliferation [START_REF] Bencze | Proinflammatory macrophages enhance the regenerative capacity of human myoblasts by modifying their kinetics of proliferation and differentiation[END_REF]. M1 macrophages, then switch to an anti-inflammatory phenotype and become M2 macrophages characterized by the production of other cytokines. This anti-inflammatory switch promotes differentiation and fusion of myocytes [START_REF] Arnold | Inflammatory monocytes recruited after skeletal muscle injury switch into antiinflammatory macrophages to support myogenesis[END_REF].

Fibro -adipogenic progenitors (FAPs) also activate and proliferate following injury and most of them are eliminated by apoptosis and go back to their basal levels [START_REF] Lemos | Nilotinib reduces muscle fibrosis in chronic muscle injury by promoting TNF-mediated apoptosis of fibro/adipogenic progenitors[END_REF].

These cells have been shown to be a source of factors that promote differentiation of myoblasts [START_REF] Joe | Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis[END_REF]. Furthermore, ablation of this population results in impaired muscle regeneration proving their important role following injury [START_REF] Wosczyna | Mesenchymal Stromal Cells Are Required for Regeneration and Homeostatic Maintenance of Skeletal Muscle[END_REF].

Endothelial cells also promote the proliferation of myoblasts by the secretion of several factors as it was shown by co-culture experiments [START_REF] Christov | Muscle satellite cells and endothelial cells: close neighbors and privileged partners[END_REF]. Reversibly, differentiating myoblasts stimulate angiogenesis through VEGF [START_REF] Rhoads | Satellite cell-mediated angiogenesis in vitro coincides with a functional hypoxia-inducible factor pathway[END_REF].

Moreover, endothelial cells secrete several factors during regeneration that promote angiogenesis and thus muscle repair [START_REF] Abou-Khalil | Regulation of myogenic stem cell behavior by vessel cells: the "ménage à trois" of satellite cells, periendothelial cells and endothelial cells[END_REF]. Periendothelial cells also play a role in muscle repair by stabilizing the regenerated vessels and promoting the self-renewal of myoblasts through ANGPT1 [START_REF] Bentzinger | Cellular dynamics in the muscle satellite cell niche[END_REF]. Pericytes are also involved in blood vessel growth, homeostasis and permeability [START_REF] Armulik | Pericytes: developmental, physiological, and pathological perspectives, problems, and promises[END_REF].

In conclusion, following injury, cells in the muscle tissue, all activate in a highly coordinated manner, all with very specific roles at very specific time points to ensure the proper regeneration and return to homeostasis of the tissue. It's interesting to know how these cells respond to the damage triggered by the injury and how this response differs from one cell type to the other. regeneration (Baghdadi and Tajbakhsh, 2018).

Muscle stem cells heterogeneity

Although all MuSCs are characterized by the expression of PAX7 and their ability to regenerate the muscle and self-renew, several studies have shown the existence of several subpopulations of MuSCs in the adult. MuSCs from different subpopulations were found to be different, not only in their gene expression profiles, but also in their regeneration and resistance potential.

Indeed, MuSCs have different developmental origins which is one of the sources of their heterogeneity. For instance, trunk, pharyngeal arches, extraocular and head MuSCs all have different origins (Sambasivan et al., 2009) [START_REF] Harel | Distinct origins and genetic programs of head muscle satellite cells[END_REF]. Consistently, this difference in origin and thus difference in transcriptional profiles have been shown to contribute to a difference in the regeneration potential of the cells as it has been shown that MuSCs from the extraocular muscle (EOM) have a better performance in transplantation experiments compared to their counterparts from the Tibialis Anterior (TA) [START_REF] Evano | Transcriptome and epigenome diversity and plasticity of muscle stem cells following transplantation[END_REF].

MuSCs can also differentially express PAX3. Interestingly, PAX3 + cells are more abundant in some muscles compared to others, as they exist in high numbers in the diaphragm and biceps for example and are very rare in the TA [START_REF] Relaix | Perspectives on skeletal muscle stem cells[END_REF]. Other than PAX3 expression, those cells are known to have higher survival potential compared to the PAX3 - cells as they have demonstrated their higher capacity to resist to environmental stress [START_REF] Der Vartanian | PAX3 Confers Functional Heterogeneity in Skeletal Muscle Stem Cell Responses to Environmental Stress[END_REF]) [START_REF] Scaramozza | Lineage Tracing Reveals a Subset of Reserve Muscle Stem Cells Capable of Clonal Expansion under Stress[END_REF].

Even though all adult MuSCs express Pax7, studies have shown that the level of Pax7 expression varies between MuSCs. Two different MuSCs populations have been identified.

The Pax7 high population of MuSCs that exhibits a more dormant state, a slower cell cycle entry, and a lower metabolic activity compared to the Pax7 low population [START_REF] Rocheteau | A subpopulation of adult skeletal muscle stem cells retains all template DNA strands after cell division[END_REF].

Consistently, a more recent single cell RNA-seq (scRNA-seq) study has identified a quiescent MuSCs population (cQ) with a high Pax7 expression, and another population that is more activated (eA) expressing biogenesis related genes (Dell'Orso et al., 2019). Another study categorized MuSCs based on the expression of CD34, as they have shown that CD34 high

MuSCs have stemness properties, whereas CD34 low MuSCs are more primed and are committed to myogenic differentiation [START_REF] García-Prat | FoxO maintains a genuine muscle stem-cell quiescent state until geriatric age[END_REF].

A study has also shown that around 90% of MuSCs express Myf5 while 10% never expressed Myf5 throughout their lifespan. The difference in Myf5 expression attributes to the fate decision of the cells, as Myf5 + MuSCs are more committed to myogenic differentiation whereas Myf5 -are more likely to self-renew [START_REF] Kuang | Asymmetric self-renewal and commitment of satellite stem cells in muscle[END_REF]. Furthermore, a histone H2B pulse chasing system allowed the identification of two distinct MuSCs populations, a label retaining LRC and a non-label retaining non-LRC population, the former inclined to self-renew while the latter is committed to differentiation [START_REF] Chakkalakal | Early forming label-retaining muscle stem cells require p27kip1 for maintenance of the primitive state[END_REF] Indeed, an obvious source of heterogeneity between MuSCs is their decision to differentiate or self-renew. Cell fate decision has been a source of controversy for a long time in the field of stem cells and several hypotheses exist to try to explain it. The decision of a cell to differentiate or self-renew is believed to be followed by a symmetric or asymmetric division. Several studies have been dedicated to explaining this phenomenon, as it was reported that the division of activated MuSCs can be planar, parallel to the myofiber, or apical-basal, perpendicular to the fiber which will lead to the asymmetric division of the cell [START_REF] Kuang | Asymmetric self-renewal and commitment of satellite stem cells in muscle[END_REF] (Le [START_REF] Grand | Wnt7a activates the planar cell polarity pathway to drive the symmetric expansion of satellite stem cells[END_REF] .

However, this became controversial after a study that was published later showing that cells mainly follow the planar model in vivo [START_REF] Webster | Intravital Imaging Reveals Ghost Fibers as Architectural Units Guiding Myogenic Progenitors during Regeneration[END_REF]. Furthermore, several studies have reported that MuSCs do not randomly segregate their DNA template and that the cell that receives the replicative strand exhibits higher stemness [START_REF] Shinin | Asymmetric division and cosegregation of template DNA strands in adult muscle satellite cells[END_REF]) [START_REF] Conboy | High incidence of non-random template strand segregation and asymmetric fate determination in dividing stem cells and their progeny[END_REF]) [START_REF] Rocheteau | A subpopulation of adult skeletal muscle stem cells retains all template DNA strands after cell division[END_REF]. It was also shown that the Pax7 high population already mentioned, is the population that conserves the replicative strand [START_REF] Rocheteau | A subpopulation of adult skeletal muscle stem cells retains all template DNA strands after cell division[END_REF]. The ability of cells to do this existed ever since the 70s and was called the immortal strand hypothesis [START_REF] Cairns | Mutation selection and the natural history of cancer[END_REF]). Yet, until today it still raises a lot of controversies and was not always supported [START_REF] Tomasetti | The (not so) Immortal Strand Hypothesis[END_REF].

Briefly, even though all MuSCs are unipotent, several data suggest a possible heterogeneity between these cells providing them with a functional diversity. It is important to mention that the relationship between these different populations was never systematically addressed and remains unclear. Furthermore, we still do not know whether some of these differences are random differences, resulting from stochastic fluctuations in biological states that have been shown to be involved in the fate decision of a cell [START_REF] Zechner | Stochasticity and determinism in cell fate decisions[END_REF]. More importantly, even though these different subpopulations might have different activation kinetics or choose different fates, it remains unclear whether they are different in terms of response to stress stimuli and whether they all follow the same activation path or have different ones based on their fate choice.

Muscle stem cells quiescence

The majority of MuSCs exist in a quiescent G0 state in the muscle during homeostasis and only get activated in response to changes in their microenvironment caused by injury or in pathological conditions. These cells share common features with other adult quiescent stem cells such as HSCs and neural stem cells (NSCs) already cited in Chapter 1. Nevertheless, due to the development of lineage tracing tools including fluorescent reporters [START_REF] Bosnakovski | Prospective isolation of skeletal muscle stem cells with a Pax7 reporter[END_REF]) (Sambasivan et al., 2009) [START_REF] Tichy | A robust Pax7EGFP mouse that enables the visualization of dynamic behaviors of muscle stem cells[END_REF] and the Cre-lox system (Lepper et al., 2009) (Murphy et al., 2011) (Mourikis et al., 2012), as well as high throughput analyses, specific mechanisms that regulate MuSCs quiescence and activation have been identified.

Several factors from the MuSCs niche have been identified in regulating MuSCs quiescence summarized in Figure 12 and Table 1. MuSCs interact with several components of their niche to maintain their quiescence. This interaction can be a direct, cell-cell interaction such as Delta/Jagged-Notch and N-Cadherin-M-Cadehrin between the myofiber and MuSC respectively. MuSCs can also interact with other components of the niche such as laminin from the basal lamina through integrins. This interaction can also be achieved through signaling pathways such as the FGF2-FGFR Quiescence maintenance, as explained in the first chapter, is very intricate. This is further highlighted in MuSCs as several pathways play essential roles in their quiescence maintenance. The Notch signaling pathway for example, is one of the main pathways required for this maintenance. Notch is a transmembrane plasma receptor (Notch 1,2,3,4) with transmembrane plasma ligands (DLL1, DLL4, JAG1, JAG2), indicating that this pathway requires cell-cell communication for its activity [START_REF] Gioftsidi | The Notch signaling network in muscle stem cells during development, homeostasis, and disease[END_REF]. Following activation, the intracellular domain of Notch (NICD) is cleaved and translocated into the nucleus where it will act as a coactivator along with RBPJ in order to stabilize its binding to the DNA of target genes and thus induce their expression (Figure 13A) [START_REF] Fortini | The suppressor of hairless protein participates in notch receptor signaling[END_REF]) [START_REF] Castel | Dynamic binding of RBPJ is determined by Notch signaling status[END_REF]. Some of these direct targets are highly conserved, the basic-helix-loophelix transcriptional repressors, HeyL, Hey1, and Hes1 [START_REF] Fukada | Hesr1 and Hesr3 are essential to generate undifferentiated quiescent satellite cells and to maintain satellite cell numbers[END_REF] (Mourikis et al., 2012a) [START_REF] Buas | The Notch effector Hey1 associates with myogenic target genes to repress myogenesis[END_REF]. [START_REF] Braune | Notch -a Goldilocks Signaling Pathway in Disease and Cancer Therapy[END_REF]. B) Notch signaling pathway in quiescence maintenance of MuSCs [START_REF] Gioftsidi | The Notch signaling network in muscle stem cells during development, homeostasis, and disease[END_REF].

The Dll ligand believed to be coming from the myofiber binds to the Notch ligand on MuSCs [START_REF] Low | Delta-Like 4 Activates Notch 3 to Regulate Self-Renewal in Skeletal Muscle Stem Cells[END_REF] . Changes in the microenvironment causing the loss of contact between the ligand and its receptor can lead to the downregulation of Notch and the quiescence loss of MuSCs and their subsequent activation, highlighting, once again, the role of the niche in the quiescence maintenance of cells. The specific deletion of Rbpj from MuSCs leads to the quiescence loss of these cells and their differentiation at the expense of self-renewal (Mourikis et al., 2012). Consistently, MuSCs with a double deletion of Hey1 and HeyL have lower numbers of quiescent MuSCs [START_REF] Fukada | Hesr1 and Hesr3 are essential to generate undifferentiated quiescent satellite cells and to maintain satellite cell numbers[END_REF]. Several mechanisms have been elucidated to understand how Notch maintains MuSCs quiescence; Notch has been shown to directly induce Pax7. Furthermore, Notch signaling has been suggested to inhibit differentiation by the direct inhibition of Myog through Hey1 (Bua et al., 2010). Another mechanism of quiescence maintenance through Notch is cell autonomous (Baghdadi et al., 2018), through which Notch induces the expression of ColV, which will in turn induce the activation of CALCR required for MuSCs quiescence maintenance through the cAMP-PKA pathway. Furthermore, the CalcR deletion from MuSCs leads to their cell death by apoptosis (Yamaguchi et al., 2015). Notch can also promote the survival of MuSCs through p53 inhibition [START_REF] Liu | Impaired Notch Signaling Leads to a Decrease in p53 Activity and Mitotic Catastrophe in Aged Muscle Stem Cells[END_REF] (Figure 13B).

The Notch pathway also plays an important role in the myogenic program through HES1 which drives the oscillatory expression of MyoD1 by binding to its regulatory elements. This oscillation of HES1 and MYOD1 is important for the proliferation of myoblasts [START_REF] Lahmann | Oscillations of MyoD and Hes1 proteins regulate the maintenance of activated muscle stem cells[END_REF]. Furthermore, Hes1 oscillation has been shown to be driven by Dll1 and is critical for the cell fate decision of MuSCs and the balance between differentiation and self-renewal [START_REF] Zhang | Oscillations of Delta-like1 regulate the balance between differentiation and maintenance of muscle stem cells[END_REF].

Other transcription factors involved in quiescence maintenance are the FoxO family.

Deletion of FoxO1/FoxO3/FoxO4 led to the spontaneous activation of MuSCs and the loss of their quiescence [START_REF] García-Prat | FoxO maintains a genuine muscle stem-cell quiescent state until geriatric age[END_REF]. Wnt4 and Wnt6 have also been shown to promote MuSCs quiescence [START_REF] Fu | Stem cell activation in skeletal muscle regeneration[END_REF]. Wnt4 derived from myofibers activates the non-canonical RhoA-YAP pathway therefore restricting MuSCs migration [START_REF] Eliazer | Wnt4 from the Niche Controls the Mechano-Properties and Quiescent State of Muscle Stem Cells[END_REF].

Post transcriptional regulation is a key participant in MuSCs quiescence maintenance and their rapid activation upon stimulation. Interestingly, both Muscle Regulatory Factors (MRFs) MyoD1 and Myf5 mRNA exist in quiescent MuSCs but are only translated upon activation. TTP and Staufen, 2 RNA binding proteins, promoting degradation and translation inhibition respectively are responsible for the post transcriptional regulation of MyoD1 and Myf5 [START_REF] Hausburg | Post-transcriptional regulation of satellite cell quiescence by TTP-mediated mRNA decay[END_REF][START_REF] De Morrée | Staufen1 inhibits MyoD translation to actively maintain muscle stem cell quiescence[END_REF]. Furthermore, the microRNA miR31 is involved in Myf5 translation inhibition by trapping Myf5 in messenger ribonucleoprotein (mRNP) and is only released once MuSCs are activated [START_REF] Crist | Muscle satellite cells are primed for myogenesis but maintain quiescence with sequestration of Myf5 mRNA targeted by microRNA-31 in mRNP granules[END_REF]. Pax3, differentially expressed in MuSCs, is also post transcriptionally regulated. This differential expression of PAX3 in different muscles is regulated through U1 nucleolar RNA-mediated alternative mRNA polyadenylation which results in differential regulation by miR-206 (de Morree et al., 2019). Remarkably, the microRNA pathway has been shown to be very important in regulating MuSCs quiescence and activation. Some examples include miR-489, expressed in quiescent MuSCs which is responsible for the inhibition of the splicing factor DEK [START_REF] Cheung | Maintenance of muscle stem-cell quiescence by microRNA-489[END_REF].

Interestingly, DEK overexpression leads to decreased intron retention and impairment in MuSCs quiescence maintenance [START_REF] Yue | Dek Modulates Global Intron Retention during Muscle Stem Cells Quiescence Exit[END_REF]. Another microRNA is the Notch induced miR-708 required for inhibiting MuSCs migration and thus maintaining their quiescence (Baghdadi et al., 2018). Furthermore, conditional knock out of Dicer from MuSCs leads to their spontaneous activation [START_REF] Cheung | Maintenance of muscle stem-cell quiescence by microRNA-489[END_REF], highlighting even more the importance of the microRNA pathway in MuSCs regulation. Additionally, quiescent MuSCs are known to have reduced protein synthesis, ensured through the inhibition by phosphorylation of the translation initiation factor eIF2α at Ser51. Consistently, phospho-eIF2α deficient MuSCs break out of their quiescence and lose their ability to self-renew [START_REF] Zismanov | Phosphorylation of eIF2α Is a Translational Control Mechanism Regulating Muscle Stem Cell Quiescence and Self-Renewal[END_REF]) [START_REF] Gugliuzza | Muscle stem cell adaptations to cellular and environmental stress[END_REF]. Interestingly, the translation of some quiescent MuSCs specific genes is phospho-eIF2α dependent [START_REF] Zismanov | Phosphorylation of eIF2α Is a Translational Control Mechanism Regulating Muscle Stem Cell Quiescence and Self-Renewal[END_REF].

As already mentioned, adult quiescent stem cells including MuSCs are epigenetically poised to allow rapid transcription of proliferation and differentiation genes upon activation.

Therefore, the balance between the expression of different epigenetic modifications is critical for MuSCs quiescence. For instance, H3K27me3, a repressive epigenetic modification, has low expression in MuSCs on the TSS of genes expressed during quiescence and is upregulated upon MuSCs activation and leads to the downregulation of the expression of these genes [START_REF] Liu | Chromatin Modifications as Determinants of Muscle Stem Cell Quiescence and Chronological Aging[END_REF]. Moreover, the permissive histone modification H3K4me3 is expressed in both quiescent and activated MuSCs, as it is deposited on the promoters of IEGs and cell cycle related genes even in quiescent cells in order to allow their rapid expression upon early activation.

Inconsistently, another permissive epigenetic modification, H3K27Ac is downregulated upon MuSCs activation resulting the downregulation of Pax7 and Hes1 (Machado et al., 2017).

Moreover, depletion of Suv4-20h1, an H4K20 dimethyltransferase involved in facultative heterochromatin, reduced the latter formation, the activation of MyoD1 and the loss of MuSCs quiescence [START_REF] Boonsanay | Regulation of Skeletal Muscle Stem Cell Quiescence by Suv4-20h1-Dependent Facultative Heterochromatin Formation[END_REF]. Consistently, deposition of H4K16Ac by SIRT1 on myogenic genes, inhibits their expression in quiescent MuSCs [START_REF] Ryall | The NAD+-Dependent SIRT1 Deacetylase Translates a Metabolic Switch into Regulatory Epigenetics in Skeletal Muscle Stem Cells[END_REF].

Metabolism also plays an essential role in quiescence maintenance of MuSCs. Those cells rely greatly on fatty acid oxidation (FAO) during homeostasis which is underlined by the expression of enzymes involved in FAO (Machado et al., 2017). Indeed, inhibition of FAO in MuSCs results in early differentiation and leads to impaired muscle regeneration [START_REF] Pala | Distinct metabolic states govern skeletal muscle stem cell fates during prenatal and postnatal myogenesis[END_REF]. Upon activation, MuSCs undergo metabolic reprogramming and switch to glycolysis, which will be further expanded later. Furthermore, MuSCs, as other quiescent stem cells, greatly rely on autophagy as an additional source of energy, which is boosted even more upon activation in order to provide the required energy for the cells to properly activate (Relaix et al., 2021).

Muscle stem cells early activation

Several pathways are greatly organized in order to maintain MuSCs quiescent during homeostasis. Remarkably, following stress, changes in the niche, or even internal stimuli can lead to the activation of these cells. MuSCs have acquired the ability to sense those signals and respond rapidly in order to adapt to these changes and subsequently activate. The activation of MuSCs is very highly orchestrated and involves highly dynamic changes from the downregulation of quiescence specific pathways while at the same time the upregulation of activation involved pathways to ensure proper entry of MuSCs into the cell cycle. Several pathways are highly induced upon MuSCs early activation. One of the earliest pathways to be induced upon MuSCs activation are the ERK1/2 and P38 MAPK pathways, as well IEGs. Furthermore, the PI3K-AKT-mTORC pathway is upregulated upon quiescence exit and required for the proper activation of MuSCs. As a matter of fact, PI3K activation is sufficient to activate MuSCs. mTORC1 on one hand, a downstream effector of PI3K is also induced in MuSCs activation and is involved in nucleotide, protein and lipid synthesis while reducing autophagy. This pathway leads to the induction of IEGs such as the AP-1 complex ensuring the proper exit of MuSCs from quiescence and their early activation (Wang et al., 2018) [START_REF] Relaix | Waking up muscle stem cells: PI3K signalling is ringing[END_REF]. mTORC2 on the other hand is involved in cytoskeletal reorganization and cell survival [START_REF] Liu | mTOR at the nexus of nutrition, growth, ageing and disease[END_REF]. Interestingly, overexpression of NICD, the intracellular domain of Notch, prevents depletion of Pten null MuSCs [START_REF] Yue | Pten is necessary for the quiescence and maintenance of adult muscle stem cells[END_REF], indicating a cross talk between these two signaling pathways. The FGF pathway is also induced upon activation and can lead to the activation of the ERK, P38 and PI3K pathways required for lineage progression of MuSCs [START_REF] Pawlikowski | Regulation of skeletal muscle stem cells by fibroblast growth factors[END_REF]. Although some WNT ligands play roles in quiescence maintenance, some others such as WNT1, WNT3a, and WNT5a were shown to promote proliferation [START_REF] Fu | Stem cell activation in skeletal muscle regeneration[END_REF]. Finally, CREB, has been shown to be involved in the entry into the first cell cycle of MuSCs [START_REF] Li | A CREB-MPP7-AMOT Regulatory Axis Controls Muscle Stem Cell Expansion and Self-Renewal Competence[END_REF].

The epigenome of a cell is highly dynamic, especially the one of stem cells in order to allow the rapid transcription of activation genes and the inhibition of quiescence specific genes. Indeed, impairment of the PRC2 complex resulted in impairment in MuSCs proliferation and muscle regeneration [START_REF] Juan | Polycomb EZH2 controls self-renewal and safeguards the transcriptional identity of skeletal muscle stem cells[END_REF]. DNA methylation is also important for MuSCs proliferation through the inhibition of Cdkn1c [START_REF] Naito | Dnmt3a Regulates Proliferation of Muscle Satellite Cells via p57Kip2[END_REF]. And finally, HIRA, a histone chaperone, has been recently shown to be important for MuSCs identity and the regulation of self-renewal by H3.3 and H3K27Ac deposition (Esteves de [START_REF] Esteves De Lima | HIRA stabilizes skeletal muscle lineage identity[END_REF]. Furthermore, a recent comparison between in situ fixed and early activated MuSCs using Hi-C shows major genome and chromatin architectural changes happening during the 2 hours of MuSCS activation [START_REF] Zhao | Multiscale 3D Genome Reorganization during Skeletal Muscle Stem Cell Lineage Progression and Muscle Aging[END_REF].

Quiescent MuSCs use FOA and switch to glycolysis upon activation. Glycolysis is mainly required upon early activation as it provides metabolic intermediates that are required for cellular growth, biosynthesis, and cell cycle re-entry of quiescent cells [START_REF] Ryall | The NAD+-Dependent SIRT1 Deacetylase Translates a Metabolic Switch into Regulatory Epigenetics in Skeletal Muscle Stem Cells[END_REF]) [START_REF] Zhou | Paxbp1 controls a key checkpoint for cell growth and survival during early activation of quiescent muscle satellite cells[END_REF]. Interestingly, the switch from FOA to glycolysis is similar to the Warburg effect described in cancer cells used for enhanced cellular growth of these cells [START_REF] Lunt | Aerobic glycolysis: meeting the metabolic requirements of cell proliferation[END_REF]. Furthermore, activated MuSCs display an increase in the number of mitochondria and OXPHOS resulting in an increase in ROS levels, all required for the proper activation of cells (L'honoré et al., 2018), as it has been shown that blocking glycolysis of the electron transport electron chain can damage the ability of MuSCs to re-enter the cell cycle [START_REF] Yucel | Glucose Metabolism Drives Histone Acetylation Landscape Transitions that Dictate Muscle Stem Cell Function[END_REF].

