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Summary The rapid turnover of the mammalian intestinal epithelium is supported by stem cells located around the base of the crypt1. Alongside Lgr5, intestinal stem cells have been associated with various markers, which are expressed heterogeneously within the crypt base region1-6. Previous quantitative clonal fate analyses have proposed that homeostasis occurs as the consequence of neutral competition between dividing stem cells7-9. However, the short-term behaviour of individual Lgr5+ cells positioned at different locations within the crypt base compartment has not been resolved. Here, we established the short-term dynamics of intestinal stem cells using a novel approach of continuous intravital imaging of Lgr5-Confetti mice. We find that Lgr5+ cells in the upper part of the niche (termed ‘border cells’) can be passively displaced into the transit-amplifying (TA) domain, following division of proximate cells, implying that determination of stem cell fate can be uncoupled from division. Through the quantitative analysis of individual clonal lineages, we show that stem cells at the crypt base, termed ‘central cells’, experience a survival advantage over border stem cells. However, through the transfer of stem cells between the border and central regions, all Lgr5+ cells are endowed with long-term self-renewal potential. These findings establish a novel paradigm for stem cell maintenance in which a dynamically heterogeneous cell population is able to function long-term as a single stem cell pool.

International audience; Notch signalling acts in virtually every tissue during the lifetime of metazoans. Recent studies have pointed to multiple roles for Notch in stem cells during quiescence, proliferation, temporal specification, and maintenance of the niche architecture. Skeletal muscle has served as an excellent paradigm to examine these diverse roles as embryonic, foetal, and adult skeletal muscle stem cells have different molecular signatures and functional properties, reflecting their developmental specification during ontology. Notably, Notch signalling has emerged as a major regulator of all muscle stem cells. This review will provide an overview of Notch signalling during myogenic development and postnatally, and underscore the seemingly opposing contextual activities of Notch that have lead to a reassessment of its role in myogenesis.

Adult skeletal muscle has a remarkable regenerative capacity, being able to recover after repeated trauma. This property depends on the presence of muscle stem cells (MuSCs), which are mostly quiescent in homeostatic conditions, re-enter the cell cycle after injury and proliferate to give rise to committed myoblasts that will eventually fuse to restore the damaged fibres. Numerous studies have investigated the cell state transitions that MuSCs undergo from cell cycle entry to differentiation. Although several genetically modified reporter mice have been generated to study these events, detailed studies on the initiation of differentiation, which is generally defined by expression of the myogenic regulatory factor Myogenin, have been hampered by the lack of a reliable reporter mouse. Therefore, we developed a fluorescent reporter line where differentiating myogenic cells expressing Myogenin are marked by the expression of a tdTomato fluorescent protein. This novel knock-in mouse line allowed us to monitor the kinetics of Myogenin expression during cell differentiation in vitro, and perform preliminary experiments on the behaviour of myogenic cells in vivo by intravital imaging. Although all mouse MuSCs are characterised by the expression of the transcription factor Pax7 and they share several properties, some studies have reported differences in proliferation, engraftment ability, and sensitivity to disease of MuSCs from cranial and limb muscles. To investigate the gene regulatory networks that govern this functional heterogeneity, we have integrated single-cell transcriptomic analyses with cell biology approaches using mouse reporter lines to identify key regulators that confer distinct properties to high performing (extraocular muscles) and lower performing (limb, Tibialis anterior muscle) MuSCs in quiescence and activated states. We identified a delayed lineage progression of extraocular MuSCs in culture that was accompanied with the expression of distinct extracellular matrix remodelling factors and membrane receptors, and we validated the expression of some of these candidates at the protein level. Advanced computational analyses highlighted the dynamics underlying the maintenance of a stem-like progenitor population in extraocular MuSCs, controlled by a singular network of transcription factors acting as a co-regulating module. Taken together, these studies provide novel insights into the mechanisms underlying the differential properties of muscle stem cells in distinct anatomical locations.; Le muscle squelettique adulte a une capacité de régénération remarquable, pouvant guérir après des traumatismes répétés. Cette propriété dépend de la présence de cellules souches musculaires (SCMu), qui sont pour la plupart quiescentes dans des conditions homéostatiques mais qui s'activent après une blessure, réintègrent le cycle cellulaire et prolifèrent pour donner naissance à des myoblastes qui fusionneront pour restaurer les fibres endommagées. De nombreuses études ont étudié les états transitoires que les SCMu empruntent de l'entrée du cycle cellulaire à la différenciation. Malgré le fait que plusieurs souris rapportrices génétiquement modifiées aient été générées pour examiner ces événements, l'initiation de la différenciation, qui est généralement définie par l'expression du facteur de régulation myogénique Myogenin, est difficilement appréciable à cause du manque de souris rapportrice fiable. Par conséquent, nous avons développé une nouvelle lignée rapportrice où la différenciation des cellules myogéniques exprimant le facteur de transcription Myogenin peut être marquée par l'expression d'une protéine fluorescente tdTomato. Cette nouvelle lignée de souris knock-in nous a permis d'analyser la cinétique de l'expression de Myogenin lors de la différenciation cellulaire in vitro et d'effectuer des expériences préliminaires in vivo par imagerie intravitale. De plus, bien que toutes les SCMu de souris soient caractérisées par l'expression du facteur de transcription Pax7, plusieurs études ont décrit des différences de prolifération, de capacité de transplantation et de sensibilité à la maladie entre les SCMu des muscles crâniens et des membres. Pour étudier les réseaux de régulation des gènes qui régissent cette hétérogénéité fonctionnelle, nous avons combiné des analyses transcriptomiques sur cellules uniques avec des approches de biologie cellulaire utilisant des lignées de souris rapportrices pour identifier les régulateurs clés qui confèrent des propriétés distinctes aux SCMu à haute-performance (extraoculaires) et à faible-performance (tibialis antérieur) en quiescence et lors de l'activation. Nous avons identifié un retard dans la différenciation des SCMu extraoculaires en culture, accompagné par l'expression de facteurs de remodelage de la matrice extracellulaire et de récepteurs membranaires distincts et nous avons validé l'expression de certains de ces candidats au niveau protéique. Des analyses informatiques avancées ont mis en évidence la dynamique sous-jacente au maintien d'une population de progéniteurs dans les SCMu extraoculaires, contrôlée par un réseau singulier de facteurs de transcription formant un module de molécules co-régulées. En conclusion, ces études apportent de nouvelles informations sur les mécanismes qui octroient des propriétés différentes des cellules souches musculaires venant d'emplacements anatomiques distincts.