The complexity of the re-entry of MuSCs into the cell cycle is also evidenced by the duration of the first G1 phase which is about 35 hours, much longer than the duration of the G1 of subsequent cell cycle of proliferating myoblasts [START_REF] Rocheteau | A subpopulation of adult skeletal muscle stem cells retains all template DNA strands after cell division[END_REF], given that MuSCs must undergo all these changes that were just described to ensure their proper activation. The complexity of the entry into the cell cycle of MuSCs is further evidenced by the44hosphtion of a new early activation state that is happening during the 2 hours of MuSCs dissociation. iSifi, an in-situ fixation protocol mentioned in Chapter1, showed that MuSCs undergo transcriptomic modifications during tissue dissociation. Those included the activation of stress pathways such as the ERK and P38 MAPK and the Rho-GTPase pathways, among other essential activation pathways that are already described (Machado et al., 2017).

Interestingly, a very recent study showed a role of the Rho-GTPase pathway in the quiescence to activation transition of MuSCs that drives the retraction of cytoplasmic projections of quiescent MuSCs and the induction of Fos expression [START_REF] Kann | An injury-responsive Rac-to-Rho GTPase switch drives activation of muscle stem cells through rapid cytoskeletal remodeling[END_REF]. Indeed, double knock out of Erk1 and Erk2 from adult MuSCs resulted in the complete depletion of the MuSCs pool upon injury [START_REF] Boyer | Satellite cell depletion in early adulthood attenuates muscular dystrophy pathogenesis[END_REF]. However, the role of the ERK1/2 MAPK pathway in the quiescence exit of MuSCs remains unclear. Of note, a role of ERK1/2 in the G1® S transition has already been described, as mentioned in Chapter 1, however, it is interesting to study its role in the G0®G1®S transition of MuSCs. Moreover, Machado et al. were able to show an induction of IEGs during these 2 hours. The role of these IEGs in MuSCs early activation is not very defined. Indeed, a role of cJun through the PI3K-AKT pathway has been investigated (Wang et al., 2018), as well as a role for Fos in the activation of MuSCs has been shown [START_REF] Almada | FOS licenses early events in stem cell activation driving skeletal muscle regeneration[END_REF].

Objectives

The main goal of my PhD was to study the different pathways that govern the quiescence exit and early activation in MuSCs and their involvement in the cell cycle entry and differentiation of those cells.

Our lab has already optimized a fixation, iSifi, protocol and uncovered the transcriptional changes happening to MuSCs upon tissue dissociation, thus uncovering a new early activation state of those cells (Machado et al., 2017). We therefore hypothesized that these changes are not exclusive to MuSCs and that cells across tissues share a common response to tissue damage. Our goal was first to identify this response.

Focusing on MuSCs as a model of stem cell quiescence, we wanted to study the relevance of this response in the early activation of these cells. We therefore wanted to investigate the role of the ERK1/2 MAPK, Notch, and the polyamine synthesis pathways, during the quiescence exit of MuSCs. We then speculated that by manipulating the pathways that we have identified we can optimize a protocol to induce quiescence of MuSCs in culture.

These results can be found in Part I and a part of them was published in Cell Stem Cell [START_REF] Machado | Tissue damage induces a conserved stress response that initiates quiescent muscle stem cell activation[END_REF].

In Part II of the results, we set to investigate other genes that we have identified during MuSCs early activation. We focused on the Krüppel-like transcription factor Klf4 as it was part of the common stress response we uncovered, and is also known for its role in regulating the cell cycle [START_REF] Ghaleb | Krüppel-like factor 4 (KLF4): What we currently know[END_REF]. We also focused on the Serum Response Factor Srf, as it is known to be a regulator of IEGs [START_REF] Bahrami | Gene regulation in the immediate-early response process[END_REF], also induced very early following the transition of MuSCs from quiescence to activation.

Results

Part I: Tissue damage induces a conserved stress response that initiates quiescent muscle stem cell activation Part II: Investigating the role of Klf4 and Srf in muscle stem cells early activation Part I: Tissue damage induces a conserved stress response that initiates quiescent muscle stem cell activation

Introduction

In all organs, cell plasticity is a fundamental feature of stem cells to respond to injury. Tissue damage causes stem/progenitor cells to activate and proliferate in order to restore homeostasis, as observed in the bone, skin, and skeletal muscle, among other tissues (Fuchs and Blau, 2020) (Jessen et al., 2015). Differentiated cells, such hepatocytes after hepatectomy (Riehle et al., 2011) or neonatal mouse cardiomyocytes (Porrello et al., 2011), can get activated and de-differentiate before eventually proliferating. When cell injury is permanent, however, cell death pathways can be triggered (Rathore et al., 2017). Understanding the features of cells when they exit homeostasis and devising methods for experimental manipulation requires figuring out their early transcriptional roadmap. Despite substantial investigation, the extent, speed, and nature of the early, cell type-specific or conserved responses to tissue damage as they occur in the body are still unknown.

As mentioned in Chapter 1, cellular isolation methods cause major transcriptomic changes to the cells. Several studies have identified these changes in MuSCs (Machado et al., 2017)(van den Brink et al., 2017)(van Velthoven et al., 2017) as well as other cell types (Adam et al., 2017) (Bakken et al., 2018) (Lacar et al., 2016) (Wu et al., 2017) (Wu et al., 2019) showing that those modifications are not exclusive to quiescent stem cells but are rather happening across cell types and cell states. However, it remains unclear whether each cell type has its own response to cellular dissociation or if there is a common response happening in all cell types and in all tissues. More importantly, whether these changes are a result of a dissociation artefact or whether they are also happening upon tissue damage in vivo and are required for the ability of cells to properly activate had not been previously investigated.

Here, we have developed diverse protocols to investigate the stress response of several cell types and tissues both ex vivo and in vivo. Furthermore, focusing on MuSCs, we have investigated the kinetics of this response and its functional importance in their activation.

Finally, implementing the tools and modulating the pathways we identified, we have done progress towards optimizing culture conditions that allow to first amplify, then induce the quiescence of MuSCs. Indeed, different studies have shown that freshly isolated MuSCs have a higher regeneration potential compared to proliferating myoblasts [START_REF] Montarras | Direct isolation of satellite cells for skeletal muscle regeneration[END_REF]) [START_REF] Ikemoto | Autologous transplantation of SM/C-2.6(+) satellite cells transduced with micro-dystrophin CS1 cDNA by lentiviral vector into mdx mice[END_REF], suggesting that early activated MuSCs (exiting quiescence) would have a higher regeneration potential compared to their proliferating counterparts. Indeed, several protocols have been published in the aim to produce quiescent-like MuSCs ex vivo [START_REF] Parker | Activation of Notch Signaling During Ex Vivo Expansion Maintains Donor Muscle Cell Engraftment[END_REF] [START_REF] Quarta | A bioengineered niche preserves the quiescence of muscle stem cells and enhances their therapeutic efficacy[END_REF] [START_REF] Sakai | Notch ligands regulate the muscle stem-like state ex vivo but are not sufficient for retaining regenerative capacity[END_REF] [START_REF] Sellathurai | A Novel In Vitro Model for Studying Quiescence and Activation of Primary Isolated Human Myoblasts[END_REF]. Although these protocols succeeded in maintaining MuSCs in a more upstream state in the myogenic program compared to myoblasts, they never managed to make quiescent-like MuSCs and thus failed to address this hypothesis.

Results

A common stress response in liver and skeletal muscle following dissociation

Since it was already established that several cell types undergo transcriptional changes during cell isolation, including MuSCs, we wanted to study the dissociation induced changes in two highly regenerative tissues, the liver and the skeletal muscle. For this aim, we decided to perform single-nucleus RNA sequencing (snRNA-seq) on intact and dissociated tissues (Figure 1A). Of note, nuclei extraction for this protocol is direct and fast preserving the transcriptional landscape of cells [START_REF] Grindberg | RNAsequencing from single nuclei[END_REF]. Using this method, we were able to identify ten main populations in the muscle and seven populations in the liver (Figure 1B). By merging nuclei from the same tissue, we noticed that even though nuclei clusters from the same cell type were close together on a UMAP they did not merge (Figure 1C), highlighting the global transcriptional changes happening during the 2 hours of dissociation.

Differential gene expression analysis revealed varying and significant changes in the ten main cell populations identified (MuSCs, tenocytes, smooth muscle cells, macrophages, FAPs, and endothelial cells from the muscle. Stellate cells, Kuppfer cells, hepatocytes, and endothelial cells from the liver) (Figure 1D). Performing gene ontology analyses on these differentially expressed genes and looking at specific pathways for some of the cell types, we saw enrichment for biological processes previously linked to the corresponding cell types.

These included TGF-β signaling in the FAPs, known to be induced in theses mesenchymal stromal cells during muscle regeneration (Contreras et al., 2019). TGF-β signaling was also enriched in both muscle and liver endothelial cells, a pathway involved in angiogenesis that can induce endothelial-to-mesenchymal transition, a process essential during tissue regeneration (van Meeteren and ten Dijke, 2012). Also, upon muscle and liver dissociation, we found a prominent response to cytokines in deferent cell-types: Interleukin (IL) -5 signaling was found in the most enriched terms of muscle endothelial cells and the FAPs, IL-6 and -2 in hepatocytes and liver sinusoidal cells, together with Interferon-gamma signaling in the latter.

Hepatocytes were also enriched for c-Met signalling, a pathway that is critical for liver regeneration (Ishikawa et al., 2012) (Figure 1E).

We then wanted to look at the common transcriptional changes happening across all cell types. We found that in both upregulated and downregulated genes, almost half the genes are cell type specific, whereas the other half are shared between at least 2 cell types (Figure 1F). We then wanted to identify a more universal stress response shared between several cell types. For this, we identified a list of 98 genes that are upregulated in at least seven out of the main ten cell types that were identified, which we called the Stress core (Figure 2A). Of note, we tested different parameters and finally chose to focus on the common genes in seven out of ten cell types because it was with this group that we were able to detect the identified Stress core in essentially all reported datasets. In fact, the stress index, defined as the average expression of these 98 genes was validated in the snRNA-seq (Figure 2B). Gene ontology analysis of this Stress core was enriched for terms linked to MAPK signaling, response to growth factors, transcription regulation and mechanical stimuli among others (Figure 2C), meaning that this identified core not only includes stress induced and growth factor induced pathways but also many other pathways involved in several functions. Consistently, we saw a rapid induction of activated 50hosphor-ERK1/2 in 15 min following muscle digestion (Figure 2D), validating the rapid dynamics of this pathway [START_REF] Amit | A module of negative feedback regulators defines growth factor signaling[END_REF].

To gain a better understanding of the biological nature of the conserved stress response, we grouped the Stress core genes according to their annotated functions. Notwithstanding that some genes have overlapping functions, we were able to divide them into 11 groups: MAPK pathway related genes (12 genes), cell cycle and proliferation (15 genes), chromatin regulation and remodeling (11 genes), cell motility and migration (10 genes), cell adhesion and cytoskeletal organization (11 genes), transcription (5 genes), metabolism (7 genes), NF-κB pathway (7 genes), ER stress and protein degradation (10 genes), cellular traffic (3 genes) and finally 7 genes with unknown functions (Figure 2E).

Stress response in vivo following tissue injury

After having identified the transcriptional changes caused by cellular dissociation ex vivo, we wanted to investigate the transcriptional response happening in vivo following tissue damage. To address this, we performed snRNA-seq on skeletal muscle and liver after chemical induced injury (Figure 3A). First, however, we assayed the kinetics of early induced changes following tetrachloride (CCl4) induced injury of the liver (Teixeira-Clerc et al., 2010) and Barium Chloride (BaCl2) induced injury of the skeletal muscle (Casar et al., 2004) by RT-qPCR on whole tissues. Even though, IEGs such as Fos, Jun, and Egr1 peaked at 2 hours post injury in both muscle and liver and start going down at 4h, we clearly saw MyoD peaking at 4h in the muscle (Figure 3B). For this reason, we decided to perform the snRNA-seq after 4 hours post injury in the muscle, in the aim to capture both the early and late responders and to perform the same time point for the liver. Similarly to our ex vivo snRNA-seq, nuclei from the same cell types tended to be close together, but intact and injured nuclei were clearly separated in UMAP plots (Figure 3C-D), once again showing the significant transcriptional changes happening very early upon tissue damage. Furthermore, our analyses showed that all nuclei from injured tissues exhibit a high stress index including myonuclei (Figure 3E) indicating that the stress core is not exclusive to activating cells, but also cells that do not activate. This observation shows that even though this core includes pathways that are not conventional stress pathways, it still represents a stress core rather than an activation core. Controversially, we noticed that not all hepatocytes expressed this stress index. We speculate that this is due to the delivery method of the CCl4, which is intraperitoneal, compared to the intramuscular injection of BaCl2, which might ultimately affect the kinetics of the injury between liver and muscle.

Focusing on quiescent muscle stem cells in our subsequent analyses, we wanted to investigate the kinetics of the early response of those cells in vivo. To address this, we performed single cell RNA-sequencing (scRNA-seq) at different time points following BaCl2 induced injury by adjusting the SMART-seq2 to the iSiFi protocol. As a matter of fact, we decided to use this method because of the incompatibility of the fixation process with the 10x Genomics pipeline at the time. MuSCs were isolated from uninjured (T0), 2 hours (T2) and 4 hours (T4) following muscle injury (Figure 4A). Principal Component Analyses (PCA) categorized the cells according to their experimental condition and revealed transcriptional consistency within clusters, but significant variations between time points. It is noteworthy that cells at T0 formed a tight and compact cluster while cells from clusters T2 and T4 even though clearly distinguishable, were more scattered (Figure 4B). These results indicate that truly quiescent MuSCs form a transcriptionally homogenous population, in contrast with what has been reported in the literature about the existence of a subpopulation of MuSCs successfuln a deeper quiescent state [START_REF] Rocheteau | A subpopulation of adult skeletal muscle stem cells retains all template DNA strands after cell division[END_REF])(Dell'Orso et al., 2019[START_REF] García-Prat | FoxO maintains a genuine muscle stem-cell quiescent state until geriatric age[END_REF], while early activated MuSCs are more heterogenous.

Pseudotime analysis of these cells showed a clear unique activation path of all MuSCs within the 4 hours after injury even though the kinetics may be variable (Figure 4B). Finally, our analyses were able to explain some of the confusing features of published single cell analyses of quiescent MuSCs (Dell'Orso et al., 2019;Giordani et al., 2019;Schaum et al., 2018) such as the low detection of Pax7 and the high expression of IEGs such as Fos, Jun, and Egr1 (Figure 4C).

Dissecting the kinetics of MuSCs early activation

In the aim of diving deeper in the early activation of MuSCs, we wanted to investigate the kinetics of the early activation on a minute scale. To do this, we performed high throughput sequencing at different time points during MuSCs dissociation. We performed bulk RNAsequencing on fixed and sorted MuSCs using the iSiFi protocol at 0-, 15-, 30-, 60-, 90-, and 120-minutes following incubation with the cell dissociation mix (collagenase/dispase) (Figure 5A). Limb muscle in this case was dissected and fixed with 0.5% PFA then digested (T0) or digested and digestion was stopped at the aforementioned time points, tissue was then fixed, MuSCs were sorted, and RNA was extracted for sequencing. PCA analyses showed clustering of the samples according to the time points, and a clear unique activation path of MuSCs (Figure 5B). Interestingly, 42 genes were already upregulated in just 15 min of digestion, even though most genes were differentially expressed after 1 hour. The 2 hours timepoint detected more than 1500 upregulated and more than 1500 downregulated genes (Figure 5C).

Hierarchical clustering analyses allowed us to group the differentially expressed genes into 5 clusters. Cluster 1 contained 36 out of the 42 upregulated genes in the first 15 min of digestion, including IEGs (Egr1, and Egr2) and ERK1/2 MAPK targets (Fos, Jun, Fosb, and Junb). These genes were characterized by a bell-shaped response as their expression peaked following 1 hour of dissociation and were then downregulated, almost to their T0 levels at 120 min, reminiscent of the IEGs response to mitogens stimulation [START_REF] Amit | A module of negative feedback regulators defines growth factor signaling[END_REF]. Cluster 2 comprised of upregulated genes with a linear trajectory. In this cluster, we identified Cdkn1a (p21), which we speculate is consistent with its role as an adaptor of kinases complex by promoting the interaction between CDKs and Cyclins and thus promoting cell cycle [START_REF] Zhang | p21-containing cyclin kinases exist in both active and inactive states[END_REF] [START_REF] Cerqueira | Genetic characterization of the role of the Cip/Kip family of proteins as cyclin-dependent kinase inhibitors and assembly factors[END_REF] . Cluster 3 included late responders of the dissociation induced stress as well as genes of the myogenic program that were upregulated following 1 hour of dissociation, such as MyoD. Finally, clusters 4 and 5 contained genes that were downregulated during the dissociation, such as known quiescence markers (Pax7, CalcR, and Spry1) and the HeyL, Hey1 and Hes1 targets of the Notch signaling pathway that is known for its role in quiescence maintenance (Figures 5D). Gene ontology analysis of the different clusters showed that the difference between those clusters was not only based on gene expression patterns but also on differences in biological functions (Figure 5E). Cluster 1 showed enrichment for cell cycle regulation, the MAPK pathway and transcription regulation that was followed by regulation of mRNA splicing and translation regulation in Cluster 2, and ribosome assembly in Cluster 3. Finally, clusters 4 and 5 comprising the downregulated genes showed a switch from the fatty acid beat oxidation to glycolysis and RNA methylation.

Functional role of the stress response in MuSCs

Having established that a common stress response happens upon tissue dissociation ex vivo and in vivo, we next wanted to investigate the function of this response in MuSCs activation. To do this, we used biological and chemical factors to modify candidate signaling pathways from the Stress core. Interestingly, we have identified Odc1 (Figure 5E) and Azin1 (Figure 2B), both elements of the polyamine synthesis pathway, known to be induced by stress and mitogens, and having a role in biogenesis and cell cycle. The products of ODC1 and AZIN1

give Spermidine which in turn will give Hypusine, a unique post-translational modification of eIF5A, playing a role in the translation elongation of cell cycle related genes [START_REF] Park | Post-translational formation of hypusine in eIF5A: implications in human neurodevelopment[END_REF].

Of note, eIF5a was also induced during the dissociation of MuSCs. To investigate the role of this pathway in the early activation, we cultured freshly isolated MuSCs with d,l-adifluoromethylornithine (DFMO), an ODC1 inhibitor, for 40h and quantified EdU incorporation, knowing that MuSCs go into a long G1 phase of about 35 hours (Rocheteau et al., 2012). Interestingly, DFMO treatment caused a decrease in the EdU incorporation in MuSCs. Surprisingly, this delay in the activation did not cause an increase in differentiation, as DFMO treatment for 72 hours maintained a more upstream PAX7+/MYOGENIN-cell population compared to the control (Figure 6A-B).

We then focused on the ERK1/2 MAPK pathway, a major component of the stress core (Figure 2B). As our data indicated a very rapid induction of P-ERK1/2 within 15 min of muscle dissociation (Figure 2E) and the gene expression of its targets in MuSCs (Figure 5E), we wanted to investigate its role in the G0-S transition of MuSCs. For this, we used SCH772984, an ERK1/2 inhibitor, during the 2 hours of skeletal muscle dissociation and muscle bulk was then cultured in normal proliferation medium (Figure 7A-C). Interestingly, ERK1/2 inhibition during dissociation caused a significant delay in the ability of MuSCs to enter the cell cycle and proliferate, however it did not cause any effect on their myogenic program as MuSCs treated with SCH772984 were MYOD+ (Figure 7D). As the ERK1/2 pathway was one of the first pathways to be induced, we asked how this pathway related to other pathways from the stress core. To do this, we looked at the expression of several genes with various functions from the stress core by RT-qPCR and found that some of them were downregulated following SCH772984 treatment during muscle dissociation (Figure 7E).

Finally, we wanted to investigate the interplay between the Notch signaling pathway and the ERK1/2 MAPK pathway, two pathways that are dynamically regulated upon MuSCs activation, and with 2 distinct yet very important functions, whereby ERK1/2 was very rapidly upregulated and required in the G0 -G1 -S transition of MuSCs whereas Notch targets were downregulated upon MuSCs early activation, and its role has already been reported in quiescence maintenance and inhibition of differentiation (Mourikis et al., 2012b) (Baghdadi et al., 2018a) (Figure 5E). We wanted to see whether by modulating these two pathways and thus blocking both activation and differentiation of MuSCs we can keep the cells quiescent ex vivo.

For this aim, we used mice that conditionally overexpress NICD, the intracellular domain of Notch, in MuSCs upon tamoxifen induction (Pax7 CreERT2 ; Rosa stop-NICD ) and cultured the muscle bulk with or without SCH772984 (Figure 8A). NICD overexpression caused a decrease in the expression of MYOD which subsequently caused a delay in the entry into the cell cycle of MuSCs (Mourikis and Tajbakhsh, 2014). However, the SCH772984 treatment did not affect the expression of MYOD but caused a severe decrease in the number of activated EdU+ MuSCs (Figure 8A), indicating that these 2 pathways are partially uncoupled in which ERK1/2 plays a role in the proliferation of MuSCs whereas Notch plays a role in their myogenic commitment.

Interestingly, the combination of NICD overexpression with ERK1/2 inhibition allowed us to obtain quiescent-like PAX7+/EdU-quiescent-like MuSCs following 72 hours in culture (Figure 8B).

Freshly isolated MuSCs exhibit a higher regeneration potential compared to myoblasts

Having managed to obtain quiescent-like MuSCs in culture conditions by combining Notch overexpression and ERK1/2 inhibition, we asked whether by manipulating the pathways we have identified, we can amplify MuSCs ex vivo while inhibiting their differentiation and then revert them back to quiescence. As a matter of fact, it's been shown that freshly isolated MuSCs exhibit a higher regeneration potential compared to myoblasts [START_REF] Montarras | Direct isolation of satellite cells for skeletal muscle regeneration[END_REF] [START_REF] Ikemoto | Autologous transplantation of SM/C-2.6(+) satellite cells transduced with micro-dystrophin CS1 cDNA by lentiviral vector into mdx mice[END_REF]. For this reason, it's been speculated that quiescent MuSCs have the highest capacity to differentiate and self-renew following grafting experiments. However, even though several protocols have been optimized to induce quiescence ex vivo, this hypothesis has never been successfully tested.

Given that in vivo engraftment experiments show a big degree of variability between biological replicates, we decided to optimize an ex vivo grafting-like system. To do this, we sorted MuSCs and the rest of the muscle cells separately. We then cultured the MuSCs-free muscle bulk and added MuSCs at their actual concentration (100%) or at lower concentrations, half (50%) or fourth (25%). Of note, the 100% concentration corresponds to the number of MuSCs we get at the FACS, which is on average 1.5% of the total number of cells in the muscle bulk. In parallel, we added myoblasts instead of MuSCs from Tg:Pax7-nGFP mice cultured for 3 days and sorted for GFP at the same concentrations in order to look at the regeneration potential of MuSCs vs myoblasts ex vivo. We left the cells in culture for 7 days for them to differentiate and make reserve cells, we also added EdU a day before fixing (Figure 9A).

Interestingly, freshly isolated cells still maintained a better ability to differentiate and make reserve cells at 25% compared to myoblasts at 100% (Figure 9B). We then wondered whether the bulk influences the activation and differentiation of the cells. To test this, we decided to repeat the same experiment on sorted MuSCs and myoblasts without the MuSCs-free bulk.

Interestingly, even though we managed to reproduce overall the same results, we noticed that in this case, both sorted MuSCs and myoblasts performed better compared to myogenic cells plated with the bulk (Figure 10A-B), however, we believe that this is due to technical issues rather than biological differences. Regardless of these differences, this system allowed us to show the higher capacity of freshly isolated MuSCs to differentiate and self-renew. Indeed, a in vivo engraftment experiment is always required to investigate the differences in the regeneration potential of those cells.

Induction of quiescent-like MuSCs ex vivo

Having obtained quiescent-like MuSCs in culture conditions by combining Notch activation and ERK1/2 inhibition. We wondered whether by manipulating these two pathways, we can optimize a protocol in which we can induce quiescence of MuSCs ex vivo following their amplification. We also wondered whether we can do this by combining Notch activation and ODC1 inhibition, since our results indicated that ODC1 is required for both the entry into the cell cycle and differentiation of MuSCs.

To do this, we plated freshly isolated MuSCs on culture dishes coated with immobilized human Dll1 for 7 days, then treated them with SCH772984 or DFMO for 2 days or left the cells untreated, we also added EdU 24 hours before fixing (Figure 11A). Interestingly, MuSCs plated on Dll for 9 days did not differentiate and form myotubes, and we can clearly notice a correlation between high GFP (PAX7) expression and low or negative MYOD expression and EdU incorporation. Furthermore, we found that following SCH772984 treatment, almost all cells stopped proliferating as very few cells were EdU+. However, DFMO had no effect on the expression of MYOD and EdU incorporation compared to the untreated cells (Figure 11B). Indeed, ODC1 might not be expressed in those cells which might explain the inefficiency of the DFMO treatment.

Even though we managed to inhibit the proliferation and differentiation of MuSCs, we noticed that we still have more than half of the cells that expressed MYOD and a small fraction that are GFP-, which we speculated are also MYOGENIN-. We wondered whether this is due to the Dll-Fc losing its efficiency following 9 days in culture. To address this, we decided to split the cells after 4 days in culture in the aim to renew the Dll1-Fc coating, treated the cells with SCH772984 at day 7 and added EdU at day 8 to assess the proliferation of the cells (Figure 11C). However, the renewal of the coating did not have any effect on the expressions of GFP (PAX7) or MYOD indicating that the Dll1-Fc coating was not the problem. (Figure 11D).

To attempt to optimize the protocol and boost the ectopic expression of Notch in the aim to increase the expression of PAX7 and decrease MYOD expression, we decided to decrease the percentage of O2 as several studies have shown that low O2 concentrations and hypoxia conditions induced Notch expression in MuSCs as well as other adult stem cells such as Neural Stem Cells (NSCs) and Hematopoietic Stem Cells (HSCs) and caused a delay in the differentiation [START_REF] Gustafsson | Hypoxia requires notch signaling to maintain the undifferentiated cell state[END_REF] [START_REF] Liu | Hypoxia promotes satellite cell self-renewal and enhances the efficiency of myoblast transplantation[END_REF] [START_REF] Endo | Loss of ARNT in skeletal muscle limits muscle regeneration in aging[END_REF] [START_REF] Araki | NOTCH-mediated ex vivo expansion of human hematopoietic stem and progenitor cells by culture under hypoxia[END_REF].

For this, we cultured freshly isolated MuSCs on Dll-Fc in 21% O2 which is the atmospheric O2 concentration that we normally use, or 3% O2, which corresponds to the O2 levels in the muscle [START_REF] Pircher | Hypoxic Signaling in Skeletal Muscle Maintenance and Regeneration: A Systematic Review[END_REF]. We fixed the cells following 4 or 8 days and assessed the differentiation of the cells (Figure 12A). We noticed a higher number of cells following 8 days in 3% O2 compared to 21% O2. However, at this time point there was no effect on the expression of MYOD even though it was lower in 3% O2 at 4 days (Figure 12B). We concluded that decreasing oxygen levels in culture only caused a small delay in the activation of the cells.

In an alternative attempt to antagonize differentiation, we cultured freshly isolated Tg:Pax7-nGFP MuSCs on Dll1-Fc coated dishes overnight, then added ColV to the cultures as it has been shown that MuSCs treated with ColV for 10 days had a decreased proliferation and differentiation (Baghdadi et al., 2018a), or treated the cells with 5mg/mL acetic acid, the solvent of COLV, as Control (Figure 13A). Furthermore, instead of inducing quiescence by inhibiting ERK1/2, we decided to try an alternative method of cell cycle arrest, that of gradual serum starvation [START_REF] Coller | A new description of cellular quiescence[END_REF]. To do that, serum concentration was decreased by half daily, going from 20% to 1.25% (Figure 13A). We speculated that inhibition of all cell cycle related genes might be a better way of quiescence induction rather than abruptly inhibiting ERK1/2, especially since the ERK1/2 pathway has been recently shown to induce fusion of myoblasts (Eigler et al., 2021, p. 2). Under this experimental setup, ColV treatment had no effect on the expression of GFP (PAX7) or EdU incorporation (24 hours EdU chase), with or without serum starvation. Furthermore, serum starvation led to an overall decrease in the percentage of GFP (PAX)+ MuSCs even though we noticed that some cells had a higher GFP expression following serum deprivation compared to cells cultured in 20% serum (Figure 13B). Based on the above observations, we concluded that ERK1/2 inhibition was a better way of quiescence induction compared to serum starvation. So, we wondered whether a more aggressive treatment of SCH772984 would increase its efficiency. We therefore decided to do a daily treatment of the inhibitor instead of leaving it for 2 days in culture (Figure 14A). Interestingly, by doing this, we managed to obtain MuSCs-like cells that were starting to express CALCR, a hallmark quiescence marker (Figure 14B). Interestingly, it has been reported that MuSCs lose CALCR expression following 24 hours in culture (Yamaguchi et al., 2015) (Baghdadi et al., 2018a). Of note, some of the CALCR + cells still expressed MYOD (Figure 14B) which we speculated might be due to the short treatment of cells with SCH772984.

We then wanted to test whether increasing the time of treatment with SCH772984 would increase the number of cells expressing CALCR. For this, we decided to do a daily SCH772984 treatment for 3 days instead of 2 days. By doing this, we managed to obtain quiescent-like PAX7 + /CALCR + /EdU -MuSCs (Figure 15A). In order to further characterize the quiescence status of the treated cells, we looked at the expression of a panel of genes that we used as reporters of true quiescent MuSCs (highly expressed in in situ fixed MuSCs and strongly downregulated upon activation) (Machado et al., 2017). We saw that 6 out of 9 reporter genes had higher expression in quiescent-like MuSCs treated with SCH772984 compared to the untreated cells (Figure 15B). In contrast, the ERK1/2 targets Fosb and Egr1 were downregulated upon SCH772984, validating our culture system. Finally, even though there was no difference in the expression of MyoD and Myogenin, treated cells showed a lower Pax7 expression compared to untreated cells (Figure 15B). Taken together, we speculated that SCH772984 treatment caused the quiescence induction of uncommitted cells and the differentiation of cells that are already committed in the myogenic program. Therefore, a putative way to optimize the protocol would be to treat the cells with ERK1/2 inhibitor at an earlier time point (before some cells become committed) to increase the fraction of the quiescent-like population.

Discussion

Using two highly regenerative tissues, the skeletal muscle and the liver, we were able to identify a conserved stress response happening upon tissue damage both ex vivo and in vivo.

Interestingly, this response seems to happen across all cell types from quiescent cells that proliferate upon activation such as MuSCs to terminally differentiated post mitotic myonuclei.

During the dissociation process, cells are subjected to enzymatic and mechanical dissociation as well as growth factors, all of which can be inducers of the response we identify. However, the source of this stress remains unclear (Figure 16). [START_REF] Marsh | Dissection of artifactual and confounding glial signatures by single-cell sequencing of mouse and human brain[END_REF]. All these studies and protocols will allow us to generate a new generation of atlases that better reflect the true transcriptional landscape of the cells in vivo.

It remains unclear whether these pathways are connected to ensure the proper activation of cells. Our results suggest that the ERK1/2 MAPK pathway is one of the first responders and its induction is indispensable for the activation of MuSCs, while Notch inhibition ensures the commitment of MuSCs in the myogenic program. This is followed by the induction of the polyamine synthesis pathway that seems to be affecting both the proliferation and differentiation of the cells.

Finally, uncovering the mechanisms that govern MuSCs quiescence exit and manipulating them in culture conditions allows us to revert proliferating MuSCs into quiescence in culture conditions. First, this protocol allows the study of quiescence ex vivo.

Second, it is a great tool for cell mediated therapy as one of its biggest obstacles is that MuSCs lose their regeneration potential when activated [START_REF] Montarras | Direct isolation of satellite cells for skeletal muscle regeneration[END_REF] [START_REF] Ikemoto | Autologous transplantation of SM/C-2.6(+) satellite cells transduced with micro-dystrophin CS1 cDNA by lentiviral vector into mdx mice[END_REF].

Finally, coupling this protocol to iPSCs differentiation into myogenic cells can help in increasing the fraction of bona fide MuSCs obtained in those protocols. In fact, several protocols of iPSCs differentiation to MuSCs have already been published. However, these protocols give mainly an activated and/or differentiated population of myogenic cells. We therefore believe that combining our protocol with these established protocols will allow the increase of the fraction of the quiescent, PAX7 expressing MuSCs population. genes as defined by hierarchical clustering. Linear regression with second-order polynomial was performed on the mean gene expression per time point for each cluster. Samples are ordered from T0 to T120. Standard error is displayed in gray.

(E) Gene ontology analysis using EnrichR for all of the genes within each of the five clusters.

Color code indicates p-value and circle size the combined enrichment score (0 to 60). Taken together, during tissue damage MuSCs are facing molecular, mechanical and toxic stimuli from all those changes. In response, MuSCs will transiently activate the ERK1/2 MAPK required for their cell cycle entry and downregulate the Notch pathway to enter the myogenic program. This is followed by the induction of the polyamine synthesis pathway (Odc1 is the rate limiting enzyme for putrescine formation) that is involved in both, the activation and differentiation of those cells. However, it remains unclear if a specific subset of those aforementioned stressors is causing the central MuSC activation response, or whether it is a combination of all of them.

Material and Methods

Animals

The following mouse lines were kindly provided by the corresponding laboratories as described: Tg:Pax7-nGFP (S. Tajbakhsh; (Sambasivan et al., 2009), R26R stop-NICD-nGFP (D.

Melton; Jackson Laboratories, stock 008159; (Murtaugh et al., 2003), Pax7-CreERT2 (G. Kardon;

Jackson Laboratories, stock 017763; (Murphy et al., 2011) The dishes were then coated with the Dll1-Fc conditioned medium for 1hour at room temperature with rocking. Finally, dishes were coated with Matrigel as already described and MuSCs were cultured in growth medium supplemented with 5ng/mL bFGF. MuSCs were then treated with SCH772984 at 1mg/mL and half of the medium was then changed for the subsequent treatment days if specified. DFMO was added at 2.5mM. For ColV treatment, ColV (Sigma; 3657) was resuspend with Acetic Acid (AcOH) (5mg/mL) at 1mg/mL and used at a final concentration of 50ug/mL.

Muscle and liver injury

For muscle injury, mice were anaesthetized with an intraperitoneal injection of 1.6:1 ketamine and xylazine (2%, VMD), 60 mL per 20 g of bodyweight. Both TA muscles were injected with 50 mL of 1.2% BaCl2 (Sigma, 202738). For liver injury, mice received an intraperitoneal injection of carbon tetrachloride (CCl4, Sigma, 289116 at 0.5 ml/kg bodyweight, 1:5 dilution in olive oil) as described previously described (Teixeira-Clerc et al., 2010).

Single-nucleus isolation

For the intact/dissociated muscle and liver atlases 6 tibialis anterior, gastrocnemius, soleus, plantaris, and extensor digitorum longus muscles as well as 3 whole liver from 3 8-week old female mice were used. For the intact/injured muscle atlas, 8 tibialis anterior mus-cles from 4 8-week old female mice were used. Tissues were dissected and pulled in cold PBS with 0.2 U/ml RNase inhibitor (Roche, 03335399001). PBS was removed and tissues were minced in 1 mL cold lysis buffer (10mM Tris-HCl, 10mM NaCl, 3mM MgCl2, and 0.1% NonidetTM P40

in Nuclease-Free Water) with scissors. After 2 min, 4 mL of cold lysis buffer was added and tissues were lysed for 3 min at 4 • C. 9 mL of cold wash buffer (PBS, BSA 2% and 0.2 U/ml RNase inhibitor) was subsequently added and the lysates were homogenized with 10 strokes of loose pestle using a 15 mL Dounce homogenizer. The homogenates were filtered with 70 mm and 40 mm cells strainers. Nuclei were pelleted by centrifugation for 5 min (500 g, 4 • C). Nuclei were washed in 1 mL of cold wash buffer, transferred in a 1.5 mL tube and centrifuged 5 min (500 g, 4 • C). The pellets were resuspended in 250 mL of wash buffer and stained during 15 min in the dark at 4 • C with DAPI at 10 mg/ml. The nuclei were therefore washed and spun down 5 min (500 g, 4 • C), resus-pended in 300 mL of wash buffer and filtered with 30 mm cell strainers.

Nuclei were then FACS sorted for Hoechst to exclude debris with a BD FACSAria III and the BD FACSDIVA software.

Staining

For cell culture, following fixation (2% paraformaldehyde (PFA), 20 min) MuSCs were washed three times in PBS, permeabilized, and blocked using a blocking solution (BS) containing 5% BSA (Jackson Laboratories 001-000-162) and 0.25% Triton X-100 (Sigma) for 1 hour at room temperature. The samples were then incubated overnight with primary antibodies. After three washes, the cells were incubated with the corresponding secondary antibodies for 1 hour at RT. The samples were subsequently washed three times with PBS and stained for EdU and Hoechst (EdU Click-iT PLUS Kit C10640, Life Technologies) following the manufacturer guidelines before a final wash and mounting.

In situ fixation treatment and reagents

For ex vivo dissociation, after mouse sacrifice muscles were dissected (hindlimb and forelimb) and minced in small pieces. Tissue pool from two mice were separated in 6 groups (T0, T15, T30, T60, T90, and T120). T0 samples were prepared according to the original iSiFi protocol (Machado et al., 2017). Briefly, the samples were fixed for 1 h in 0.5% PFA, washed 3 times in ice-cold PBS and digested for 2 h at 37 • C with agitation in 2X digestion solution: Dispase II, 6 U/ml (Roche, 4942078001), Collagenase A 1 U/ml (Roche, 10103586001) and 0.2% BSA:DMEM. T15, T30, T60, T90 and T120 samples were digested for 15, 30, 60, 90 and 120 min respectively in 1X digestion solution: Dispase II, 3 U/ml (Roche, 4942078001), Collagenase A 0.5 U/ml (Roche, 10103586001) and 0.2% BSA/ DMEM at 37 • C with agitation. The samples were then spun down (600 g, 5 min) and resuspended in ice-cold 0.5% PFA and incubated for 1 h at 4 • C with gentle agitation to fix the cells. The samples were washed 3 time with ice-cold PBS (600 g, 5 min) and resuspended in 2X digestion solution to terminate digestion for 105, 90, 60 and 30 min for T15, T30, T60 and T90 samples respectively. T120 samples were not redigested.

At this point, all the samples were filled with DMEM (GIBCO, 41966-029) and filtered successively through 100 mm and 70 mm cell strainers (MACS, 130-110-917 and 130-110-916 respectively) then span down (600 g, 5 min), resuspended in ice-cold DMEM and filtered through 40 mm cell strainer (Corning, 352340) before a last centrifugation (600 g, 5 min) and resuspension in 0.2% BSA/DMEM for cell sorting.

For the experiment comparing T60 MuSCs in the supernatant to intact tissue pieces, T0, T60 and T120 samples were obtained from the dissection of hindlimbs and forelimbs from the same mice. For this experiment T0 and T120 samples were processed as indicated above. T60 samples were incubated for 60 min in 1X digestion solution. The samples were then separated simply with gravity by incubating the tube (50 mL conical) vertically at RT for 5 min to pellet intact tissue pieces and to carefully pass the supernatant successively through 100, 70 and 40 mm filters (references above) to obtain dissociated cells. Both samples were filled with icecold DMEM, span down (600 g, 5 min) and resuspended in ice-cold 0.5% PFA for 1 h at 4 • C with agitation. The T60-pellet samples were then re-digested for 60 min in 2X digestion solution. All samples were then filtered as indicated above.

For in vivo activation, after mouse sacrifice injured TA muscles were dissected and minced in small pieces. The samples were pre-pared according to the original iSiFi protocol (Machado et al., 2017), with the following modification: 0.5 M glycine: PBS was used in the first post-PFA wash.

RNA extraction

For ex vivo dissociation time-course, in vivo activation control and ex vivo ERK1/2 inhibition during dissociation RT-qPCR, the cells were sorted directly in lysis buffer from the Quick RNA FFPE kit (Zymo, R1008) and the RNA extracted following the manufacturer guidelines (without the tissue digestion step). For T60 supernatant/pellet RT-qPCR, the cells were sorted directly in lysis buffer from the RecoverAll FFPE kit (Thermo, AM1975) and the RNA extracted following the manufacturer guidelines (without the tissue digestion step, without the incubation step at 80 • C and with the incubation step at 50 • C for 1 h instead of 15 min). For in vivo activation time-course, Pax7-nGFP cells were sorted single-cell by FACS in 96 well plates (4titude, 4ti-0960). Each well contained 2.5 ml PKD buffer (QIAGEN, 1034963) supplemented with 1:16 proteinase K (QIAGEN, 19131). Prior to RNA separation, plates were decrosslinked for 1 h at 56 • C. RNA separation was performed on a Hamilton STAR according to the G&T-seq protocol (Macaulay et al., 2015). Briefly, oligo-dt30VN-labeled beads and wash buffer were prepared. 10 ml of beads were added to every well with the liquid-handling robot and incubated for 20 min at RT while mixing (2,000 rpm). Thereafter, the beads were washed twice with wash buffer and 5 ml of Reverse Transcriptase master mix was added to every well of the bead-containing 96-well plate.

RT-qPCR

Reverse transcription was performed using the SuperScript IV VILO Master Mix (Thermofisher, 11756050) following the manufacturer's guidelines. RT-qPCR was performed using the Power SYBR Green PCR Master Mix (Applied Biosystems, 4367659).

Statistical analysis

For comparison between two groups, unpaired Student's t test was performed to calculate p values and to determine statistically significant differences (*p < 0.05; **p < 0.01; ***p < 0.001).

Library preparation

Libraries preparations and data analysis are detailed in [START_REF] Machado | Tissue damage induces a conserved stress response that initiates quiescent muscle stem cell activation[END_REF] List of qPCR primers

Gene (mouse)

Forward sequence 5' -3' Reverse sequence 5' -3'

Pax7

GACAAAGGGAACCGTCTGGAT TGTGAACGTGGTCCGACTG

Myod1

CACTACAGTGGCGACTCAGATGCA CCTGGACTCGCGCGCCGCCTCAC

Fos

CTACCATTCCCCAGCCGAC GTTGGCACTAGAGACCGACA

Jun

GGAACAGGTGGCACGCTTA GCTGCGTTAGCATGAGTTGG

Fosb

AGCTAAGTGCAGGAACCGTC ACTCAAGCTCTGCCTTTTCC

Fosl1

AGCTCCGGGGCTGGC ATGCTTGGCACAAGGTGGAA

Fosl2

CGAGTCCTACTCCAGCGGT ATGGCGTGGATTGTGGGGAT

Ets1

TCTCAAGCCGACTCTCACCA GCACATTCCATGTCCGGGG

Ets2

CAGTCGAGCAAGGCAAACCA TGGATTGGTCCGCTTCCAGT

Myc

GATTCCACGGCCTTCTCTCC GCCTCTTCTCCACAGACACC

Egr1

GAGCACCTGACCACAGAGTC CGAGTCGTTTGGCTGGGATA

Egr2

TGACCAGATGAACGGAGTGG GCGAAGCTACTCGGATACGG

Igf-bp

TGTTCAGCTCCCAGCATGAA TGGGCTCTCAGAAAGCTCAT

Azin1

TCCTAACTTGTCCATGGAGGTC CCACTTGACACCAAGCTGGA

Odc1

AAGTCGACCTTGTGAGGAGC GTTCTCGATGTGCTTACAGGGA

Runx1

CGGCCATGAAGAACCAGGTA TGAAGCTCTTGCCTCTACCG

Stat3

GAGACAGTCGAGACCCCTGAC TGCTCCAGGTAGCGTGTGT

Ppp1r15

CGGAAGGTACACTTCGCTGA GGCTTCGATCTCGTGCAAAC

Actb

CACTGTCGAGTCGCGTCC TCATCCATGGCGAACTGGTG

Klf4

GATGCAGTCACAAGTCCCCT CCTTCTTCCCCTCTTTGGCTT

Klf6

CCCACTGTGACAGGTGTTTCT GCCTACAGGATTCGTCCCTC

p21

GTACTTCCTCTGCCCTGCTG GGGCACTTCAGGGTTTTCTC

Med13

TCCCTACTTCTGCTTCTGTGC TCCGTCTGCTCCATCATTGT

Nfkbia

TAGCAGTCTTGACGCAGACC AGACACGTGTGGCCATTGTA

Myogenin

GTGAATGCAACTCCCACAGC CGCGAGCAAATGATCTCCTG

CalcR

TCATCATCCACCTGGTTGAG GCTCGTCGGTAAACACAGC

Ron1

GCCTACCTCGTCCTGGAACA TTCATCGGCTCATCAAGGGT

Pde10a

GCCGACGGATTTGCACTGTA TCCTTCATCCCGGGTGGTAT

Dtx4

CACTGGCCACTTTGAATCGG CACTGGCCACTTTGAATCGG

Sema6a

CATTGAGCGTGGCAATACGG CGAATCACTCCATTCAGTGCC

Pde7b

TGGCTCCTACCCATTCATTGAC CTTGGTGCCAATTTCCCCTGA

Abr

CTGACCCCAACCTCATCACC ATGGGCTCCTTCTCAGCAAC

Zbtb6

AGCGTTTCCTTCCAGGCTAA AGCCATGGTCACGATCGAC

Prkq

CACCATGCGTACCAACACCT GCTCACTTCGGGTTCAGGAG

Clmn

AGAGAATGGCCCTGTGGAAAG TCTAATGGCTTTGTGGGCAG

Tbp

ATCCCAAGCGATTTGCTG CCTGTGCACACCATTTTTCC

List of antibodies

Antibodies Reference Dilution

IgY chicken polyclonal anti-GFP Abcam ; ab13970 IF 1/1 000 pathways. The ERK1/2 MAPK and polyamine synthesis pathways from this stress core have been shown to be required for MuSCs quiescence exit and their entry into the cell cycle [START_REF] Machado | Tissue damage induces a conserved stress response that initiates quiescent muscle stem cell activation[END_REF]. However, given the variety of genes and their functions from this stress core, it remains unclear what role each of these genes is playing during the early steps of MuSCs activation.

IgG1
One gene from this early stress response that we identified is the Krüppel like factor Klf4 [START_REF] Machado | Tissue damage induces a conserved stress response that initiates quiescent muscle stem cell activation[END_REF] , a highly conserved zinc finger containing transcription factor, coming from a large family of Krüppel like transcription factors. KLF4 is one of the four factors required for reprogramming differentiated cells into iPSCs [START_REF] Takahashi | Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors[END_REF]. After this function of Klf4 was uncovered, it gained a lot of attention, and its role has been intensively investigated in various tissues and cell types [START_REF] Ghaleb | Krüppel-like factor 4 (KLF4): What we currently know[END_REF]. Klf4 is known to be involved in cell cycle regulation, mainly as an antagonist by activating Cdkn1a (p21) [START_REF] Chen | Transcriptional profiling of Krüppel-like factor 4 reveals a function in cell cycle regulation and epithelial differentiation[END_REF]) [START_REF] Yoon | Krüppel-like Factor 4 Mediates p53-dependent G1/S Cell Cycle Arrest in Response to DNA Damage[END_REF]) [START_REF] Zhang | The gut-enriched Kruppel-like factor (Kruppel-like factor 4) mediates the transactivating effect of p53 on the p21WAF1/Cip1 promoter[END_REF]. However, the role of Klf4 has been shown to be context dependent, as it can also activate the cell cycle in other conditions [START_REF] Rowland | KLF4, p21 and context-dependent opposing forces in cancer[END_REF]) [START_REF] Foster | Increase of GKLF messenger RNA and protein expression during progression of breast cancer[END_REF].

The function of Klf4 has been investigated in various tissues such as the gut, where it was first described as it is important for intestinal homeostasis [START_REF] Shields | Identification and Characterization of a Gene Encoding a Gut-enriched Krüppel-like Factor Expressed during Growth Arrest[END_REF] [START_REF] Katz | The zinc-finger transcription factor Klf4 is required for terminal differentiation of goblet cells in the colon[END_REF]. Klf4 is also required for the formation of the skin barrier, as Klf4 null mice die shortly after birth because of defects in the skin and rapid loss of body fluids [START_REF] Segre | Klf4 is a transcription factor required for establishing the barrier function of the skin[END_REF]. However, the role of Klf4 in the muscle and the muscle stem cells has never been investigated, except for one study that was performed on C2C12 cells where it has been shown to be required for myocytes fusion [START_REF] Sunadome | ERK5 Regulates Muscle Cell Fusion through Klf Transcription Factors[END_REF].