Skeletal muscles are found throughout the body and they display a surprising level of heterogeneity in properties and function. For example, some muscles are specifically susceptible to diseases, and some have better regenerative potential or different metabolic capacities. Diversity is also found during embryonic development where myogenic and non-myogenic cells establish the musculoskeletal system. The head and neck are comprised of a wide variety of muscles that perform essential functions such as feeding, breathing and vocalising, yet little is known about craniofacial muscle biology. Novel structures are associated with the emergence of neural crest cells (NCC) which give rise to most craniofacial connective tissue, cartilage and bone and are crucial for muscle morphogenesis. However, some cranial muscles are deprived of NCC, and it is unclear how myogenic and non-myogenic cells contribute to those domains. This thesis provides evidence demonstrating that upstream progenitors redirect from the myogenic program to give rise to the muscle-associated connective tissue that supports the formation of muscular structures. We employed unbiased and lineage-restricted single-cell RNAseq using different mouse transgenic lines at distinct embryonic stages, in situ labelling, and new analytical methods, and show that bipotent progenitors expressing the muscle determination gene Myf5 give rise to skeletal muscle and anatomically associated connective tissue in distinct muscle groups spatiotemporally. Notably, this property was restricted to muscles with only partial contribution from NCCs suggesting that in their absence, the balance of myogenic and connective tissue cells is undertaken by somite-derived or cranial-derived mesoderm. This transition is characterised by a complementarity of tyrosine kinase receptor signalling between muscle and non-muscle cells, as well as distinct regulatory modules. Cranial muscles also originate from different lineages that involve the activity of specific gene regulatory cascades. Here, we used an all-inclusive unbiased approach to uncover specific regulatory modules that underlie different myogenic cell populations in the head and across multiple developmental stages. Some of these unique “genetic birthmarks” are specific transcription factors, and are retained in adult muscle stem cells pointing to their potential importance is delivering the unique properties that have been reported for different muscle stem cell populations. Finally, these studies employ novel computational methods that benefit from the latest algorithmic advancements and they provide prospects for the discovery of new biological processes from high throughput data.; Les muscles squelettiques sont présents dans tout le corps et présentent un niveau surprenant d'hétérogénéité, dans leur susceptibilité aux maladies, potentiel de régénération ou capacités métaboliques. Cette diversité est également retrouvée au cours du développement embryonnaire où les cellules myogéniques et non myogéniques établissent le système musculo-squelettique. La tête et le cou sont constitués d'une grande variété de muscles qui remplissent des fonctions essentielles, mais nous en savons peu sur la biologie des muscles craniofaciaux. Ces structures sont associées à l'émergence de cellules de la crête neurale (CCN) qui donnent naissance à la plupart des tissus non myogéniques crâniens et qui sont cruciales à la formation des muscles. Cependant, certains muscles crâniens sont privés de CCN, et nous ignorons comment les cellules myogéniques et non myogéniques contribuent à ces domaines. Cette thèse fournit des preuves démontrant que les progéniteurs en amont du muscle se détournent du programme myogénique pour donner naissance au tissu conjonctif. Nous avons utilisé une approche de single-cell RNAseq non biaisée et restreinte avec différentes lignées transgéniques de souris à des stades embryonnaires distincts, des marquages in situ et de nouvelles méthodes analytiques, et avons montré que les progéniteurs bipotents issus du mésoderme exprimant le gène de détermination musculaire Myf5 donnent naissance au muscle squelettique et au tissu conjonctif anatomiquement associé dans les muscles partiellement privés de CCN. Cette transition est caractérisée par une complémentarité de signalisation de récepteurs tyrosine kinase entre les cellules musculaires et non musculaires, ainsi que par des modules régulateurs distincts. Les muscles crâniens proviennent également de différentes lignées qui impliquent l'activité de cascades de régulation génique spécifiques. Ici, nous avons utilisé une approche non biaisée et large pour découvrir des modules de régulation spécifiques qui sous-tendent différentes populations de cellules myogéniques dans la tête et à travers plusieurs stades de développement. Certaines de ces « tâches de naissance génétiques » uniques sont des facteurs de transcription spécifiques et sont conservées dans les cellules souches musculaires adultes, ce qui indique que leur importance potentielle est de fournir les propriétés uniques qui ont été signalées pour différentes populations de cellules souches musculaires. Enfin, ces études utilisent des méthodes analytiques inédites qui bénéficient des dernières avancées algorithmiques et offrent de nouvelles perspectives pour la découverte de processus biologiques à partir de données à haut débit.