Furthermore, the stress core includes several IEGs such as Egr1, Fos, Jun among many others. A known master regulator of these IEGs, is the Serum Response Factor SRF [START_REF] Lee | Activation and Repression of Cellular Immediate Early Genes by Serum Response Factor Cofactors[END_REF]. This role of SRF was already described in Chapter 1. Srf is required during development as Srf null mice fail to form the mesoderm and die shortly after gastrulation [START_REF] Arsenian | Serum response factor is essential for mesoderm formation during mouse embryogenesis[END_REF]. In the muscle, Srf has been shown to be required for myoblasts fusion, as Myo:Cre;

Srf flox/flox mice, having the Cre recombinase expressed under the control of the Myogenin promoter and the myocyte-specific enhancer factor 2, MEF2C enhancer, die during perinatal development due to severe hypoplasia [START_REF] Li | Requirement for serum response factor for skeletal muscle growth and maturation revealed by tissue-specific gene deletion in mice[END_REF]. And in adults, it has been shown that Srf is required for primary myocytes fusion [START_REF] Randrianarison-Huetz | Srf controls satellite cell fusion through the maintenance of actin architecture[END_REF]. However, its function in the early activation of MuSCs has never been studied.

Here, we investigated the function of Klf4 during the early activation as well as the differentiation of MuSCs by conditionally deleting Klf4 specifically from MuSCs.

Furthermore, we examined the role of Srf during the quiescence exit of MuSCs. Our data showed that neither Klf4 nor Srf were required for the ability of quiescent MuSCs to go into the cell cycle following activation, and in contrast to what has been reported for C2C12 cells [START_REF] Sunadome | ERK5 Regulates Muscle Cell Fusion through Klf Transcription Factors[END_REF], Klf4 was not required for the fusion of primary myocytes.

Results

Dynamics of Klf4 expression in MuSCs

Following tissue damage and dissociation, cells react to these changes by undergoing transcriptional changes. This stress response is important for MuSCs to exit quiescence, enter the cell cycle, and induce the myogenic program. Klf4 is a part of this stress core, expressed in all cell types in skeletal muscle upon dissociation (Figure 1A). Interestingly, Klf4 is also induced in the first 15 minutes in MuSCs following tissue dissociation along with other IEGs such as the AP-1 complex and before MyoD (Figure 1B), it also has a bell-shaped response, reminiscent of the response of IEGs to mitogens stimulation [START_REF] Amit | A module of negative feedback regulators defines growth factor signaling[END_REF]. Moreover, KLF4 protein is absent in quiescent MuSC but is detected 2 hours following activation and remains during their proliferation. Interestingly, KLF4 expression is slightly increased during differentiation and in myotubes (Figure 1C-E) in line to its role in C2C12 myoblasts fusion [START_REF] Sunadome | ERK5 Regulates Muscle Cell Fusion through Klf Transcription Factors[END_REF]. The expression pattern of KLF4 during the different steps of MuSCs activation and differentiation hint to a dual role of Klf4, first during the early activation of MuSCs and then latter during differentiation and fusion of myoblasts.

Loss of the myogenic identity of myoblasts following KLF4 overexpression

As we wanted to investigate the role of Klf4 in myoblasts, and data from our lab (not shown here) had suggested a loss of the myogenic identity of MuSCs upon KLF4 overexpression, we wanted to see whether we can reproduce these results and understand the resulting phenotype. For this aim, we transduced C2C12 cells, an established myogenic cell line, with a KLF4-GFP or MOCK-GFP retroviruses and sorted the GFP+ cells (Figure 2A).

We then stained the cells with KLF4 and GFP and noticed that the intensity of the GFP expression in the C2C12 cells transduced with the KLF4-GFP retrovirus correlates with the intensity of KLF4 expression, indicating that our system is effective in overexpressing Klf4 in cells (Figure 2B). Furthermore, Klf4 expression was 17 times higher in C2C12 cells transduced with the KLF4-GFP retrovirus compared to the C2C12 cells transduced with the control Mock-GFP retrovirus (Figure 2C). In order to study the effect of Klf4 overexpression in myogenic cells, we assessed the proliferation of these cells with a 2 hours EdU chase and their differentiation by scoring for MYOGENIN and saw that Klf4 overexpression did not have any effect on the proliferation and differentiation of those cells (Figure 2D).

However, when we transduced myoblasts enriched cultures with the KLF4-GFP or MOCK-GFP retroviruses (Figure 3A), remarkably, myoblasts overexpressing Klf4 lost the expression of all three myogenic markers (PAX7, MYOD, and MYOGENIN). Furthermore, when left in culture for 7 days to differentiate and fuse, these cells failed to form myotubes unlike the MOCK-GFP control, indicating that myoblasts overexpressing Klf4 lost their myogenic identity (Figure 2B-D). This difference in the phenotypes following Klf4 overexpression between C2C12 cells and primary myoblasts is an indication of the huge difference between those two cell types. Furthermore, this phenotype might be consistent with the role of Klf4 as a reprogramming factor of differentiated cells to iPSCs [START_REF] Takahashi | Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors[END_REF]. Of course, a screen for other pluripotent factors such as Nanog and Sox2 is required to prove this hypothesis.

Moreover, cells that overexpressed Klf4 were bigger in size compared to the control and some of them were binucleated. To see whether these cells were cycling, we added EdU in the culture for 8 hours. Interestingly, Klf4 overexpressing cells did not show a decrease in the percentage of EdU+ cells as we expected. Moreover, around 50% of the binucleated cells were also EdU+ which was the same percentage of the total number of EdU+ cells indicating that even the binucleated cells were still cycling (Figure 3B-D). We therefore found that myoblasts overexpressing Klf4 even though undergo the S-phase, failed to complete cytokinesis, and became binucleated. This phenotype might be indicative of a role of Klf4 in the cell cycle of MuSCs. Of note, a checkpoint for cytokinesis and tetraploidy has always been controversial in the literature. Interestingly, a study showed that such a checkpoint does not exist, and rather tetraploid cells are eventually arrested in the subsequent G1 phase because of aberrant DNA damage [START_REF] Wong | Mammalian cells lack checkpoints for tetraploidy, aberrant centrosome number, and cytokinesis failure[END_REF].

Klf4 is not required for MuSCs activation and differentiation

Since published data indicated a dual role of Klf4 in MuSCs, the first one during their quiescence exit and early activation [START_REF] Machado | Tissue damage induces a conserved stress response that initiates quiescent muscle stem cell activation[END_REF] Murphy et al., 2011). We dissected and dissociated the limb muscle of those mice and sorted the early activated GFP + MuSCs. In fact, the cKO cells were efficiently targeted since we could not detect the induction of the transcript (95% decrease). Interestingly, we also saw an increase in the expression of Cdkn1a (p21) in the early activated MuSCs depleted from Klf4

(Figure 4D). This increase in p21 is in contrast with the role of KLF4 as its activator (Chen et al., 2003) [START_REF] Yoon | Krüppel-like Factor 4 Mediates p53-dependent G1/S Cell Cycle Arrest in Response to DNA Damage[END_REF] [START_REF] Zhang | The gut-enriched Kruppel-like factor (Kruppel-like factor 4) mediates the transactivating effect of p53 on the p21WAF1/Cip1 promoter[END_REF]. Furthermore, we know that p21 is highly induced during the early activation of MuSCs (Machado et al., 2017) [START_REF] Machado | Tissue damage induces a conserved stress response that initiates quiescent muscle stem cell activation[END_REF], which is contradictory with its known role as a cell cycle inhibitor. It has been speculated that this induction of p21 is required for the proper regulation of the cell cycle entry, as it can bridge CDKs and cyclins, thus promoting their assembly [START_REF] Zhang | p21-containing cyclin kinases exist in both active and inactive states[END_REF]) [START_REF] Cerqueira | Genetic characterization of the role of the Cip/Kip family of proteins as cyclin-dependent kinase inhibitors and assembly factors[END_REF]. Of note, KLF4 has both an activation and a repression domain,

indicating that KLF4 might be, in this case, a repressor of p21 to keep its expression at the required level. Regardless, Klf4 cKO MuSCs were able to enter the cell cycle following activation shown by their ability to incorporate EdU after 43 hours, go in the myogenic program, evident by the expression of MYOD at 43 hours and MYOGENIN at 72 hours and eventually differentiate and fuse to form myotubes with the same number of myonuclei per myotube as the Control (Figure 4A-C). Finally, we investigated the ability of Klf4 cKO MuSCs to self-renew ex vivo by culturing cells for 7 days, quantifying the number of PAX7+ reserve cells and looking at their cell cycle status by an overnight EdU chase. Klf4 cKO MuSCs were able to self-renew similarly to the Control (Figure 4E).

In order to study the role of Klf4 in MuSCs in vivo, we injured TAs of Klf4 cKO mice and looked at the ability of these cells to enter the cell cycle following BaCl2 induced injury (Figure 5A-C). MuSCs lacking Klf4 were able to properly go through the G1 phase following activation (Figure 5A). We also looked at the morphology of the muscle and quantified the number of PAX7 + MuSCs 14 days following injury (Figure 5B). Even though we were lacking a suitable number of biological replicates for statistical analyses, we decided to push the system and perform 2 consecutive injuries of the TAs of Control and Klf4 cKO mice and found that Klf4 depleted MuSCs were able to regenerate the muscle after 2 rounds of injury and properly self-renew as there was no difference in the number of PAX7 + MuSCs in the cKO compared to the Control (Figure 5C). All these data indicated that Klf4 is not essential for any of the major functions of MuSCs both ex vivo and in vivo.

Klf4 is a member of a big family of Krüppel-like transcription factors, all of which are highly conserved and share structural similarities, giving them, in some cases, common and overlapping functions (McConnell and Yang, 2010) [START_REF] Yamane | Overlapping functions of Krüppel-like factor family members: targeting multiple transcription factors to maintain the naïve pluripotency of mouse embryonic stem cells[END_REF]. Furthermore, several Klf factors other than Klf4 are also induced during the early activation of MuSCs (Figure 5D). For this reason, we wondered whether another Klf factor is compensating the depletion of Klf4 from MuSCs. To address this, we looked at the expression of Klf1, Klf2, Klf5, and Klf6 that are from the same group and thus structurally close to Klf4 (McConnell and Yang, 2010) during MuSCs early activation as well as Klf9 and Klf16 as they are activated upon MuSCs quiescence exit (Figure 5D). However, the expression of these Klfs was not further induced following Klf4 depletion compared to the Control (Figure 5E). This result does not exclude that there is functional redundancy but suggests that the absence of Klf4 does not trigger a compensatory overexpression of related genes. Of course, a double mutant of Klf4 and each of these Klf factors would be needed to test their overlapping functions.

SRF is not the main regulator of IEGs during MuSCs activation

Several IEGs are induced within 15 minutes of MuSCs activation, they are also a part of the identified stress response happening in all tissues upon dissociation [START_REF] Machado | Tissue damage induces a conserved stress response that initiates quiescent muscle stem cell activation[END_REF]. SRF is known as the main regulator of several of these genes such as Egr1/2, Fos, Jun, and Fosl1 upon mitogen and stress signals [START_REF] Bahrami | Gene regulation in the immediate-early response process[END_REF]. Srf is also induced very early during MuSCs dissociation (Figure 6A). For this reason, we decided to look at the role of Srf in the ability of MuSCs to enter the cell cycle following activation. To do this, we generated a Srf cKO in MuSCs by crossing the Tg:Pax7-CreER T2 ; Rosa26 mTomato- STOP-mGFP to the Srf Flox allele harboring loxP sites flanking exon 2 of Srf, thus generating the Tg:Pax7-CreER T2 ;Srf flox/flox Rosa26 mTomato-STOP-mGFP and Control Tg:Pax7-CreER T2 ;

Rosa26 mTomato-STOP-mGFP . Following tamoxifen induction, we sorted the early activated GFP+ MuSCs and saw a 97.5% decrease in the expression of Srf in the cKO confirming the efficiency of the recombination. We also looked at the expression of several IEGs known to be Srf targets in the Srf cKO early activated MuSCs (Figure 6B-C). However, with the exception of Egr1 and Egr2, Srf cKO did not affect the expression of several genes known to be regulated by SRF or myogenic genes. Furthermore, we cultured the sorted MuSCs in the presence of EdU for 43 hours to look at the ability of the cells to enter the S phase. Nevertheless, there was no difference between the Control and the Srf cKO MuSCs to enter the cell cycle, nor in the expression of MYOD (Figure 6B). All these results indicated that Srf is not required for the induction of all IEGs and for the quiescence exit of MuSCs.

Discussion

We have identified IEGs and Klf4 as part of the tissue damage induced stress response.

And as part of investigating the role of this response in MuSCs activation, we wanted to investigate the role of Klf4 and Srf, an activator of IEGs, during the early activation of MuSCs.

Even though overexpression of Klf4 in primary myoblasts indicated a potential role of Klf4 in regulating the cell cycle, knock out of Klf4 in MuSCs did not affect the ability of these to activate, proliferate and eventually differentiate and fuse to form myotubes. Similarly, Srf was not required for the induction of several IEGs as well as the entry of MuSCs into the cell cycle.

As we previously stated, several Klf factors have been shown to play similar or compensatory functions as Klf4. Of note, Klf6 and Klf9 are highly induced during MuSCs activation, and Klf6 has the same activation kinetics as Klf4 and is also a part of the stress response we identified (Machado et al., 2021a). Moreover, even though Klf2 is not induced during MuSCs quiescence exit, it is the closest paralog to Klf4 and has been shown to have similar functions in some cases (McConnell and Yang, 2010) (Sunadome et al., 2011, p. 5) [START_REF] Chiplunkar | The Krüppel-like factor 2 and Krüppel-like factor 4 genes interact to maintain endothelial integrity in mouse embryonic vasculogenesis[END_REF] [START_REF] Sangwung | KLF2 and KLF4 control endothelial identity and vascular integrity[END_REF]. A double deletion of those candidate Klf factors along with Klf4 is required for testing this hypothesis. Furthermore, a role of Klf4 in the fusion of myocytes has been uncovered in C2C12, that we were not able to reproduce in primary cells, whether this is due to the difference between primary cells and cell lines or the difference between a gene knock-out and knock-down remains unknown [START_REF] El-Brolosy | Genetic compensation triggered by mutant mRNA degradation[END_REF].

Furthermore, deletion of Srf from MuSCs was not required for their early activation.

However, since the expression of several IEGs was not affected following Srf deletion, it seems that Srf is not the regulator of these genes following stress induced dissociation. panel). Data from snRNA-seq [START_REF] Machado | Tissue damage induces a conserved stress response that initiates quiescent muscle stem cell activation[END_REF].

(B) Klf4, Fos, Jun, and MyoD normalized expression from bulk RNAseq data [START_REF] Machado | Tissue damage induces a conserved stress response that initiates quiescent muscle stem cell activation[END_REF]

Material and methods

Animals

The following mouse lines were kindly provided by the corresponding laboratories as described: Tg:Pax7-nGFP [START_REF] Tajbakhsh | Skeletal muscle stem cells in developmental versus regenerative myogenesis[END_REF](Sambasivan et al., 2009), Klf4 flox [START_REF] Katz | The zinc-finger transcription factor Klf4 is required for terminal differentiation of goblet cells in the colon[END_REF] and Srf flox (generously provided by Athanassia Sotiropoulos) were crossed to the Tg:Pax7-CreER T2 mouse line [START_REF] Tajbakhsh | Skeletal muscle stem cells in developmental versus regenerative myogenesis[END_REF](Mourikis et al., 2012b), and the Cre reporter line

Rosa26 mTomato-STOP-mGFP [START_REF] Muzumdar | A global double-fluorescent Cre reporter mouse[END_REF]. Pax7 CreERT2 (G. Kardon; Jackson Laboratories, stock 017763; (Murphy et al., 2011) mice were crossed to Rosa26 Stop-tdTomato [START_REF] Madisen | A robust and highthroughput Cre reporting and characterization system for the whole mouse brain[END_REF]. Animals were handled as per French and European Community guidelines and protocols were approved by the ethics committee at the French Ministry (Project No: 20-027 #24357).

Tamoxifen induction and muscle injury

Cre recombination was induced in both control Tg:Pax7-CreER T2 ; Rosa26 mTomato-STOP- mGFP and Klf4 cKO Tg:Pax7-CreER T2 ;Klf4 flox/flox ;Rosa26 mTomato-STOP-mGFP with 2 weeks of tamoxifen diet.

For muscle injury, mice were anesthetized with 3% isoflurane and both TAs were injected with 50mL of 1.2% BaCL2 (Sigma, 202738). BrdU (Sigma, #B5002) at 1mg/mL was added in the drinking water supplemented with 2.5% sucrose.

Cell culture of primary muscle cells

MuSCs were isolated by FACS from male and female Tg:Pax7-nGFP mice and plated on Matrigel-coated (Corning, 354248), 8-chamber slides (Sarstedt, 94.6140.802) in growth medium (GM) composed of DMEM (GIBCO) with 20% fetal bovine serum (FBS), 1% penicillin/streptomycin (GIBCO), and supplemented with 5 ng/ml basic FGF (Peprotech, 450-33) at 37 • C. Whole muscle bulk preparations were cultured on Matrigel-coated 8-chamber Sarstedt slides in GM at 37 • C in the presence of 10 mM EdU when specified. Muscles from mice were dissected and chopped on ice, then digested following standard procedures i. Digested muscle was then filtered through 100 mm and 70 mm cell strainers, spun down at 600 g/ 5 min, then resuspended in cold DMEM and filtered through a 40 mm cell strainer and eventually spun down at 600 g for 5 min. All filtering steps were done on ice.

For overexpression experiments, muscle bulk from tamoxifen induced Pax7 CreERT2 ; Rosa26 stop- tdTomato was pre-plated on non-coated dishes in growth medium overnight. Non-adherent myoblasts were then transferred to Matrigel coated 8-chamber slides and transduced with KLF4-GFP or MOCK-GFP retroviruses produced by transfecting Hek-293T cell lines and collecting the retrovirus containing medium 3 days post transfection.

KLF4-GFP and MOCK-GFP plasmids contained an eGFP sequence under the control of a MiG promoter. For the KLF4-GFP plasmid, the ORF of Klf4 was added and was separated from eGFP by an IRES sequence.

Single fibers isolation

Single fibers were isolated from the EDL as previously described [START_REF] Moyle | Isolation, culture and immunostaining of skeletal muscle fibres to study myogenic progression in satellite cells[END_REF]. Briefly, EDL Tg:Pax7-nGFP was dissected and digested for 2 hours at 37°C in a filtered solution of 0.2% collagenase (Sigma) in DMEM supplemented with 1% Penicillin/Streptomycin. Mechanical dissociation was used after connective tissue digestion to release individual myofibers. After that, single myofibers were fixed with 4% PFA for 10 minutes (T2h). Before immunostaining, myofibers were washed three times with 1x PBS and kept at 4°C. Freshly isolated myofibers were cultured in myofiber growth media comprising DMEM, 20% serum and and 1% chicken embryo extract (MP-Biomedical) at 37°C, 5% CO2

and then recovered for fixation 2h or 4h following dissociation (T4h and T6h respectively).

For T0h fibers, EDL from Tg:Pax7-nGFP mice was dissected and fixed with 0.5%PFA for 30 minutes, myofibers were then mechanically pealed and kept at 4°C for immunostaining.

Staining

For cell culture, following fixation (2% paraformaldehyde (PFA), 20 min) MuSCs, muscle sections or myofibers were washed three times in PBS, permeabilized, and blocked using a blocking solution (BS) containing 5% BSA (Jackson Laboratories 001-000-162) and 0.25% Triton X-100 (Sigma) for 1 hour at room temperature. The samples were then incubated overnight with primary antibodies. After three washes, the cells were incubated with the corresponding secondary antibodies for 1 hour at RT. The samples were subsequently washed three times with PBS and stained for EdU, when added, and Hoechst (EdU Click-iT PLUS Kit C10640, Life Technologies) following the manufacturer guidelines before a final wash and mounting.

For PAX7 immunostaining on sections, muscle sections were permeabilized with methanol (-20°C) for 6 minutes, then antigen retrieval was performed by boiling the samples for 3 minutes in Citric acid (1mM, pH 6) and incubated at RT for 20 min. Samples were then blocked and immunostaining was completed as previously described.

RNA extraction

A minimum of 40 x10 3 FACS isolated MuSCs from Tg:Pax7-nGFP mice per replicate were used per time point. And RNA was extracted using the Qiagen RNeasy Micro Kit following the manufacturer's guidelines.

RT-qPCR

Reverse transcription was performed using the SuperScript IV VILO Master Mix (Thermofisher, 11756050) following the manufacturer's guidelines. RT-qPCR was performed using the Power SYBR Green PCR Master Mix (Applied Biosystems, 4367659).

Statistical analyses

For comparison between two groups, unpaired Student's t test was performed to calculate p values and to determine statistically significant differences (*p < 0.05; **p < 0.01; ***p < 0.001).

List of qPCR primers

Gene (mouse)

Forward sequence 5' -3' Reverse sequence 5' -3'

Pax7

GACAAAGGGAACCGTCTGGAT TGTGAACGTGGTCCGACTG

Myod1

CACTACAGTGGCGACTCAGATGCA CCTGGACTCGCGCGCCGCCTCACT

Fos

CTACCATTCCCCAGCCGAC GTTGGCACTAGAGACCGACA

Jun

GGAACAGGTGGCACGCTTA GCTGCGTTAGCATGAGTTGG

Fosb

AGCTAAGTGCAGGAACCGTC ACTCAAGCTCTGCCTTTTCC

Klf4

GATGCAGTCACAAGTCCCCT CCTTCTTCCCCTCTTTGGCTT

p21

GTACTTCCTCTGCCCTGCTG GGGCACTTCAGGGTTTTCTC

Myc

GATTCCACGGCCTTCTCTCC GCCTCTTCTCCACAGACACC

Hes1

ACACCGGACAAACCAAAGAC AATGCCGGGAGCTATCTTTC

HeyL

GTCTTGCAGATGACCGTGGA CTCGGGCATCAAAGAACCCT

Fosl1

AGCTCCGGGGCTGGC ATGCTTGGCACAAGGTGGAA

Junb

AGGCAGCTACTTTTCGGGTC TTGCTGTTGGGGACGATCAA

Klf1

ACTTTGGCACCTAAGAGGCAG TTCTCTCCCGTGTGCGTG

Klf2

GCGTACACACACAGGTGAGA TTTCGGTAGTGGCGGGTAAG

Klf5

GAGGACTCATACGGGCGAGA CTTCCTGTAGTGGCGGGTC

Klf6

CCCACTGTGACAGGTGTTTCT GCCTACAGGATTCGTCCCTC

Klf9

AGTGCATACAGGTGAACGGC ATCCGAGCGCGAGAACTTTT

Klf16

GACTCACACAGGTGAGCGG CTCATCAGAACGGGCGAACT

Srf

TGGGGAAACCAAGGACACAC GGTGCTGTCTGGATTGTGGA

Egr1

GAGCACCTGACCACAGAGTC CGAGTCGTTTGGCTGGGATA

Egr2

TGACCAGATGAACGGAGTGG GCGAAGCTACTCGGATACGG

Tbp

ATCCCAAGCGATTTGCTG CCTGTGCACACCATTTTTCC

List of genotyping primers

Gene (mouse) Sequence 5' -3'

Klf4 exon 1

Discussion

1-Summary

In all tissues, each cell type resides in its own home or niche, receiving and producing all the necessary queues to maintain its comfort and the overall tissue homeostasis. These queues include growth factors, cell-cell and cell-ECM interactions, and mechanical stimuli.

One can imagine that any damage to the home of these cells, let alone a complete destruction, that can be caused by the dissociation of the tissue using harsh mechanical and enzymatic methods or injury causing the necrosis of the tissue, will stress the cells and lead to their breakage from their comfortable state.

In the present work, we have showed that cells from all tissues exhibit a common response to stress following tissue damage both ex vivo in response to dissociation and in vivo in response to chemical induced injury ultimately leading to their activation. This response included several transcription factors such as the ERK1/2 MAPK, the polyamine synthesis pathway, several IEGs, Krüppel-like factors such as Klf4 and Klf6, transcriptional regulation, cytoskeletal organization, inflammation pathways, and so on. Furthermore, each cell type, in parallel to this common stress response, induces its own specific response to adapt to these vast sudden alterations.

When it comes to MuSCs, residing in a dormant quiescent state in the skeletal muscle, we were able to dissect the kinetics of the dissociation induced changes, uncovering the ERK1/2 MAPK pathway and IEGs as one of the first genes to be induced, as early as 15 min of dissociation, followed by the induction of myogenic genes such as MyoD and the downregulation of quiescence specific genes such as Pax7 and the Notch signaling pathway.

Interestingly, we also showed that following injury in vivo, MuSCs exhibit the same response but with slower kinetics. Moreover, we managed to show that, at least in the TA muscle, all cells cluster in one homogenous truly quiescent cluster and ultimately activate following the same trajectory of activation. This comes in contrast to some studies showing that following dissociation a more quiescent subpopulation of MuSCs remains quiescent (van den Brink et al., 2017) Furthermore, we were able to show that this stress response in important for these cells to be able to activate. Indeed, we showed that the ERK1/2 MAPK pathway is required in the G0 -G1 -S transition of the cells, while the Notch pathway inhibition is required for their

2-Methods to preserve the transcriptome of the cells

In addition to uncovering a common stress response happening in several tissues following dissociation. This work, as well as previous work from our laboratory have managed to optimize protocols in order to isolate cells from tissues while preserving their in vivo state.

In parallel, several labs are working on protocols to preserve the transcriptional landscape of the cells. Here, I will discuss some of these protocols with their advantages and weaknesses.

These have also been discussed in much more details in a recent review (Machado et al., 2021) These protocols include paraformaldehyde-based tissue fixation prior to dissociation also known as iSiFi (Machado et al., 2017). As a matter of fact, paraformaldehyde (PFA) has been shown to preserve RNA integrity for bulk RNA-seq techniques (Machado et al., 2017), however, cells do not survive PFA fixation which is a limiting step of the protocol.

Another method is nascent RNA tagging, to specifically tag cell specific nascent RNA before isolation. Unlike tissue fixation, this method preserves cells viability, and has the advantage of capturing the transcriptome of cells at a specific time in vivo [START_REF] Gay | Mouse TU tagging: a chemical/genetic intersectional method for purifying cell type-specific nascent RNA[END_REF].

This technique is not commonly used due to its complexity and the difficulty of its application in some tissues. Cell viability can also be preserved with the use of inhibitors of transcription during dissociation in the aim of preserving the transcriptional integrity of cells (van Velthoven et al., 2017). Nevertheless, this method doesn't consider the epigenetic as well as the post translational modifications, both, highly dynamic phenomena that happen very early on during quiescence exit and activation. Moreover, it is impossible to ensure homogenous diffusion of the inhibitors through dissociating tissues (Machado et al., 2021).

Some groups have worked on cold tissue dissociation using cold active proteases [START_REF] Mattei | Enzymatic Dissociation Induces Transcriptional and Proteotype Bias in Brain Cell Populations[END_REF]. Although this method preserves the transcriptional and viability of cells, it fails to take into consideration the cold induced changes that can happen to cells, modifications that remain very ill defined until today [START_REF] Fuller | Gene expression in response to low temperatures in mammalian cells: a review of current ideas[END_REF]. In brief, even though this method succeeds in preventing dissociation induced changes that are already very well described, it doesn't necessarily manage to conserve the transcriptional integrity of the in vivo cells.

It is now feasible to perform high throughput transcriptomics in situ, making it possible to capture the transcriptome of the cells while preserving tissue integrity (Eng et al., 2019). It is also possible to perform micro-dissections on frozen samples followed by next generation sequencing [START_REF] Song | Transcriptomic comparison of human and mouse brain microvessels[END_REF]. These techniques are still fairly new and lack, until today, the feasibility and simplicity to be an ideal substitution for cell dissociation techniques but have the potential to become it in the future [START_REF] Liao | Uncovering an Organ's Molecular Architecture at Single-Cell Resolution by Spatially Resolved Transcriptomics[END_REF].

Given the fact that the alterations happening following dissociation have been identified. It is now possible to "clean" existing datasets form stress induced genes or remove identified stress genes or even stress genes and stressed cells in silico [START_REF] Romanov | Molecular design of hypothalamus development[END_REF].

This cleaning method doesn't consider all the alterations happening in cells, and even worse, can end up adding even more alterations rather than removing them.

Until today, the only method that allows transcriptomic analyses on a large scale while preserving the in vivo state of cells is the use of single nucleus RNA sequencing (snRNA-seq) in which nuclei are directly and rapidly extracted from tissues [START_REF] Grindberg | RNAsequencing from single nuclei[END_REF]. Even though this method only allows the analyses of nuclear RNA, it still reflects the transcriptome of the entire cell [START_REF] Gaidatzis | Analysis of intronic and exonic reads in RNA-seq data characterizes transcriptional and post-transcriptional regulation[END_REF]. Applying snRNA-seq in various tissues has been shown to circumvent dissociation induced genes (Machado et al., 2021a).

3-Triggering events of the stress response

Indeed, our use of the term stress response to define the transcriptional changes happening during dissociation might be misleading, as the word "stress" is usually associated with an uncontrolled response following adverse events. However, having uncovered the importance of this response during the early activation of MuSCs, it becomes clear that cells have adapted this response to be able to overcome all the challenging events during dissociation and eventually properly activate, differentiate, or self-renew. As accurately described in a recent review about adaptation to stress, "MuSCs are comfortable being uncomfortable" [START_REF] Gugliuzza | Muscle stem cell adaptations to cellular and environmental stress[END_REF].

Nevertheless, it remains unclear what is the triggering event of this stress response in MuSCs. During dissociation, cells are extracted from their microenvironment, losing all interactions with its components. Furthermore, they are subjected to a burst of growth factors coming from the damaged muscle fibers and other cells. Indeed, growth factors play an important role in the activation of MuSCs [START_REF] Montarras | Lying low but ready for action: the quiescent muscle satellite cell[END_REF]. Additionally, cells are subjected to mechanical dissociation, that might also play a role in their activation. Of note, recent studies have shown that MuSCs have the ability to mechanically sense their microenvironment [START_REF] Ma | Piezo1 regulates the regenerative capacity of skeletal muscles via orchestration of stem cell morphological states[END_REF]) [START_REF] Kann | An injury-responsive Rac-to-Rho GTPase switch drives activation of muscle stem cells through rapid cytoskeletal remodeling[END_REF].

Although this work has managed to uncover the steps of the early activation events of MuSCs at a minute scale at the transcriptional level, it does not address the triggering events of all these changes. Seeing that the mechanical changes and the burst of growth factors happen simultaneously during dissociation, it is not so simple to separate the two and study their implication in the early activation of MuSCs.

Furthermore, our data suggest that the ERK1/2 MAPK pathway is one of the first pathways to be induced upon quiescence exit, as it is upregulated in the first 15 minutes of dissociation. However, it remains unclear whether it is really the first pathway to be activated upon quiescence exit and is the one responsible for the activation of all the pathways that are induced later. In fact, a small-scale RT-qPCR of stress core genes in MuSCs treated with an ERK1/2 inhibitor during dissociation, showed that not all these genes were affected by this inhibition, indicating that even though this pathway is induced very early, it is probably not the one triggering the induction of all the pathways that we see upregulated during early activation.

It is also noteworthy that the ERK1/2 pathway is implicated in several functions such as

4-Deciphering the chronological chain of events upon

MuSCs quiescence exit

It is noteworthy, that even though the stress core we have identified is comprised of 98 genes only, this core is a very small list of common genes upregulated in several tissues.

Focusing on MuSCs, we managed to identify around 1,500 genes that are upregulated upon dissociation and 1,500 downregulated ones. We also succeeded in following the activation trajectory of these genes during the two hours of dissociation. Our gene ontology analysis has showed a chronological order of events happening from MAPK pathway activation and cell cycle regulation, transcriptional regulation, followed by splicing regulation and translation, ribosome biogenesis and finally to metabolism switch.

Indeed, our work has showed that the ERK1/2 MAPK pathway is one of the first pathways to be activated upon MuSCs quiescence exit and is required for their G0 -G1 -S transition. This pathway functions in parallel with the Notch pathway regulating their myogenic program. We also showed that the polyamine synthesis probably comes up later and plays a dual role for both activation and differentiation. However, these pathways comprise only a small number of genes and pathways from the thousands of upregulated and downregulated genes. It remains unclear the interplay between these pathways and their chronological at the functional level.

5-The elixir of quiescence

Even though it is believed that quiescent MuSCs have a higher capacity to differentiate and self-renew compared to cultured myoblasts, this hypothesis has never been proven, because all the studies compare freshly isolated, early activated MuSCs to proliferating myoblasts.

Of course, several protocols have been published that tried to maintain the quiescence of MuSCs in culture conditions in the aim to test this hypothesis. However, none of these protocols managed to come up with the perfect cocktail that maintains the quiescence of those cells. During my thesis, having uncovered two important pathways, one implicated in the cell cycle of MuSCs and the other one implicated in their myogenic commitment, we tried to test whether by manipulating these two pathways would give the cells the kickstart they need to revert to quiescence. Of course, our protocol is still not perfect, but our data suggests that we were on the right path in amplifying MuSCs and then inducing their quiescence.

Combining ectopic Notch expression to inhibit the differentiation of MuSCs ex vivo followed by ERK1/2 inhibition to stop them from proliferating allowed us to obtain a small fraction of quiescent-like PAX7 +/ EdU -/CALCR + MuSCs. Indeed, the next challenge would be to increase this fraction. Our observations lead us to believe that inhibition of ERK1/2 is resulting in an heterogenous population in which committed cells would differentiate with ERL1/2 inhibition and non-committed cells would go back to quiescence considering that it has been recently shown that ERK1/2 is involved in the fusion of myoblasts (Eigler et al., 2021, p. 2). Furthermore, we noticed that prolonged SCH772984 treatment is causing cell death which is consistent with a recent study showing that ERK1/2 is required in MuSCs viability [START_REF] Yin | Satellite Cells and the Muscle Stem Cell Niche[END_REF]. In the light of these observations, we wondered whether an earlier treatment with the inhibitor but for the same amount of time would be helpful in increasing the efficiency of our protocol.

Furthermore, we believe that coating our culture plated with Dll1-Fc is not the most efficient way to robustly induce the Notch pathway. This also brings forward the disadvantage of working in a 2D system. Indeed, we believe that applying our protocol in a 3D system in which cells are plated in a gel containing immobilized Dll1 would help in increasing its efficiency.

After establishing the optimization steps this protocol is still lacking, it is also important to mention that we cannot consider that cells are quiescent-like just based on a few markers.

Actually, a sc-RNA sequencing would be needed to better characterize the different expression profiles of the cells. Furthermore, at the functional level, it would be interesting to measure the length of the G1 phase of our quiescent-like MuSCs, knowing that quiescent MuSCs take about 35 hours to go through the G1 phase [START_REF] Rocheteau | A subpopulation of adult skeletal muscle stem cells retains all template DNA strands after cell division[END_REF]. Ultimately, we still need to test the regeneration potential of those cells. We have optimized an ex vivo protocol to investigate the differentiation and self-renewal of MuSCs that we can use, before eventually performing engraftment experiments to look at their ability of those cells to form new fibers and self-renew in vivo.

Finally, it is important to highlight that the advantage of optimizing such a protocol is not limited to testing the regeneration potential of quiescent MuSCs. This system has great advantages especially in the field of cell-based therapy. For instance, it can be combined with differentiation protocols of iPSCs into MuSCs. As a matter of fact, even though recent protocols managed to get a pure population of myogenic cells (Bou Akar et al., under revision), combining our system with the differentiation protocol of iPSCs can help enrich this myogenic population with a true MuSCs population. This increase of the quiescent, PAX7 expressing MuSCs population might help in the use of those cells for a better understanding of muscle stem cells in diseases and drug screening. Moreover, as we have discussed the differentiation in the regeneration potential of MuSCs in comparison to their activated counterparts, an enrichment of the MuSCs population in those protocols would increase their potential for engraftment purposes. Finally, as one of the biggest limitations of cell-based therapies is the loss of the regeneration potential of cells in culture, this protocol can be used to bypass this limitation.

6-Klf4 and Srf induced but not required during MuSCs quiescence exit

During my thesis, I was able to show that Klf4 even though highly induced during MuSCs early activation, its deletion did not affect the ability of the cells to activate or differentiate. However, we did not appropriately address the possibility of a redundant function of another Klf factor that is compensating the absence of Klf4. As already discussed, there are several candidate Klf factors to be tested such as Klf2 and Klf6. However, since the window of early activation is very short, the only way to test the redundancy of those factors is to generate a conditional double knock out mouse line to induce the deletion of both Klf4 and Klf2 or Klf6 from MuSCs.

Furthermore, we wanted to investigate the role of the IEGs we see induced during the first 15 minutes of dissociation. For this, we decided to conditionally knock-out Srf from MuSCs as it is known to be a regulator of those IEGs. However, Srf deletion did not affect the expression of many of those IEGs and had no effect on the activation of MuSCs. Interestingly, a recent study has showed that the RhoA-Act pathway is required for MuSCs quiescence exit through MRTF, the cofactor of SRF [START_REF] Kann | An injury-responsive Rac-to-Rho GTPase switch drives activation of muscle stem cells through rapid cytoskeletal remodeling[END_REF]. In contrast, another study on Srf has shown that conditional deletion of Srf from MuSCs did not affect the proliferation of myoblasts but their ability to fuse and form myotubes [START_REF] Randrianarison-Huetz | Srf controls satellite cell fusion through the maintenance of actin architecture[END_REF]. Furthermore, our RNA-seq data during MuSCs dissociation shows that Srf is induced slightly later than IEGs.

Additionally, published data showed that even though all MuSCs express SRF following 24 hours of culture, only 20% of them expressed SRF in early activated MuSCs right after dissociation [START_REF] Randrianarison-Huetz | Srf controls satellite cell fusion through the maintenance of actin architecture[END_REF]. All these data had led us to believe that Srf deletion could cause a small delay in MuSCs activation, which did not turn out to be the case.

Briefly, even though Srf is known as a main activator of several IEGs [START_REF] Bahrami | Gene regulation in the immediate-early response process[END_REF], our data showed that this might not be the case of MuSCs. Whether there is another factor that might be compensating the deletion of Srf, or the expression of those IEGs was not affected because they can be regulated by several other pathways remains unclear.

7-Concluding remarks

Our work has demonstrated that a common stress response is induced in all tissues both ex vivo and in vivo following tissue damage. This highlights the worrisome impact of contamination by dissociation induced genes in big, published atlases. Furthermore, focusing on MuSCs, we have managed to identify a new cellular state of early activation at a minute scale and show that a cellular event requires only hours, even minutes.

Furthermore, we have managed to identify several pathways such as the ERK1/2

MAPK and polyamine synthesis pathways that are required for this early activation. However, as mentioned above, given the big number of induced genes, we are far from uncovering all the pathways implicated in this step and the interaction between all of them. It is also noteworthy that there are almost the same number of downregulated genes as there are induced ones during quiescence exit. During my PhD, we mainly focused on the induced pathways, and with the exception of the Notch signaling pathway, we neglected the quiescence specific pathways that still need to be investigated.

Finally, we also showed the great implications of uncovering these different pathways, as we showed that by manipulating them, we can optimize a protocol that allows us to induce quiescence of MuSCs ex vivo following their amplification. Even though we did not manage to fully optimize it, I would like to believe that we were on the right track to doing so.

Indeed, this protocol would have vast implications in studying quiescence, increasing the efficiency of iPSCs differentiation into MuSCs and eventually cell-based therapies.

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

INTRODUCTION

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, 2020;Jessen et al., 2015). Differentiated cells too can get activated and eventually proliferate following de-differentiation, as is the case for hepatocytes after hepatectomy (Riehle et al., 2011) or neonatal mouse cardiomyocytes (Porrello et al., 2011). Cell death pathways, however, can also be instigated when cell injury is irreversible (Rathore et al., 2017). 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 (F) Gene Ontology analysis of the stress core using the biological process category. Selected terms with corrected p values < 0.05 are displayed and ranked by fold change.

(legend continued on next page) Machado et al., 2017;van Velthoven et al., 2017). 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., 2017;Bakken et al., 2018;Lacar et al., 2016;[START_REF] O'flanagan | Dissociation of solid tumor tissues with cold active protease for single-cell RNA-seq minimizes conserved collagenase-associated stress responses[END_REF]Wu et al., 2017Wu et al., , 2019)). 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.

RESULTS

Definition of a core stress signature in muscle and liver in response to cell dissociation In order to test the impact of cell dissociation across cell types and tissues, we performed single-nucleus RNA sequencing (snRNAseq) 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) [START_REF] Bohmann | Human proto-oncogene c-jun encodes a DNA binding protein with structural and functional properties of transcription factor AP-1[END_REF]. 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., 2017), validating the single-nucleus approach (Figures S1I andS1J). Analysis of the upregulated genes showed enrichment for cell-specific biological processes, as described in Figures S1K-S1N (Ishikawa et al., 2012;van Meeteren and ten Dijke, 2012;Contreras et al., 2019). We then examined the extent to which the dissociationinduced 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 andS2B; 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., 2018), (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 andS2K).

Analysis of the cellular response to in vivo tissue injury

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 snRNAseq atlas from injured livers 4 h post-carbon tetrachloride (CCl 4 ) injection (Teixeira-Clerc et al., 2010) and compared with to uninjured liver . 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 (BaCl 2 ) (Casar et al., 2004). We first determined the stress-response kinetics (Figure S3E) and generated snRNA-seq atlases from muscles collected 4 h post-BaCl 2 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-BaCl 2 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 [START_REF] Street | Slingshot: cell lineage and pseudotime inference for single-cell transcriptomics[END_REF] 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 [START_REF] Camps | Interstitial cell remodeling promotes aberrant adipogenesis in dystrophic muscles[END_REF]Dell'Orso et al., 2019;Giordani et al., 2019;Schaum et al., 2018), 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., 2018a).

Time course analysis of MuSC quiescence exit reveals modules of co-regulated genes

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., 2017) (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 [START_REF] Barton | Defective thymocyte proliferation and IL-2 production in transgenic mice expressing a dominant-negative form of CREB[END_REF].

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., 2000), Spry1 [START_REF] Chakkalakal | The aged niche disrupts muscle stem cell quiescence[END_REF], and Calcr (Yamaguchi et al., 2015) (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.

(B) UMAP plots displaying the different populations inferred from intact and injured liver (top) and muscle (bottom) single-nucleus RNA-seq after data integration using Seurat3. Endo, endothelial cells; T, T cells; B, B cells; FAP, fibro-adipogenic progenitor; Teno, tenocytes; SM, smooth-muscle cells; Macro, macrophages; MuSC, skeletal muscle stem cell; Lymph, lymphatic cells; Adipo, adipocytes; Chol, cholangiocytes. (C) UMAP plots displaying the different populations inferred from intact (blue) or injured (red) liver (top) and muscle (bottom) single-nucleus transcriptomes without data integration. Red box inset in muscle UMAP designates the MuSC and tenocyte populations. (D) UMAP plots representing the stress index among liver (top) and muscle (bottom) intact and injured cells. (E) Graphical scheme of single-MuSC RNA-seq post-injury experiment: TA muscles of Tg:Pax7-nGFP mice were injected with BaCl 2 and single MuSCs were isolated at 0, 2, and 4 h post-injury using iSiFi methodology. Two hundred eighty cells were obtained from six mice (two biological replicates per time point). (F) PCA of resting (T0) and in vivo activated MuSCs (T2 and T4). PC1 and PC2 represent 4% and 3% of variance explained, respectively. (G) Pseudotime-analysis (Slingshot) shows the differentiation trajectory of MuSCs during in vivo activation. Pseudotime values are color-graded from dark blue to dark red for undifferentiated to differentiated cellular state, respectively. (H) Kinetics of the stress response during muscle injury. Representation of the stress index (log 2 ) from the shared stress core genes into each single MuSC (gray dot), ordered by pseudotime value. Polynomial second order regression shown in red and standard error in gray.

Functional role of early response to MuSC activation

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 and3E), whose products form a complex for the synthesis of the polyamine spermidine that is important in cell proliferation, growth, translation initiation and elongation [START_REF] Mandal | Depletion of cellular polyamines, spermidine and spermine, causes a total arrest in translation and growth in mammalian cells[END_REF]. In turn, spermidine is the source of the amino acid hypusine, a unique post-translational addition to the eukaryotic translation initiation factor eIF5A [START_REF] Schuller | eIF5A functions globally in translation elongation and termination[END_REF], which was also found to be transcriptionally induced during activation. To test the role of polyamine biosynthesis on the G 0 -to-S transition of quiescent cells, we cultured freshly isolated MuSCs with the ODC1 inhibitor d,l-a-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+/MyogeninÀ cell population, compared with control (Figures 4A and4B).

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 (Pax7 CreERT2 ; Rosa stop-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, 2014) (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 quiescentlike state for at least 72 h in culture (EdUÀ, Pax7+, MyogÀ) with spindle-shaped nuclei, characteristic of quiescent MuSCs (Machado et al., 2017) (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 andS4P).

DISCUSSION

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 activation of MuSC points toward growth factors' playing a prominent role in this process.

Using specialized protocols, we have generated single-cell atlases that accurately capture the transcriptional states of cells as they exist in the context of their native tissue. Recent studies have shown that dissociation affects the transcriptome of freshly isolated neurons (Bakken et al., 2018;Lacar et al., 2016;Wu et al., 2017), kidney cells (Adam et al., 2017;Wu et al., 2019),

EXPERIMENTAL MODEL AND SUBJECT DETAILS Animals

The following mouse lines were kindly provided by the corresponding laboratories as described: Tg:Pax7-nGFP [START_REF] Tajbakhsh | Skeletal muscle stem cells in developmental versus regenerative myogenesis[END_REF]Sambasivan et al., 2009), R26R stop-NICD-nGFP (D. Melton; Jackson Laboratories, stock 008159; Murtaugh et al., 2003), Pax7-CreERT2 (G. Kardon; Jackson Laboratories, stock 017763; Murphy and Kardon, 2011). Animals were handled as per French and European Community guidelines and protocols were approved by the ethics committee at the French Ministry (Project No: 20-027 #24357).

Cell culture of primary muscle cells MuSCs were isolated by FACS from male and female Tg:Pax7-nGFP mice and plated on Matrigel-coated (Corning, 354248), 8-chamber slides (Sarstedt,94.6140.802) in growth medium (GM) composed of DMEM (GIBCO) with 20% fetal bovine serum (FBS), 1% penicillin/streptomycin (GIBCO), and supplemented with 5 ng/ml basic FGF (Peprotech, 450-33) at 37 C. Whole muscle bulk preparations were cultured on Matrigel-coated 8-chamber Sarstedt slides in GM at 37 C in the presence of 10 mM EdU. Muscles from Tg:Pax7-nGFP mice were dissected and chopped on ice, then digested following standard procedures in the presence or absence of 2 mg/mL SCH772984 or DMSO control. Digested muscle was then filtered through 100 mm and 70 mm cell strainers, spun down at 600 g/ 5 min, then resuspended in cold DMEM and filtered through a 40 mm cell strainer and eventually spun down at 600 g for 5 min. All filtering steps were done on ice. For Pax7-CreERT2; R26 Stop-NICD-nGFP and Tg:Pax7-nGFP bulk muscle preparations, growth medium was supplemented with 5 ng/mL of basic FGF and cells were cultured in the presence of DMSO (control) or 1 mg/ml SCH772984. For ERK inhibition, cells were plated in the presence of the ERK1/2 inhibitor SCH772984 (Selleckchem, S7101) at 1 mg/ml or DMSO, in the presence of 10 mM 5-ethynyl-20-deoxyuridine (EdU), (Click-iT PLUS Kit C10640, Life Technologies). For ODC1 inhibition, cells were plated in the presence or absence of 2.5 mM DFMO (Tocris, 2761) resuspended in DMEM with 10 mM EdU for the first time point.

METHOD DETAILS

Muscle and liver injury

For muscle injury, mice were anaesthetized with an intraperitoneal injection of 1.6:1 ketamine and xylazine (2%, VMD), 60 mL per 20 g of bodyweight. Both TA muscles were injected with 50 mL of 1.2% BaCl2 (Sigma, 202738). For liver injury, mice received an intraperitoneal injection of carbon tetrachloride (CCl 4 , Sigma, 289116 at 0.5 ml/kg bodyweight, 1:5 dilution in olive oil) as described previously described (Teixeira-Clerc et al., 2010).

Single-nucleus isolation

For the intact/dissociated muscle and liver atlases 6 tibialis anterior, gastrocnemius, soleus, plantaris, and extensor digitorum longus muscles as well as 3 whole liver from 3 8-week old female mice were used. For the intact/injured muscle atlas, 8 tibialis anterior muscles from 4 8-week old female mice were used. Tissues were dissected and pulled in cold PBS with 0.2 U/ml RNase inhibitor (Roche, 03335399001). PBS was removed and tissues were minced in 1 mL cold lysis buffer (10mM Tris-HCl, 10mM NaCl, 3mM MgCl2, and 0.1% NonidetTM P40 in Nuclease-Free Water) with scissors. After 2 min, 4 mL of cold lysis buffer was added and tissues were lysed for 3 min at 4 C. 9 mL of cold wash buffer (PBS, BSA 2% and 0.2 U/ml RNase inhibitor) was subsequently added and the lysates were homogenized with 10 strokes of loose pestle using a 15 mL Dounce homogenizer. The homogenates were filtered with 70 mm and 40 mm cells strainers. Nuclei were pelleted by centrifugation for 5 min (500 g, 4 C). Nuclei were washed in 1 mL of cold wash buffer, transferred in a 1.5 mL tube and centrifuged 5 min (500 g, 4 C). The pellets were resuspended in 250 mL of wash buffer and stained during 15 min in the dark at 4 C with DAPI at 10 mg/ml. The nuclei were therefore washed and spun down 5 min (500 g, 4 C), resuspended in 300 mL of wash buffer and filtered with 30 mm cell strainers. Nuclei were then FACS sorted for Hoechst to exclude debris with a BD FACSAria III and the BD FACSDIVA software.

Staining

For cell culture, following fixation (2% paraformaldehyde (PFA), 20 min), MuSCs were washed three times in PBS, permeabilized, and blocked using a blocking solution (BS) containing 5% BSA (Jackson Laboratories 001-000-162) and 0.25% Triton X-100 (Sigma) for 60 min at room temperature. The samples were subsequently washed three times with PBS and stained for EdU and Hoechst (EdU Click-iT PLUS Kit C10640, Life Technologies) following the manufacturer guidelines before a final wash and mounting. For tissue sections, dissected and injured TA muscles were immediately frozen in liquid-nitrogen-cooled isopentane and sectioned transversely at 8 mm. The sections were fixed for 10 min with 4% PFA, washed three times, permeabilized and blocked with BS for 1 h at RT before being incubated with anti-laminin primary antibody (Sigma, L9393, 1:500) overnight. After three washes, the sections were incubated with the corresponding secondary antibodies and Hoechst (1:1000) for 1 h at RT.

Western blot

Hind limb muscles were dissected and directly processed or digested for 15 min at 37 C in 1X digestion solution. Before lysis, tissues were fixed in 0.5% PFA for 1 h at 4 C and then washed 3 times in cold PBS (600 g, 5 min). The samples where then re-digested for 2 h in 2X digestion solution, minus the initial digestion time, before being filtered successively through 100 mm and 70 mm cell strainers

In situ fixation treatment and reagents For ex vivo dissociation, after mouse sacrifice muscles were dissected (hindlimb and forelimb) and minced in small pieces. Tissue pool from two mice were separated in 6 groups (T0, T15, T30, T60, T90, and T120). T0 samples were prepared according to the original iSiFi protocol (Machado et al., 2017). Briefly, the samples were fixed for 1 h in 0.5% PFA, washed 3 times in ice-cold PBS and digested for 2 h at 37 C with agitation in 2X digestion solution: Dispase II, 6 U/ml (Roche, 4942078001), Collagenase A 1 U/ml (Roche, 10103586001) and 0.2% BSA:DMEM. T15, T30, T60, T90 and T120 samples were digested for 15, 30, 60, 90 and 120 min respectively in 1X digestion solution: Dispase II, 3 U/ml (Roche, 4942078001), Collagenase A 0.5 U/ml (Roche, 10103586001) and 0.2% BSA/ DMEM at 37 C with agitation. The samples were then spun down (600 g, 5 min) and resuspended in ice-cold 0.5% PFA and incubated for 1 h at 4 C with gentle agitation to fix the cells. The samples were washed 3 time with ice-cold PBS (600 g, 5 min) and resuspended in 2X digestion solution to terminate digestion for 105, 90, 60 and 30 min for T15, T30, T60 and T90 samples respectively. T120 samples were not re-digested. At this point, all the samples were filled with DMEM (GIBCO, 41966-029) and filtered successively through 100 mm and 70 mm cell strainers (MACS, 130-110-917 and 130-110-916 respectively) then span down (600 g, 5 min), resuspended in ice-cold DMEM and filtered through 40 mm cell strainer (Corning, 352340) before a last centrifugation (600 g, 5 min) and resuspension in 0.2% BSA/DMEM for cell sorting.

For the experiment comparing T60 MuSCs in the supernatant to intact tissue pieces, T0, T60 and T120 samples were obtained from the dissection of hindlimbs and forelimbs from the same mice. For this experiment T0 and T120 samples were processed as indicated above. T60 samples were incubated for 60 min in 1X digestion solution. The samples were then separated simply with gravity by incubating the tube (50 mL conical) vertically at RT for 5 min to pellet intact tissue pieces and to carefully pass the supernatant successively through 100, 70 and 40 mm filters (references above) to obtain dissociated cells. Both samples were filled with ice-cold DMEM, span down (600 g, 5 min) and resuspended in ice-cold 0.5% PFA for 1 h at 4 C with agitation. The T60-pellet samples were then re-digested for 60 min in 2X digestion solution. All samples were then filtered as indicated above.

For in vivo activation, after mouse sacrifice injured TA muscles were dissected and minced in small pieces. The samples were prepared according to the original iSiFi protocol (Machado et al., 2017), with the following modification: 0.5 M glycine:PBS was used in the first post-PFA wash.

RNA extraction

For ex vivo dissociation time-course, in vivo activation control and ex vivo ERK1/2 inhibition during dissociation RT-qPCR, the cells were sorted directly in lysis buffer from the Quick RNA FFPE kit (Zymo, R1008) and the RNA extracted following the manufacturer guidelines (without the tissue digestion step). For T60 supernatant/pellet RT-qPCR, the cells were sorted directly in lysis buffer from the RecoverAll FFPE kit (Thermo, AM1975) and the RNA extracted following the manufacturer guidelines (without the tissue digestion step, without the incubation step at 80 C and with the incubation step at 50 C for 1 h instead of 15 min). For in vivo activation time-course, Pax7-nGFP cells were sorted single-cell by FACS in 96 well plates (4titude, 4ti-0960). Each well contained 2.5 ml PKD buffer (QIAGEN, 1034963) supplemented with 1:16 proteinase K (QIAGEN, 19131). Prior to RNA separation, plates were decrosslinked for 1 h at 56 C. RNA separation was performed on a Hamilton STAR according to the G&T-seq protocol (Macaulay et al., 2015). Briefly, oligo-dt30VN-labeled beads and wash buffer were prepared. 10 ml of beads were added to every well with the liquid-handling robot and incubated for 20 min at RT while mixing (2,000 rpm). Thereafter, the beads were washed twice with wash buffer and 5 ml of Reverse Transcriptase master mix was added to every well of the bead-containing 96-well plate.

RT-qPCR

Reverse transcription was performed using the SuperScript IV VILO Master Mix (Thermofisher, 11756050) following the manufacturer's guidelines. RT-qPCR was performed using the Power SYBR Green PCR Master Mix (Applied Biosystems, 4367659).

Library preparation

d For snRNA-seq: Single-cell Gel Bead-In-EMulsions (GEMs) were generated using a Chromium Controller instrument (10x Genomics). Sequencing libraries were prepared using Chromium Single Cell 3 0 Reagent Kits (10x Genomics, 1000074), according to the manufacturer's instructions. Briefly, GEM-RT was performed in a thermal cycler: 53 C for 45 min, 85 C for 5 min. Post GEM-RT Cleanup using Dyna-Beads MyOne Silane Beads (Thermofisher, 37002D) was followed by cDNA amplification (98 C for 3 min, cycled 12 3 98 C for 15 s, 67 C for 20 s, 72 C for 1 min, and 72 C 1 min). After a cleanup with SPRIselect Reagent Kit (Beckman, C10640) and fragment size estimation with High Sensitivity HS DNA kit runned on 2100 Bioanalyzer (Agilent, 5067-4626), the libraries were constructed by performing the following steps: fragmentation, end-repair, A-tailing, SPRIselect cleanup, adaptor ligation, SPRIselect cleanup, sample index PCR, and SPRIselect size selection.

The fragment size estimation of the resulting libraries were assessed with High Sensitivity HS DNA kit runned on 2100 Bioanalyzer and quantified using the Qubit dsDNA High Sensitivity HS assay (Thermofisher, Q32851). Libraries were then sequenced by pair with a HighOutput flowcel using an Illumina Nextseq 500 with the following mode: 26 base-pairs (bp) (10X Index + UMI), 8 bp (i7 Index) and 57 bp (Read 2).

d For Bulk RNA-seq: Directional libraries were prepared using the Smarter Stranded Total RNA-Seq kit-Pico Input Mammalian following the manufacturer's instructions (Clontech,635005). The quality of all libraries was checked with the DNA-1000 kit (Agilent) on a 2100 Bioanalyzer and quantification was performed with Quant-It assays on a Qubit 3.0 fluorometer (Invitrogen). Clusters were generated for the resulting libraries, with Illumina HiSeq SR Cluster Kit v4 reagents (Illumina, GD-401-4001). Sequencing was performed with the Illumina HiSeq 2500 system and HiSeq SBS kit v4 reagents. Runs were carried out over 65 cycles, including seven indexing cycles, to obtain 65 bp single-end reads. Sequencing data were processed with Illumina Pipeline software (Casava version 1.9).

d For scRNA-seq: cDNA libraries were generated based on the Smart-seq2 protocol [START_REF] Picelli | Full-length RNA-seq from single cells using Smartseq2[END_REF]. To increase RNA extraction from fixed cells, we sorted single cells into 96-well tubes containing 5 mL of PKD Buffer (QIAGEN) with 1:16 Proteinase K Solution (QIAGEN), as previously described [START_REF] Thomsen | Fixed single-cell transcriptomic characterization of human radial glial diversity[END_REF]. Briefly, mRNA was reverse transcribed and cDNA was amplified via PCR for 27 cycles. Amplification was done with KAPA HIFI Hot Start ReadyMix (Roche, 07958919001) and purification by magnetic beads (CleanNA CPCR). Quantity and quality of cDNA was assessed with the quantifluor RNA system (Promega E3310) and Agilent 2100 BioAnalyzer with a high-sensitivity chip. Library preparation was done with the Nextera XT library prep and index kit (Illumina FC-131-1096 andFC-131-2001). 100 pg of cDNA was tagmented by transposase Tn5 and amplified with dual-index primers (i7 and i5, Illumina, 12 cycles). Reagents were mixed together by the Echo 555 (Labcyte) and pooled-nextera XT libraries were purified. 384 single-cell libraries were pooled together and sequenced single-end 50 bp on a single lane of a HiSeq4000 (Illumina). All results related to scRNA-seq are based on 2 pooled biological replicates per group, experiments were performed on the same day.

QUANTIFICATION AND STATISTICAL ANALYSIS

High-throughput datasets processing d snRNA-seq

The sequencing data was processed into transcript count tables with the Cell Ranger Single Cell Software Suite 3.0.2 by 10X Genomics. Raw base call files from the Nextseq 500 were demultiplexed with the cellranger mkfastq pipeline into library-specific FASTQ files. The FASTQ files for each library were then processed independently with the cellranger count pipeline. This pipeline used STAR to align cDNA reads to the Mus musculus transcriptome (Sequence: GRCm38, Mouse reference provided by 10X.). Once aligned, barcodes associated with these reads -cell identifiers and Unique Molecular Identifiers (UMIs), underwent filtering and correction. Reads associated with retained barcodes were quantified and used to build a transcript count table. We re-run this same pipeline with the pre-mRNA reference, build with cellranger mkgtf and cellranger mkref. Quality control on aligned and counted reads was performed keeping cells with > 500 reads, > 250 detected genes and less than 15% mitochondrial genes. The subsequent visualizations, clustering and differential expression tests were performed in R (v 3.4.3) using Seurat (v3.0.2) [START_REF] Stuart | Comprehensive integration of single-cell data[END_REF], Pooling of intact and dissociated nuclei was performed by merging the raw data per tissue followed by normalization.

d Bulk RNA-seq datasets processing Bioinformatics analysis was performed using the RNA-seq pipeline from Sequana [START_REF] Cokelaer | Sequana': a set of Snakemake NGS pipelines[END_REF]. Reads were cleaned of adaptor sequences and low-quality sequences using cutadapt version 1.11 [START_REF] Martin | Cutadapt removes adapter sequences from highthroughput sequencing reads[END_REF]. Only sequences at least 25 nt in length were considered for further analysis. STAR version 2.5.0a [START_REF] Dobin | STAR: ultrafast universal RNA-seq aligner[END_REF], with default parameters, was used for alignment on the reference genome (Mus musculus GRCm38 assembly from Ensembl release 87). Genes were counted using featureCounts version 1.4.6-p3 (Liao et al., 2014) from Subreads package (parameters: -t exon -g gene_id -s 1) under gencode mm10 annotation version M11.

Count data were analyzed using R version 3.4.3 and the Bioconductor package DESeq2 version 1.18.1 [START_REF] Love | Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2[END_REF]. Due to putative genomic DNA contamination, only genes with an average number of reads greater than 150 were used as input of the differential analysis (N = 8059 genes). The normalization was performed using the ''shorth'' parameter and dispersion estimation was performed using the default parameters. A generalized linear model was set in order to test for the differential expression between the different time points. For each pairwise comparison, the independent filtering algorithm was applied and raw p values were adjusted for multiple testing according to the Benjamini and Hochberg (BH) procedure [START_REF] Benjamini | Controlling the false discovery rate: a practical and powerful approach to multiple testing[END_REF]. Genes with an adjusted p value lower than 0.05 and an absolute fold-change higher than 2 were then considered differentially expressed.

d scRNA-seq downstream analysis Fastq files contained 50-bp-long single-end sequenced tags (reads) from 384 cells. Each were trimmed with cutadapt 1.5 with the parameters '-q 20,20-minimum-length 35 '' and the nextera adapter sequence. The retained tags were aligned to the Ensembl 84 [START_REF] Yates | and 130-110-916, respectively), then spun down (600 g, 5 min), resuspended in ice-cold DMEM and filtered through 40 mm cell strainer (Corning, 352340) before a last centrifugation (600 g, 5 min). For ERK inhibition during the dissociation, hind limb muscles from Tg:Pax7-nGFP mice were dissected and digested in the presence of 2 mg/mL SCH772984. DMSO was added in control samples. The cells were lysed using RIPA buffer: 50 mM Tris Hcl pH 8,150 mM NaCl, 1% NP40, 0.5% Sodium Deoxycholate, 0.1% SDS, 1 mM EDTA pH 8, protease inhibitor cocktail (Merck, 4693159001) and phosSTOP[END_REF] gene annotation of the NCBI38/mm10 mouse genome using STAR 2.4.0 with the parameters '-outSAMunmapped Within -runThreadN 44 -genomeDir {reference_genome}-readFilesCommand zcat-outSAMtype BAM SortedByCoordinate'. The number of tags per gene was calculated using htseq-count 0.6.0 [START_REF] Anders | HTSeq-a Python framework to work with high-throughput sequencing data[END_REF] with the parameters -'format=bam-order= pos-mode=intersection-strict-stranded=no-type=exon-idattr=gene_id {input.bam} {reference_gtf} > {output.count}'. Quality control on alignment and counting was done with ngsplot 44 with parameters '-G mm10 -R genebody -C {input.bam} -O {results_dir}/ genecoverage/ {wildcards.sample}.gene -F rnaseq' and multiqc [START_REF] Ewels | MultiQC: summarize analysis results for multiple tools and samples in a single report[END_REF]. Quality control on aligned and counted reads was performed with Scater 1.8.4 (McCarthy et al., 2017), cells with < 200,000 reads, < 900 detected genes, > 6 percent mitochondrial genes and > 0.75 percent of spike-in ERCCs. Read normalization was done with Scran 1.8.4. Reads were normalized separately from ERCCs. For each gene, expression estimates per gene were expressed as log-transformed counts by taking the log 2 (counts + 1). We used the top 500 highest variable genes to obtain a 2D representation of the cells, while maintaining the similarity relationships between them using PCA as implemented in the package Scater 1.8.4 (McCarthy et al., 2017). The trend line and IC-75 were calculated using the function geom_smooth() in R using the following parameters: method=''glm,'' formula = y poly(x, 2), level = 0.75, alpha = 0.04. All data analysis was conducted in Python v3.6 (Python software foundation) or R v3.5.1 (CRAN). Differential expression was performed using MAST version 1.10 (Finak et al., 2015) and Monocle2 version 2.12.0 (Qiu et al., 2017) across all time-points.

Pseudotime analysis

Satellite cells are reported to display a linear differentiation trajectory during the first hours after injury [START_REF] De Micheli | Single-cell analysis of the muscle stem cell hierarchy identifies heterotypic communication signals involved in skeletal muscle regeneration[END_REF]. Therefore, we performed single-cell trajectory analysis with Slingshot v1.4 [START_REF] Street | Slingshot: cell lineage and pseudotime inference for single-cell transcriptomics[END_REF], shown in benchmarks to be the most optimal trajectory inference method for linear single-cell trajectories [START_REF] Saelens | A comparison of single-cell trajectory inference methods[END_REF]. A matrix containing log normalized counts from 280 single satellite cells over three different time points was used to calculate the transcriptional differentiation path. Slingshot consists of three main steps. First a minimum spanning tree on the predefined clusters resembling different time points is constructed, this was done on the top five principal components. It builds a rough representation of the lineage trajectory that is then smoothened out in the second step, here, simultaneous principal curves are used to obtain smooth representations. Third, pseudotime values for each cell were calculated by orthogonal projection onto the smoothened lineage curve.

Comparison of in vivo scRNA-seq with ex vivo bulk RNaseq data In order to compare the in vivo single-cell RNaseq data with ex vivo bulk RNaseq data, we exported the gene loadings of the first two principal components from the single-cell RNaseq data and multiplied this by the scaled normalized expression values of the bulk RNaseq data. The single-cell RNaseq matrix consisted of the principal components as rows and genes as columns, while the bulk RNaseq matrix (N) consisted of genes as rows and replicates as columns. Matrix multiplication yielded a matrix with components as rows and replicates as columns. The first two projected principal components were then plotted per time point of the bulk RNaseq data (Figure 3G).

For comparison of gene cluster modules of the bulk RNaseq data described in Figure 3H, we summed raw counts of the single-cell RNaseq data per replicate yielding two pseudobulks per time point. Bulk and pseudobulk data was combined and normalized for counts per million (CPM), genes contained in the different gene clusters were then averaged per replicate and visualized as a linear model (avg CPM Time grouped for Experiment and gene clusters). To find out if gene clusters portrayed differences between experiments independently on time we performed an analysis of covariance test (ANCOVA) and computed the pairwise comparison between experiment groups per gene cluster by avg CPM Experiment*Time with Bonferroni correction as post hoc test (Figure S4M).

Associations with Tabula-Muris scRNA-seq

The Tabula Muris droplet and Smart-Seq2 datasets were loaded from the TabulaMuris R package (v1.0) analyzed and visualized using Seurat. The Stress Index was calculated using the Seurat function ''AddModuleScore'' using the Stress response core from Figure S2B as a feature set. The Tabula Muris tissue digestion time were inferred from the original paper supplemental methods (Table S1). An arbitrary conversion factor of 0.5 and 0.2 were applied to correct kinetics of digestion steps at RT or on ice, respectively. The sum of all corrected sequential enzymatic digestion times for each cell was used to define the digestion time at 37 C. The relative gene expression on muscle stem cells in each dataset shown in Figure S4J (iSiFi single-cell RNA-seq, snRNA-seq and Tabula Muris scRNA-seq) was analyzed and visualized using Seurat.

Statistical analysis

For comparison between two groups, unpaired Student's t test was performed to calculate p values and to determine statistically significant differences (*p < 0.05; **p < 0.01; ***p < 0.001).

sinusoidal cells (M) and Interleukin (IL) -5 signaling highly enriched in muscle endothelial cells and FAPs, and IL-6, IL-2 (not shown). Hepatocytes were also enriched with terms for IL-6 and IL-2 in addition to c-Met signaling, a pathway that is critical for liver regeneration (N), (Ishikawa et al., 2012). (B) UMAP plot of intact (blue) and injured (red) liver cells after data integration using Seurat3.

(C) Quality controls of intact and injured liver and muscle cells showing the number of UMI (left), the number of detected genes (middle), and the % of mitochondrial genes (right).

(D) Heatmap displaying the expression of common cellular populations markers used to annotate the nuclei from integrated intact and injured liver. (F) UMAP plot of intact (blue) and injured (red) muscle cells after data integration using Seurat3.

(G) Heatmap displaying the expression of common cellular populations markers used to annotate the nuclei from integrated intact and injured muscle.

Independent student t-test was performed; * p<0.05, ** p<0.01, *** p<0.001. MAPK cascade. This was followed by mRNA processing, including splicing and initiation of translation (cluster 2), and in a later phase by ontologies related to ribosome assembly and biosynthesis (cluster 3). Of note, late cluster 3 (maximum at T120) also contained ontologies related to inactivation of MAPK signaling, which coincides with the attenuation of the corresponding genes of early cluster 1 (drop at T120). The quiescence-to-activation transition is nevertheless characterized also by a downregulation of a significant number of genes. Indeed, we validated previous findings on T0-MuSCs enriched ontologies, including a switch from fatty-acid beta-oxidation to glycolysis, and processes related to cilium disassembly, (cluster 4). Of interest, cluster 5 was enriched in ontologies related to RNA methylation and mitochondrial RNA metabolic genes, yet the significance of these processes in quiescence-exit remains to be uncovered.

(M) Pairwise comparison between the gene clusters in ex vivo and in vivo activated MuSCs using an analysis of covariance test (ANCOVA).

(N) Western blot for total ERK1/2 and p-ERK1/2 from whole muscle extracts right after 2 h dissociation in the presence of DMSO (Control) or SCH772984. On the bottom, the ratio of intensity of p-ERK1 over total ERK1 is shown (upper bands).

(O) RT-qPCR on myogenic and stress core genes on freshly isolated MuSCs of Tg:Pax7-nGFP mice, from muscles dissociated in the presence or absence of SCH772984 (n=3 mice).

(P) Percentage of EdU + myogenic cells (Pax7 and/or Myog) in whole muscle bulk preparations cultured for 72 h; EdU was added 24 h before fixation.

Independent student t-test was performed. ns not statistically significant * p<0.05, ** p<0.01, *** p<0.001.

MuSCs) and eMyHC+ myofibers (i.e. newly formed, regenerated fibers; Figure 1C-1E).

However, two weeks of tamoxifen treatment increased eMyHC+ fibers in Pax7 CreERT2(Fan)/+ and Pax7 LacZ/+ but not Pax7 CreERT2(Gaka)/+ muscles, suggesting a combinatorial regeneration delay due to Pax7 haploinsufficiency in the presence of tamoxifen (Figure 1F-1H). This phenotype was even more evident with five weeks of tamoxifen treatment (Figure 1I-M). In this tamoxifen regimen, MuSCs were reduced in Pax7 CreERT2(Fan)/+ and Pax7 LacZ/+ mice, but not Pax7 CreERT2(Gaka)/+ , as revealed by PAX7+ counts in all models and confirmed by mCadherin+ cells in Pax7 CreERT2(Fan)/+ (Figure 1I-M). In addition, at five weeks of tamoxifen diet, hematoxylin-eosin staining revealed considerable regions that were not repaired in Pax7 CreERT2(Fan)/+ samples, as opposed to their controls or other lines (data not shown). Given the severe D7 regenerative phenotype in the five-week tamoxifen regimen, we then evaluated the injured muscles 28 days post-injury, when the damaged muscles are expected to display a complete recovery. At this late stage, Pax7 CreERT2(Fan)/+ TAs still showed a reduced number of PAX7+ MuSCs compared to their controls, while we did not observe significant differences in Pax7 LacZ/+ and Pax7 CreERT2(Gaka)/+ TAs compared to their respective controls (Figure 1N).

In conclusion, we report that Pax7 +/-mice (i.e. Pax7 CreERT2(Fan)/+ or Pax7 LacZ/+ ) present with a defect in regeneration following prolonged tamoxifen treatment. Our study therefore highlights the critical importance of selecting the correct genetic tools with appropriate controls to study biological mechanisms and diseases. This is recognized by the lab that created the Pax7 CreERT2(Fan)/+ mice and that consistently compares conditional knock-outs to Pax7-CreERT controls. However, attention should be drawn since the main commercial provider of these mice recommends wild-type littermates as controls (jax.org/strain/012476).

Moreover, our analysis revealed that approximately half of the published studies using this allele have not included the appropriate controls. Pax7 null MuSCs display enhanced cell death and differentiation (Lepper and Fan, 2010;Relaix et al., 2006), yet it is unclear whether the reduction that we observed in regenerating Pax7 heterozygote animals -especially Pax7 CreERT2(Fan)/+ -is a result of cell death, precocious activation and inappropriate differentiation, or impaired proliferation, ineffective self-renewal, or even additive effects between inherent Pax7 heterozygous phenotypes and either Cre (Schmidt-Supprian and Rajewsky, 2007) or tamoxifen treatment. Regardless of the mechanism, Pax7 heterozygosity results in defective muscle regeneration in the presence of tamoxifen, and, thus, our study

Tamoxifen treatment and injury

Mice were placed on a tamoxifen diet (Envigo; TD.55125.I) for the indicated time points. For muscle injuries, mice were anesthetized with 3% isoflurane, and injured by intramuscular injection of 50uL cardiotoxin at 10 umol (Latoxan; L8102).

Immunostaining

Muscles were sectioned at 8um with a Leica cryostat and sections were first fixed with 4% paraformaldehyde for 10 min and washed 3 times with PBS. The sections were then permeabilized with 0.5% triton-X (Sigma) for 8 minutes, washed, then blocked with 5% BSA (Jackson Laboratories 001-000-162) for 1 hour at room temperature. Sections were then incubated with the primary antibodies overnight. The next day, samples were washed, incubated with the secondary antibodies for 1 hour at room temperature, washed and mounted.

For PAX7 staining, samples were permeabilized with cold methanol for 6 min instead of Triton, washed, then boiled in citric acid (10 mM, pH6) for 3 min, then let cool down at RT for 30 minutes. Sections were then washed, blocked with 5% BSA and incubated with Fab (Interchim; 115-007-003) for 30 minutes at RT. The staining was then completed as described above.

Statistical analyses

For comparison between two groups, unpaired Student's t test was performed to calculate p values and to determine statistically significant differences (*p < 0.05; **p < 0.01; ***p < 0.001).

List of antibodies

Antibodies

  ACKNOWLEDGMENTS .................................................................................................................................... ABSTRACT ......................................................................................................................................................

Figure 1 :

 1 Figure 1: Different transitional states of adult stem cells (Urbán and Cheung, 2021).

Figure 2 :

 2 Figure 2: Expression profile of growth factors induced genes in HeLa cells (Amit

Figure 3 :

 3 Figure 3: Main stress and mitogen induced signaling pathways. A simplified scheme of stress or mitogen induced pathways. Following stress and growth factor stimulation, several pathways can be induced, including the ERK1/2 and the p38 MAPK pathways, through the Ribosomal S6 Kinase (RSK) receptor or the RhoA pathway (Integrins, GPRCs, RTKs). These pathways, through two distinct co-factors (TCF or MRTF) will lead to the activation of SRF already bound to the promoter of IEGs, which will lead to their transcriptional activation.

Figure 4 :

 4 Figure 4: ERK1/2 signaling pathway and subcellular localization (Lavoie et al., 2020). A) The activation of the receptor tyrosine kinase (RTK)-RAS-induced extracellular signalregulated kinase (ERK) results in the phosphorylation of a wide range of substrates important in cell proliferation, survival, growth, metabolism, migration, and differentiation. The several isoforms that make up the main RAS-ERK pathway in mammals are shown. B) ERK can either passively diffuse to the nucleus or be actively transported by a RAN-GTP dependent

Figure 5 :

 5 Figure 5: The polyamine synthesis pathway.

Figure 6 :

 6 Figure 6: Dissociation induced transcriptional changes in MuSCs and microglial cells. A) MA plot showing deregulated transcripts of quiescent vs freshly isolated MuSCs (Machado et al., 2017). B) Scatterplot showing the activation score (based on DEGs from pseudobulk analysis) in microgial cells caused by tissue dissociation from scRNA-seq of the brain tissue performed using a cold mechanical dissociation protocol vs enzymatic dissociation protocol.

Figure 8 :

 8 Figure 8: The different cell types of the skeletal muscle tissue. A) UMAP of scRNA-seq data colored by metaclusters from uninjured and injured Tas at 0.5, 2, 3, 3.5, 5, 10, 21 days following injury. B) The fraction of each cell type during each time point, based on the scRNAseq data. (Oprescu et al., 2020).

Figure 9 :

 9 Figure 9: The myogenic program. The evolution of the myogenic lineage is described in this diagram. Within minutes after the disruption of the quiescence niche, satellite cells undergo drastic transcriptional and epigenomic changes (early activation), before becoming fully activated MYOD+ cells. Then they begin to proliferate, becoming myoblasts that differentiate into Myogenin expressing myocytes, which merge to create myotubes that express Myosin

Figure 10 :

 10 Figure 10: Muscle injury. A) H&E staining showing the histology of the skeletal muscle

Figure 11 :

 11 Figure 11: Cell populations of the skeletal muscle during regeneration. A) The presence of different cell types on the regenerating site following injury. Modified from (Bentzinger et al., 2013). B) The different cell types of the skeletal muscle during homeostasis and

Figure 12 .

 12 Figure 12. MuSCs in their quiescent niche. Modified from (Sousa-Victor et al., 2022).

Figure 13 :

 13 Figure 13: The Notch signaling pathway. A) An overview on the Notch signaling pathway

Figure 14 :

 14 Figure 14: Signalling pathways regulating quiescence maintenance and activation of MuSCs[START_REF] Zhou | A Long Journey before Cycling: Regulation of Quiescence Exit in Adult Muscle Satellite Cells[END_REF]. These mechanisms include signaling pathways such as the Notch signaling pathway expressed in quiescence and is downregulated at the expense of the PI3K pathway induced upon activation. The epigenome of MuSCs is also different between quiescent

  Using different tools such as iSiFi, snRNA-seq, transcription inhibitors during dissociation, cold digestion, and spatial transcriptomics, several studies have shown the same response in MuSCs as well as several other tissues(van Velthoven et al., 2017)(van den Brink et al., 2017)

Figure 1 .

 1 Figure 1. Dissociation induced transcriptional changes in skeletal muscle and liver

Figure 2 .

 2 Figure 2. Identification of a conserved stress core upon tissue dissociation

Figure 3 .

 3 Figure 3. Stress response during in vivo tissue damage

Figure 4 .

 4 Figure 4. Time course of MuSCs stress response in vivo

Figure 5 .

 5 Figure 5. Kinetics of MuSCs early activation ex vivo

Figure 6 .

 6 Figure 6. Investigation of the role of the polyamine synthesis pathway in the early activation of MuSCs

Figure 7 .

 7 Figure 7. Inhibition of the ERK1/2 MAPK pathway during the early activation of MuSCs

Figure 8 .

 8 Figure 8. Combination of ERK1/2 inhibition and Notch overexpression

Figure 9 .

 9 Figure 9. Ex vivo system for testing the regeneration potential of MuSCs (A) (Top) Skeletal muscle from Tg:Pax7-nGFP mice was dissected and digested. MuSCs and the MuSCs-free bulk were sorted separately. MuSCs-free bulk was plated on 8 wells chambered slides and freshly isolated MuSCs or Myoblasts that had been cultured for 3 days and sorted for GFP were added to the bulk at different concentrations. (Bottom) FACS plots of the sorted cells. (B) Immunostaining and quantifications of GFP (PAX7), EdU, added 24 hours before fixing, and MYHC (n=3 per condition).

Figure 10 .

 10 Figure 10. Ex vivo system for testing the regeneration potential of MuSCs

Figure 11 .

 11 Figure 11. Protocol optimization for quiescent-like MuSCs induction ex vivo

Figure 12 .

 12 Figure 12. Test of the effect of Oxygene levels on the differentiation of MuSCs plated on Dll1-Fc (A) Freshly isolated MuSCs from Tg:Pax7-nGFP mice were plated on Dll1-Fc coated 8-well chambered slides in 3% or 21% O2 and fixed at 4 or 8 days following culture. (B) Immunostaining and quantification of GFP (PAX7), MYOD and MYOGENIN of MuSCs cultured on Dll1-Fc at different O2 concentrations (n=3 per condition).

Figure 13 .

 13 Figure 13. Test of ColV and Serum starvation on the induction of quiescent-like MuSCs ex vivo (A) MuSCs from Tg:Pax7-nGFP mice were cultured on Dll1-Fc coated chambered slides overnight, ColV was then added and refreshed every other day, acetic acid at 5mg/mL was added to the non-treated wells. At 4 days, serum was reduced by half daily going from 20%, 10%, 5%, 2.5% to 1.25%. EdU was added 24 hours before fixing. (B) MuSCs were immunostained and quantified for GFP (PAX7) and EdU (n=3 per condition).

Figure 14 .

 14 Figure 14. Protocol optimization for quiescent-like MuSCs induction ex vivo (A) MuSCs were sorted from Tg:Pax7-nGFP mice and cultured on Dll1-Fc coated 8-well chambered slides for 7 days, cells were then treated daily with SCH772984 (Dll+SCH) or left untreated for 2 days (Dll). (B) Immunostaining and quantification for GFP (PAX7), MYOD and CALCR of MuSCs treated or not with SCH772984 (n=3 per condition).

Figure 15 .

 15 Figure 15. Induction of quiescent-like MuSCs ex vivo

Figure 16 .

 16 Figure 16. Model for tissue damage and MuSCs quiescence exit.

  . Animals were handled as per French and European Com-munity guidelines and protocols were approved by the ethics committee at the French Ministry (Project No: 20-027 #24357).Cell culture of primary muscle cellsMuSCs were isolated by FACS from male and female Tg:Pax7-nGFP mice and plated on 354248), 94.6140.802) in growth medium (GM) composed of DMEM (GIBCO) with 20% fetal bovine serum (FBS), 1% penicillin/streptomycin (GIBCO), and supplemented with 5 ng/ml basic FGF (Peprotech, 450-33) at 37 • C. Whole muscle bulk preparations were cultured on Matrigel-coated 8-chamber Sarstedt slides in GM at 37 • C in the presence of 10 uM EdU when specified. Muscles from Tg:Pax7-nGFP mice were dissected and chopped on ice, then digested following standard procedures in the presence or absence of 2 mg/mL SCH772984 or DMSO control when indicated. Digested muscle was then filtered through 100 mm and 70 mm cell strainers, spun down at 600 g/ 5 min, then resuspended in cold DMEM and filtered through a 40 mm cell strainer and eventually spun down at 600 g for 5 min. All filtering steps were done on ice. For Pax7-CreERT2; R26Stop-NICD-nGFP and Tg:Pax7-nGFP bulk muscle preparations, growth medium was supplemented with 5 ng/mL of basic FGF and cells were cultured in the presence of DMSO (control) or 1 mg/ml SCH772984. For ERK inhibition, cells were plated in the presence of the ERK1/2 inhibitor SCH772984 (Selleckchem, S7101) at 1 mg/ml or DMSO, in the pres ence of 10 uM 5-ethynyl-20-deoxyuridine (EdU), (Click-iT PLUS Kit C10640, Life Technologies). For ODC1 inhibition, cells were plated in the presence or absence of 2.5 mMDFMO (Tocris, 2761) resuspended in DMEM with 10 uM EdU for the first time point.For Notch induction, conditioned medium was collected from a HEK293-T cell line that stably expresses the human Dll1-Fc fusion protein. Culture dishes were first coated with 10µg/mL anti-Fc (Jackson ImmunoResearch 109-005-098) for 1hour at room temperature with rocking.

  and then a later role during the fusion of myocytes(Sunadome et al., 2011, p. 5), we wanted to investigate Klf4 during the different steps of MuSCs activation. To do this, we generated an inducible conditional knockout mouse model to specifically delete Klf4 in MuSCs following tamoxifen induction (Figure4A-C). A conditional null allele of Klf4 targeting exons 3 and 4 of Klf4[START_REF] Katz | The zinc-finger transcription factor Klf4 is required for terminal differentiation of goblet cells in the colon[END_REF] was coupled with the tamoxifen-inducible Cre recombinase that is under the control of the Pax7 regulatory elements and an inducible GFP reporter to produce the Tg:Pax7-CreER T2 ;Klf4 flox/flox ;Rosa26 mTomato-STOP-mGFP (Klf4 cKO). The Tg:Pax7-CreER T2 ;Rosa26 mTomato-STOP- mGFP mouse line was used as Control. Of note, we used the BAC transgenic Tg:Pax7-CreER T2 because Pax7 and Klf4 are both on chromosome 4, making it impossible to use the Pax7 CreERT2

(

Figure 1 :

 1 Figure 1: Klf4 dynamics in MuSCs during activation, proliferation, and differentiation

  from quiescent T0 MuSCs, or at different time points during dissociation ; 15 min (T15), 30 min (T30), 60 min (T60), 90 min (T90), 120 min (T120). (C) KLF4 immunostaining and quantification in MuSCs on isolated myofibers from Tg:Pax7-nGFP mice from fixed EDL (T0), following digestion of live EDL (T2), 2 hours (T4) or 6 hours (T6) following digestion (n=3 mice per condition). (D) KLF4 immunostaining on sorted MuSCs from Tg:Pax7-nGFP mice and cultured for 16h (activation), 48h (proliferation), 72h (differentiation), and 112h (fusion) (n=3 mice per condition). (E) RT-qPCR on Klf4 from MuSCs isolated from Tg:Pax7-nGFP mice right after isolation (3h), cultured for 16h, 48h, 72h and 112h (n=3 mice per condition). Independent Student's t test was performed. ns, not statistically significant; *p < 0.05, **p < 0.01, and ***p < 0.001. Scale bar, 15 um. Error bars indicate standard deviation.

Figure 2 :

 2 Figure 2: KLF4 overexpression in C2C12 cells (A) C2C12 cells were transduced with Mock-GFP or KLF4-GFP retroviruses. GFP + cells were then sorted and plated on 8 wells chambered slides and fixed a day later. (B) Immunostaining for KLF4 and GFP on Mock and Klf4 overexpressing C2C12 cells. Arrowheads show C2C12 cells that are GFP high and KLF4 high (n=1 per condition). (C) RT-qPCR of Klf4 from control and C2C12 cells overexpressing KLF4. Klf4 expression in C2C12-KLF4 cells was normalized to C2C12-Mock cells (n=1 per condition). (D) C2C12 cells were transduced with Mock-GFP or KLF4-GFP retroviruses. GFP + C2C12 cells were then sorted and plated for 24 hours on 8 wells chambered slides then fixed after a 2 hours EdU chase or plated for 48 hours and fixed. (E) Immunsotaining and quantifications for EdU and MYOGENIN on Mock and KLF4 C2C12 cells fixed at 24 hours or 48 hours respectively (n=3 per condition for the 24 hours time point and n=4 per condition for the 48 hours time point).

Figure 3 .

 3 Figure 3. KLF4 overexpression in primary myoblasts. (A) Dissociated muscle bulk from tamoxifen induced Pax7 CreERT2 ; Rosa26 Stop-tdTomato mice was pre-plated on non-coated dishes overnight, then myoblasts enriched medium was moved to Matrigel coated 8-chamber slides and transduced with Mock-GFP and KLF4-GFP retroviruses 8 hours later. Transduced cultures were then fixed 3 days or 6 days later. (B) Immunostaining for GFP, Myogenic markers (PAX7, MYOD, MYOGENIN) or EdU following 8 hours of EdU chase on Mock-GFP and KLF4-GFP transduced myoblasts enriched cultures cultured for 4 days (n=3 mice per condition). (C) Representative images of Mock-GFP and KLF4-GFP transduced cells following 7 days in culture and immunostained for GFP and MyHC (n=3 mice per condition). (D) Quantifications of immunostaining from (B) and (C).

Figure 4 :

 4 Figure 4: Role of Klf4 in MuSCs ex vivo (A) GFP + MuSCs from tamoxifen induced Control Tg:Pax7-CreER T2 ; Rosa26 mTomato-STOP- mGFP and Klf4 cKO Tg:Pax7-CreER T2 ; Klf4 flox/flox ; Rosa26 mTomato-STOP-mGFP mice were sorted and plated on 8-wells chambered slides and fixed at 43h, 72h, and 110h. (B) Immunostaining and quantification for EdU and MYOD (43h), and MYOGENIN (72h), on sorted GFP + MuSCs from tamoxifen induced Control and Klf4 cKO mice and cultured in growth medium for 43h in the presence of EdU, 72h. (n=4 mice per genotype). (C) Immunostaining for GFP and MyHC from Control and Klf4 cKO MuSCs following 110h in culture. Nuclei per MyHC + cell were quantified. (n=4 per genotype). (D) RT-qPCR on quiescence specific and early induced genes from MuSCs from tamoxifen induced Control and Klf4 cKO right after sorting. Genex expresseion was normalized to Tbp (n=4 mice per genotype).(E) GFP + MuSCs from Control and Klf4 cKO mice were cultured for 7 days and EdU was added 16 hours before fixing. Cells were then immunostained and quantified for PAX7 and EdU (n=4 per genotype).

Figure 5 :

 5 Figure 5: Role of Klf4 in MuSCs in vivo

Figure 6 :

 6 Figure 6: Investigation of the role of Srf during the early activation of MuSCs (A) Srf, Fos, Jun, and MyoD normalized expression from bulk RNAseq data (Machado, Geara et al., 2021) from quiescent T0 MuSCs, or during dissociation at 15 min (T15), 30 min (T30), 60 min (T60), 90 min (T90), 120 min (T120). (B) GFP + MuSCs were sorted from tamoxifen induced Control Tg:Pax7-CreER T2 ; Rosa26 mTomato-STOP-mGFP and Srf cKO Tg:Pax7-CreER T2 ; Srf flox/flox ; Rosa26 mTomato-STOP-

  Machado L*, Geara P*, Camps J, Dos Santos M, Teixeira-Clerc F, Van Herck J, Varet H, Legendre R, Pawlotsky JM, Sampaolesi M, Voet T, Maire P, Relaix F, Mourikis P. Tissue damage induces a conserved stress response that initiates quiescent muscle stem cell activation. Cell Stem Cell. 2021Jun 3;28(6):1125-1135.e7. doi: 10.1016/j.stem.2021.01.017. Epub 2021 Feb 19. PMID: 33609440.

Figure 1 .

 1 Figure 1. Definition of a core stress signature in response to cell dissociation (A) Liver and hindlimb skeletal muscle single nuclei were FACS-isolated on the basis of DAPI from either intact tissues or after 2 h enzymatic dissociation at 37 C. Intact muscle: 7,951 nuclei from two biological replicates. Digested muscle: 8,640 nuclei from one biological replicate. Each muscle biological replicate is a pool of TAs from four mice. Intact liver: 8,996 nuclei from one biological replicate. Digested liver: 3,762 nuclei from one biological replicate. Each liver biological replicate is a pool of livers from three mice. (B) Uniform manifold approximation and projection (UMAP) plots displaying the different cell populations in intact and digested muscle and liver. Endo, endothelial cells; Hepato, hepatocytes; T, T cells; B, B cells; FAP, fibro-adipogenic progenitor; Teno, tenocytes; SM, smooth-muscle cells; Macro, macrophages; MuSC, skeletal muscle stem cell; Lymph, lymphatic cells; Adipo, adipocytes; Chol, cholangiocytes. (C) UMAP plots displaying the different populations inferred from intact (blue) or dissociated (red) muscle (left) and liver (right) single-nucleus transcriptomes without data integration. Differences were observed in the relative abundance of the populations: myonuclei: digested 6%, intact 64%; endothelial muscle: digested 29%, intact 1%; endothelial liver: digested 36%, intact 4%. (D) Histogram representing the distribution of genes induced by dissociation in the ten main cell populations. Number of genes within each segment is indicated. (E) Genes upregulated by at least seven populations composed the 98-gene stress core. Selected examples are displayed in the table.(F) Gene Ontology analysis of the stress core using the biological process category. Selected terms with corrected p values < 0.05 are displayed and ranked by fold change.

  (G) UMAP plot representing the stress index of every cell from the published Tabula Muris Smart-Seq2 dataset. (H) UMAP plot representing the calculated digestion time at 37 C (see STAR methods) of every cell from the Tabula Muris Smart-Seq2 dataset (53,760 cells). Note the correlation between high digestion time and high stress index (Spearman correlation coefficient = 0.51). (I) Median stress index in different cell types. Median digestion time at 37 C is color-coded (gray/blue gradient).

Figure 2 .

 2 Figure 2. Stress signature during in vivo tissue injury (A) Nuclei from intact and 4 h injured tibialis anterior (TA) muscle and liver were FACS-isolated on the basis of DAPI and analyzed using 10x Genomics. Intact muscle: 7,951 nuclei from two biological replicates. Injured muscle: 1,618 nuclei from two biological replicates. Each muscle biological replicate is a pool of TAs from four mice. Intact liver: 8,996 nuclei from one biological replicate. Injured liver: 6,271 nuclei from one biological replicate. Each liver biological replicate is a pool of livers from three mice.

Figure 3 .

 3 Figure 3. Time course analysis reveals the kinetics of MuSC quiescence exit (A) Graphical scheme of the time course experiment: MuSCs from forelimbs and hindlimbs of Tg:Pax7-nGFP mice were fixed with ice-cold 0.5% paraformaldehyde at different time points during the enzymatic digestion and sorted for GFP for RNA extraction and sequencing. Three or four biological replicates were used per time point for a total of 24 mice analyzed for the experiment. (B) Principal-component (PC) analysis during MuSCs ex vivo dissociation delineating the transcriptional trajectories during early activation. Trend line and IC-75 (gray zone) have been added for visualization. PC1, PC2, and PC3 represent 27%, 10%, and 8% of the variance explained, respectively. (C) MA plots during MuSC ex vivo dissociation. Colored dots represent statistically differentially expressed genes with p < 0.05 and absolute FC > 2 (red dots) or <0.5 (blue dots), using DEseq2 against T0 for each time point. The number of up-and down-regulated genes is indicated in each panel. (D) Hierarchical clustering of gene expression during MuSC ex vivo dissociation reveals transcriptional modules of co-expressed genes. The clusters were extracted from the row dendrogram using k = 5. (E) Mean expression of specific genes per time point. Gene symbols in red are members of the stress core.(F) Clusters of upregulated (clusters 1, 2, and 3) and downregulated (clusters 4 and 5) genes as defined by hierarchical clustering. Linear regression with secondorder polynomial was performed on the mean gene expression per time point for each cluster. Samples are ordered from T0 to T120. Standard error is displayed in gray. (G) Projection of the ex vivo dissociated MuSCs using gene loadings of the first two Principal components from the injury-activated MuSCs; dissociated MuSCs are positioned according to their digestion time from T0 to T120. (H) Mean normalized counts per million genes contained in the five cluster modules of the ex vivo bulk RNA-seq dataset (D) compared with the mean normalized counts per million of the same genes into the in vivo injury-activated MuSCs. The gray zone indicates the 95% confidence interval.

Figure 4 .

 4 Figure 4. Functional assessment of the stress response in MuSC exit from quiescence (A) Effect of DFMO on the activation and differentiation of freshly isolated MuSCs from Tg:Pax7-nGFP mice. Cell cycle entry was quantified by EdU incorporation for 40 h after plating, and MuSC differentiation by PAX7 (GFP) and myogenin positive cells at 72 h after plating (n = 4 mice per condition). (B) Left: representative images of EdU and nuclear GFP after 40 h in culture in the absence (control) or presence of 2 mM DFMO. Right: MuSCs stained against GFP and the differentiation myogenin after 72 h in culture. (C) Immunostaining and quantification for MYOD and EdU on dissociated muscle cells after 45 h in culture in the presence of the ERK1/2 inhibitor SCH772984 or DMSO vehicle control. MuSCs were detected by GFP in control (Tg:Pax7-nGFP) and NICD-ires-GFP expression (Pax7 CreERT2 ; R26 stop-NICD-ires-nGFP ) (n = 4 mice per genotype and per condition). (D) Immunostaining and quantification for PAX7, EdU (24 h chase) and NICD (GFP) on cultured muscle cells from Pax7 CreERT2 ; R26 stop-NICD-ires-nGFP mice with or without SCH772984 after plating for 72 h; asterisk indicates EdU -cell (n = 4 mice per genotype and per condition). (E) Inhibition of ERK1/2 signaling during the dissociation process. Efficiency of inhibition was evaluated in MuSCs using qRT-PCR for MAPK target genes on freshly isolated GFP + cells. Gene expression was normalized to Tbp (n = 3 mice per condition). (F) Representative images of cells from Tg:Pax7-nGFP mice stained for MYOD, EdU, and GFP after 45 h in culture; EdU added at t = 0 (n = 4 mice per condition). Independent Student's t test was performed. ns, not statistically significant; *p < 0.05, **p < 0.01, and ***p < 0.001. Scale bar, 15 mm. Error bars indicate standard deviation.

  For panels A-D: DL: Digested Liver; DM: Digested Muscle; IL: Intact Liver; IM: Intact Muscle.

  FigureS2 Machado et al., 2020

Figure S3 .

 S3 Figure S3. Stress response kinetics and quality controls related to single-nuclei RNA-seq experiments on intact and injured muscle and liver. Related to Figure 2.(A) Measurement of early stress response to liver injury by RT-qPCR at 0, 2, 4 and 6 h postinjection of CCl4. Transcripts were measured from bulk liver extracts. Statistical t-tests are T0/T2 for Fos, Jun, and Egr1 and T0/T6 for Stat3,

(

  E) Measurement of early MuSCs response to muscle injury (1.2% BaCl2) by RT-qPCR at 0, 2, 4 and 6 h post-injury. Transcripts were measured in FACS-isolated, iSiFi-treated MuSCs. Statistical t-tests are T0/T6 for Myod1 and Pax7 and T0/T2 for Jun.

  FigureS4 Machado et al., 2020

  in the differentiation of ESCs during development. ESCs are maintained in a pluripotent state by several pluripotency factors, one of which is KLF4. Phosphorylation of KLF4 by ERK1/2

: Niche factors contributing to MuSCs quiescence.

	Niche factors	Source	Functions	Reference
	DLL4	Muscle fibers	Quiescence maintenance through Notch signalling	(Low et al., 2018)
	CalcR	MuSCs	Quiescence maintenance	(Yamaguchi et al., 2015)
	Notch1	MuSCs	Quiescence maintenance	(Fujimaki et al., 2018)
	COLV	MuSC	Interaction with CalcR to maintain quiescence	(Baghdadi et al., 2018a)
	OSM	Muscle fibers	Quiescence induction	(Sampath et al., 2018)
	Wnt4	Muscle fibers	Quiescence maintenance of MuSCs	(Eliazer et al., 2019)
	NCAD/MCAD Muscle fibers	Adhesive junctions formation for quiescence maintenance	(Goel et al., 2017)
	Angiopoietin1	Circulation	Quiescence maintenance through Tie-2 receptor	(Abou-Khalil et al., 2009)
	ß1-integrin	MuSC	Cell-matrix adhesion leading to quiescence maintenance	(Rozo et al., 2016)
				(Cornelison et al.,
	Syndecan-3	MuSC	MuSC-myofiber adhesion	2001) (Pisconti et al.,
				2010)
	Table1
	modulated by Sprouty1 and Wnt4-Frizzeld between the myofiber and MuSCs. MuSCs can also
	interact with factors coming from the niche such as ColV produced by the MuSC itself that
	interacts with CalcR. It also expresses Tie2 that interacts with ANG1 coming from the
	circulation.

Goat anti-rabbit IgG, Alexa Fluor 555 conjugated

	Introduction
	Upon damage, cells have acquired the ability to respond to the stress induced by this
	damage and undergo major transcriptional changes. These changes include various genes
	involved in different functions, such as the MAPK pathway, immediate early genes,
	cytoskeletal organization, chromatin regulation, polyamine synthesis, among many other
	mouse monoclonal anti-Pax7	Santa cruz ; sc-61648	IF 1/200
	IgG1 mouse monolonal anti-MyoD	Dako ; M3512	IF 1/200
	IgG2a rat Anti-MyoD	Active motif ; 39991	IF 1/200
	IgG1 mouse monoclonal anti-Myogenin	Santa cruz ; sc-52903	IF 1/200
	Rabbit polyclonal anti-CalcR IgG2b mouse monoclonal Anti-MyHC Part II: Investigating the role of Klf4 and Srf in muscle Active motif ; AHP635 IF 1/100 DSHB ; AB-2147781 IF 1/400 Rabbit polyclonal Anti-ERK1/2 EMD Millipore ; 06-182 WB 1/10 000 stem cells early activation
	IgG1 Mouse Anti-phospho-ERK1/2	Abcam ; ab-500011	WB 1/1 000
	Rabbit polyclonal Anti-phospho-ERK1/2	Cell Signaling Technology ; 9101	WB 1/1 000
	Goat anti-chicken IgY	Jackson ImmunoResearch Labs; 103-155-155	IF 1/ 1 000
	Goat anti-mouse IgG1, alexa fluor 555
	conjugated	Molecular Probes; A-21127	IF 1/ 1 000
		Molecular Probes; A-21429	IF 1/ 1 000
	Goat anti-mouse IgG2b, Alexa Fluor 555 conjugated	Molecular Probes ; A-21147	IF 1/ 1 000
	Goat anti-rat IgG, Alexa Fluor 555 conjugated	Molecular Probes ; A-23414	IF 1/ 1 000
	Goat anti-rabbit IgG, Alexa Fluor 647 conjugated	Molecular Probes ; A-21244	IF 1/ 1 000
	Goat anti-mouse IgG, Alexa Fluor 647 conjugated	Molecular Probes ; A-21235	IF 1/ 1 000
	Goat anti-rabbit, peroxidase	Vector Laboratories; PI-1000	WB 1/5 000
	Horse anti-mouse, peroxidase	Vector Laboratories; PI-2000	WB 1/5 000

  ; Rosa26 mTomato-STOP- mGFP mice and cultured for 43 hours in the presence of EdU. Cells were immunostained and quantified for EdU and MYOD (n=4 mice per condition). (C) RT-qPCR on Srf, myogenic and immediate early genes from sorted Control and Srf cKO

	MuSCs (n=4 per genotype).
	Independent Student's t test was performed. ns, not statistically significant; *p < 0.05, **p <
	0.01, and ***p < 0.001. Scale bar, 15 um. Error bars indicate standard deviation.

Goat anti-mouse IgG2b, Alexa Fluor 555 conjugated

	Antibodies	Reference	Dilution
	IgY chicken polyclonal anti-GFP	Abcam ; ab13970	IF 1/1 000
	IgG1 mouse monoclonal anti-Pax7	Santa cruz ; sc-61648	IF 1/200
	IgG1 mouse monolonal anti-MyoD	Dako ; M3512	IF 1/200
	IgG2a rat Anti-MyoD	Active motif ; 39991	IF 1/200
	IgG1 mouse monoclonal anti-Myogenin	Santa cruz ; sc-52903	IF 1/200
	IgG2b mouse monoclonal Anti-MyHC	DSHB ; AB-2147781	IF 1/400
	IgG1 mouse monoclonal Anti-Klf4	Santa cruz ; sc-166238	IF 1/50
	IgG2b sheep polyclonal Anti-BrdU	Abcam; ab1893	IF 1/100
	Rabbit polyclonal Anti-Fra1	Santa cruz ; sc-183	IF 1/200
	Goat anti-chicken IgY	Jackson ImmunoResearch Labs; 103-155-155	IF 1/ 1
	Goat anti-mouse IgG1, alexa fluor 555
	conjugated	Molecular Probes; A-21127	IF 1/ 1
	Goat anti-rabbit IgG, Alexa Fluor 555 conjugated	Molecular Probes; A-21429	IF 1/ 1
		Molecular Probes ; A-21147	IF 1/ 1

GATGCAGTCACAAGTCCCCT

Klf4 exon 2

GATGCAGTCACAAGTCCCCT

Klf4 intron 3

GATGCAGTCACAAGTCCCCT Srf 1 CTGTAAGGGATGGAAGCAGA Srf 2 TAAGGACAGTGAGGTCCCTA Cre 1 TGATGGACATGTTCAGGGATC Cre 2 CAGCCACCAGCTTGCATGA List of antibodies

Goat anti-rat IgG, Alexa Fluor 555 conjugated

	Molecular Probes ; A-23414	IF 1/ 1

Goat anti-rabbit IgG, Alexa Fluor 647 conjugated

		Molecular Probes ; A-21244	IF 1/ 1
	Goat anti-mouse IgG, Alexa Fluor 647 conjugated	Molecular Probes ; A-21235	IF 1/ 1
	Donkey anti-sheep IgG, Alexa Fluor 647 conjugated	Molecular Probes ; A-21448	IF 1/ 1

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ACKNOWLEDGMENTS

We would like to thank Hanne Grosemans for performing the muscle injuries. We thank Brigitte Izac and Benjamin Saintpierre, Institut Cochin (Universite ´de Paris, Institut Cochin, CNRS, INSERM, Paris, France) for single-nucleus sequencing libraries and data processing. We also thank Erica Kimber for her excellent editing of the manuscript. The F.R. laboratory receives funding from Association Franc ¸aise contre les Myopathies (AFM) via TRANSLAMUSCLE (PROJECT 22946), Labex REVIVE (ANR-10-LABX-73), Agence Nationale pour la Recherche (ANR) grants Bone-muscle-repair (ANR-13-BSV1-0011-02), Satnet (ANR-15-CE13-0011-01), BMP-MyoStem (ANR-16-CE14-0002-03), MyoStemVasc (ANR-17-CE14-0018-01), muscleXTRA (ANR-19-CE13-0010), Fondation pour la Recherche Me ´dicale (FRM) ECO201806006793, and RHU CARMMA (ANR-15-RHUS-0003). The P. Maire laboratory receives funding from grants ANR-16-CE14-0032-01 and AFM 21711. T.V. and J.V.H. are supported by KU Leuven (SymBioSys, C14/18/092) and the Research Foundation Flanders (FWO; G092415N and I001818N).

Data and code availability

All single-cell, single-nuclei and bulk RNA-sequencing datasets generated during this study are available at Gene Expression Omnibus (GEO), accession number GEO: GSE163856.

Abbreviations

MuSCs: muscle stem cells PCNA: proliferating cell nuclear antigen Contribution to the project: My initial Thesis project was the characterization of the role of the Krüppel-like Factor Klf4 in MuSCs (Part II of the results section). Klf4 was identified as an early response gene in MuSCs and previous data from the host laboratory produced by Dr Sonia Alonso-Martin suggested that KLF4 could play a role in MuSCs reprogramming. Based on these aspects, we decided to focus on this transcription factor and generate conditional Klf4 flox knock-out mice to study its role in vivo, with a focus on early MuSC activation.

In parallel, another PhD student in the team Mourikis/Relaix, Léo Machado, was pursuing a project on the transcriptional response of cells to tissue damage, using specialized high throughput protocols. Since the beginning of my Thesis I worked with Léo and helped him with different technical aspect that his project required. Later, given the exciting finding and progress of the project, I became fully focused on the early activation state and its functional relevance in MuSCs quiescence exit where I performed all the functional part of the study. The study was published in 2021 and I share first co-authorship with LM (Annex Part I) (Machado, Geara, et al., Cell Stem Cell 2021). entry in the myogenic program, followed by the polyamine synthesis pathway involved in both, the cell cycle and differentiation of the cells. However, Klf4 and Srf did not seem to be required for either the activation or differentiation of MuSCs.

Finally, using the pathways we have identified, we have attempted to optimize a protocol that allows us to amplify MuSCs ex vivo by the ectopic expression of Notch and revert them back to quiescence by inhibiting ERK1/2 in order to produce quiescent-like MuSCs ex vivo.

proliferation, differentiation, survival, migration, growth, and metabolism. Indeed, we do not know whether the only function of ERK1/2 is the entry into the cell cycle of MuSCs and why it is induced so early. In fact, a recent study showed that ERK1/2 is also required for MuSCs viability and their double knock out leads to the loss of the MuSCs pool following injury [START_REF] Boyer | Depletion of skeletal muscle satellite cells attenuates pathology in muscular dystrophy[END_REF].

Appendix

Publications: Part I: Tissue damage induces a conserved stress response that initiates quiescent muscle stem cell activation Machado L*, Geara P*, Camps J, Dos Santos M, Teixeira-Clerc F, Van Herck J, Varet H, Legendre R, Pawlotsky JM, Sampaolesi M, Voet T, Maire P, Relaix F, Mourikis P. Tissue damage induces a conserved stress response that initiates quiescent muscle stem cell activation. Cell Stem Cell. 2021 Jun 3;28(6):1125-1135.e7. doi: 10.1016/j.stem.2021.01.

Short Article

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

Le ´o Machado, 1,14 Perla Geara, 1,14 Jordi Camps, 2,3,15 Matthieu Dos Santos, 4,15 Fatima Teixeira-Clerc, 1 Jens Van Herck, 5 Hugo Varet, 6,7 Rachel Legendre, 6,7 Jean-Michel Pawlotsky, 1,8 Maurilio Sampaolesi, 2,9 Thierry Voet, 5,10 Pascal Maire, 4 Frederic Relaix, 1,11,12,13,16, * and Philippos Mourikis 1, *

Limitations of study

In this study we have demonstrated the advantages of implementing specialized protocols for capturing accurately the molecular profile of cells both in homeostatic tissue and in response to injury. The two protocols we propose (in situ paraformaldehyde fixation of cells [Machado et al., 2017] and instant nuclei extraction from muscle [Dos Santos et al., 2020]), demonstrate great advantages but have certain limitations. Because of their chemical properties, aldehyde-fixed cells were incompatible with high-throughput 10x Genomics single-cell analysis. To overcome that, we performed snRNAseq, which results in lower yields and decreased gene coverage compared with single-cell approaches. Moreover, despite producing precise homeostatic atlases, snRNA-seq lacks spatial information.

In addition, we investigated the effect of dissociation on many cell types and focused on MuSCs as a paradigm for stem cell fate transition. The functional role of this cellular response should be studied in other cell types, including stem cells and terminally differentiated cells. Moreover, we analyzed two highly regenerative tissues from one mammalian organism. To strengthen the conservation aspect of the stress core, our field would benefit from a systematic study of this phenomenon across tissues and organisms, including humans, under normal and pathological conditions.

STAR+METHODS

Detailed methods are provided in the online version of this paper and include the following:

DECLARATION OF INTERESTS

The authors declare no competing interests.

STAR+METHODS KEY RESOURCES

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Frederic Relaix (frederic.relaix@inserm.fr).

Materials availability

This study did not generate new unique reagents. (middle) combined expression of upregulated genes from the selected signaling pathways visualized using the Seurat AddModuleScore() function in the selected cell-types; (right) UMAPplots of the merged intact and dissociated selected cell-types. Analysis of the upregulated genes showed enrichment for cell-specific biological processes. These included TGF-β signaling in the FAPs (K), known to be induced in the mesenchymal stromal cells during muscle regeneration (Contreras et al., 2019). TGF-β signaling is also involved in angiogenesis and was found enriched in muscle endothelial cells (L) (Van Meeteren and Ten Dijke, 2012). Moreover, we found a prominent response to cytokines in different cell-types with Interferon-gamma signaling in liver (A) Histogram representing the distribution of downregulated genes by dissociation in the 10 main populations (sinusoidal cells, hepatocytes, Kupffer cells, stellate cells, FAPs, tenocytes, muscle endothelial cells, smooth muscle cells, MuSCs, and macrophages) that we found in the muscle and liver snRNA-seq atlases (see Figure 1B).

(B) Heatmap displaying the expression of the Stress core genes in all cells from muscle and liver snRNA-seq, grouped by dissociation status (digested/intact).

(C) Stress index in UMAP plots of all cells from intact and dissociated muscle and liver (dissociation status blue/red in Fig. 1C). The Stress Index was calculated by the average expression of the 98 Stress core genes.

(D) Western blot analysis of ERK1/2 phosphorylation on intact (0 min) and 15 min digested whole muscle, showing early MAPK activation. (right) Quantification of pERK1\2 over total ERK ration (student t-test, * p<0.05).

(E) Graphical representation of the analysis: Stress core genes were used to calculate the Stress Index into the Tabula Muris datasets using more than 120,000 cells from the droplet (> 50,000 cells) and the Smart-Seq2 (> 70,000) datasets across 20 tissues.

(F) UMAP plots representing the Stress Index across 2,102 muscle single-cells (left) and 981 liver single-cells (right) from the published Tabula Muris Smart-Seq2 dataset. The digestion times at 37 °C is indicated adjacent to the panels: 90 min for muscle and < 20 min for liver.

(G) UMAP plot representing the Stress Index of every cell from the published Tabula Muris droplet dataset (70,118 cells).

(H) UMAP plot representing the digestion time at 37 °C of every cell from the published Tabula Muris droplet dataset (70,118 cells). Note the correlation between digestion time and Stress Index (Spearman correlation index: 0.54).

(I) Correlation between tissue digestion time at 37 °C and median tissue Stress Index calculated from the Tabula Muris Smartseq2 dataset. Spearman correlation index: 0.71.

(J) Correlation between tissue digestion time at 37 °C and median tissue Stress Index calculated from the Tabula Muris droplet dataset. Spearman correlation index: 0.71.

(K) Median Stress Index in different cell types calculated from the Tabula Muris droplet dataset. Median cell-type digestion time at 37 °C is color-coded (grey/blue gradient). (K) Comparison of the T0/T120 fold changes (Fig. 3) and the T0/T3 (Machado et al., 2017) fold changes for shared genes, showing strong reproducibility between the two RNA-seq experiments. Note that T3 refers to MuSCs digested for 120 min.

(L) Gene ontology analysis using EnrichR for all of the genes within each of the five clusters.

Color code indicates p-value and circle size the combined enrichment score (0 to 60). The ontology terms that emerged during MuSCs dissociation represented a sequence of coherent biological processes: the triggering events (cluster 1) were initiation of transcription and stimulation of the Skeletal muscle displays an incredible capacity to fully regenerate after severe injuries, thanks to a dedicated pool of muscle stem cells (MuSCs, also called satellite cells). Four groups showed independently that in the absence of MuSCs, skeletal muscle cannot recover from injuries [START_REF] Relaix | Satellite cells are essential for skeletal muscle regeneration: the cell on the edge returns centre stage[END_REF]. MuSCs have attracted much attention as a stem cell and regeneration model thanks to this extraordinary repair potential, but also because their malfunctioning could lead to (or fail to protect from) degenerative muscle disorders progression. To develop effective gene or cell therapies for these diseases, it is fundamental to elucidate the factors and pathways that ensure the correct function of the MuSCs. The Crelox genetic tool has been widely used for this purpose since it allows controlled spatiotemporal deletion of factors of interest, notably when using the estrogen receptor (ER) fusion alleles, CreER or CreERT2 (Feil et al., 1997), in which recombination is induced by the administration of exogenous estrogens (e.g. tamoxifen) that drive translocation of Cre recombinase to the nucleus.

Pax7 regulatory elements have been commonly used as the driver of Cre recombinase for lineage tracing and genetic alterations in MuSCs. Several muscle labs generated Pax7-Cre, Pax7-CreER (or Pax7-CreER TM ) and Pax7-CreERT2 alleles [START_REF] Relaix | Satellite cells are essential for skeletal muscle regeneration: the cell on the edge returns centre stage[END_REF]. The Pax7-CreERT2 generated by the Fan lab (Lepper et al., 2009) is the first MuSC-specific CreERT2 to be produced and thus has been widely used for a long time. However, CreERT2 is inserted offframe in exon 1 of the Pax7 locus rendering it null and thus generating a Pax7 heterozygous mouse, a parameter which has been disregarded in many studies (see below).

Pax7 CreERT2(Fan)/+ mice are heterozygous for Pax7 and PAX genes often show heterozygous phenotypes in mouse models and human disease (Buckingham and Relaix, 2007). Given that Pax7 is important for proper MuSC function, it is imperative to compare Pax7 CreERT2(Fan) -inducible mutants to Pax7 CreERT2(Fan)/+ controls rather than Pax7 +/+ controls.

Thus, we first mined the literature for studies with the Pax7 CreERT2(Fan) allele and we reviewed the selected controls. We listed all papers that cited the original publication of the Pax7 CreERT2(Fan) allele (Lepper et al., 2009), but also the three follow-up studies that could be mistaken for the original publication (Lepper et al., 2011;Lepper andFan, 2012, 2010). We then removed reviews, duplicates, papers that cited the study without using the Pax7 CreERT2(Fan) mice, and six publications that we could not access. This left us with 71 papers in which a floxed gene was recombined by Pax7 CreERT2(Fan) . Interestingly, more than half of those studies were using inappropriate controls (i.e. wild type mice) and only 31 of them compared mutant phenotypes to control Pax7 CreERT2(Fan)/+ allele (Figure 1A).

Strikingly, most studies that used a Pax7-CreERT2 allele to delete a gene of interest performed muscle injury and analyzed the capacity of the conditional mutant muscle to regenerate, with the aim of revealing the gene(s) that drive MuSC function and could be used in a therapeutic context. Thus, an inherent regenerative problem of the Pax7-CreERT2 mouse line could be wrongly attributed as a phenotype caused by targeted deletion of the floxed gene. We, therefore, wanted to investigate the regeneration capacity of the two most used Pax7-CreERT2 alleles generated by the Fan and Kardon labs (hereafter Pax7 CreERT2(Fan) and Pax7 CreERT2(Gaka) , respectively). In targeted alleles expressing CreERT2, Cre translocation to the nucleus is induced by tamoxifen; the effect of tamoxifen has been investigated in muscle and muscle diseases, but studies are lacking for MuSCs. In addition to untreated animals, we therefore analyzed cohorts under two (short induction) or five weeks of tamoxifen diet, which achieve 95% recombination efficiency (data not shown). We chose tamoxifen diet over other administration methods driven by the: i) need for less interventional animal treatments amid growing concerns on experimental animal welfare and increasingly strict regulations on experimental conditions, and ii) lack of side effects on mouse physiology by tamoxifen diet, as opposed to intraperitoneal injections [START_REF] Ceasrine | Tamoxifen Improves Glucose Tolerance in a Delivery-, Sex-, and Strain-Dependent Manner in Mice[END_REF]. To evaluate the interactions between tamoxifen treatment, the Pax7-CreERT2 alleles, and muscle tissue repair, we injected cardiotoxin into the Tibialis anterior (TA) muscle, a standard model of muscle injury and regeneration of tamoxifen-treated mice. Regenerating muscles were collected seven days post-injection (D7), when they are expected to have newly formed myofibers and MuSCs renewal.

We analyzed the regeneration capacity of the Pax7 CreERT2(Fan)/+ and Pax7 CreERT2(Gaka)/+ mice, which respectively contain a mutant or an intact (even though Pax7 expression has not been properly quantified in this allele) Pax7 allele, because the Cre was inserted in Exon 1 or as a bicistronic IRES transcript in the 3' UTR, respectively (Figure 1B). As an internal control of Pax7 loss, our analysis includes the Pax7 LacZ mouse that also harbors a Pax7 null allele (Figure 1B). Because these alleles come from slightly different genetic backgrounds, we used Pax7 +/+ littermates as controls for each mouse line. Histological analysis of regenerating muscles did not reveal gross morphological differences in any line (data not shown). In the absence of tamoxifen treatment, there was no significant difference in the number of PAX7+ cells (i.e.

highlights the importance of carefully designing mechanistic studies of stem cells with appropriate Cre alleles and respective controls. A. Scheme of literature mining to identify studies using the Pax7 CreERT2(Fan) allele and used controls. Briefly, a list of all studies citing the Pax7 CreERT2(Fan) allele, and all associated studies was made. Reviews, duplicates, studies that do not use the allele, and that we couldn't access were removed. Finally, out of the remaining studies, Controls were divided

Material and methods

Study analysis

In June 2022 we mined pubmed for all papers that cited one or more of the four publications that describe the Pax7 CreERT2(Fan) allele by the group that generated it (Lepper et al., 2011(Lepper et al., , 2009;;Lepper andFan, 2012, 2010). We then removed reviews and duplicates. The remaining papers were categorized as shown in the following table and as summarized in Figure 1A.

Category PMID numbers of publications Publications to which we had no full text access 22562803, 23708921, 30852688, 31734897, 32364045, 22290419 (Total: 6 Publications in which the Pax7 CreERT2(Fan) allele was used for lineage tracing rather than conditional knock-out 33131406, 20641127, 22869749, 23760954, 25133429, 26686466, 27918914, 28218909, 29091764, 29643389, 31545169 (Total: 11 publications) Publications in which the Pax7 CreERT2(Fan) allele was used for conditional knock-out and there is no control or ambiguous control 27548913, 25761890, 32433961, 24239359, 24276242, 25503558, 25636474, 29156133 (also

Mice

The following mouse lines were provided by the corresponding laboratories as described: (Lepper et al., 2009), Pax7 +/LacZ [START_REF] Mansouri | Dysgenesis of cephalic neural crest derivatives in Pax7-/-mutant mice[END_REF], Pax7 CreERT2 (G. Kardon; Jackson Laboratories, stock 017763; (Murphy et al., 2011). Animals were handled as per