Adult neural stem cells in vertebrates are maintained at long term through a hierarchy of subfunctionalized stem cell pools., The cellular basis and extent of neural stem cell (NSC) self-renewal in adult vertebrates, and their heterogeneity, remain controversial. To explore the functional behavior and dynamics of individual NSCs, we combined genetic lineage tracing, quantitative clonal analysis, intravital imaging, and global population assessments in the adult zebrafish telencephalon. Our results are compatible with a model where adult neurogenesis is organized in a hierarchy in which a subpopulation of deeply quiescent reservoir NSCs with long-term self-renewal potential generate, through asymmetric divisions, a pool of operational NSCs activating more frequently and taking stochastic fates biased toward neuronal differentiation. Our data further suggest the existence of an additional, upstream, progenitor population that supports the continuous generation of new reservoir NSCs, thus contributing to their overall expansion. Hence, we propose that the dynamics of vertebrate neurogenesis relies on a hierarchical organization where growth, self-renewal, and neurogenic functions are segregated between different NSC types.

Muscle stem (satellite, MuSC) cells acquire different cell states as they need to pass from quiescence to proliferation and differentiation to support muscle homeostasis. Some of these changes are accompanied by changes in energy demands. However, it is currently unclear whether modulation in the energy metabolism pathways can in turn influence the commitment to a specific cell state. A central focus of my thesis project is to characterise the energy metabolism pathways that act in the different phases of lineage progression and how their modulation can influence the state of the cell. We show that quiescent cells have low energetic demands and OxPhos is perturbed during aging, as well as in cells that survive after death. We also compared different proliferative states, both during muscle growth and regeneration, and our results indicate a surprising difference in their metabolic requirements. Gene expression profiling and bioenergetics analysis showed that foetal cells have a low respiration demand and rely mostly on glycolysis when compared to regenerating MuSCs. Furthermore, we show distinct requirements for peroxisomal and mitochondrial mediated fatty acid oxidation (FAO) in myogenic cells. Altering peroxisomal but not mitochondrial FAO promotes early differentiation of satellite cells. Experiments using acute muscle injury and pharmacological block show differential requirements for these organelles during regeneration. These observations indicate that changes in the cell state of muscle stem cells lead to significant changes in metabolic requirements and altering specific metabolic pathways can have an impact on myogenic cell fate and the regeneration process.; Les cellules souches musculaires, ou cellules satellites, adoptent différents états en transitant de quiescence à prolifération et différentiation. Ces transitions s'accompagnent de variations des demandes énergétiques. Il demeure cependant incertain comment la modulation du métabolisme énergétique peut dicter la spécification d'un état cellulaire donné. Mon projet de thèse a eu pour objectif principal la caractérisation des voies du métabolisme énergétique à l’œuvre dans les différents états cellulaires, et comment leur modulation peut influencer ces états. Nous montrons ainsi que les cellules satellites quiescentes ont de faibles besoins énergétiques et que la phosphorylation oxydative est altérée au cours du vieillissement ainsi que dans les cellules survivant après la mort de l'animal. Au cours de la formation du tissu en croissance ou en régénération chez l'adulte, nos résultats indiquent de larges différences dans leurs demandes énergétiques. Les cellules fœtales ont une faible demande respiratoire et reposent essentiellement sur la glycolyse par rapport aux cellules adultes en cours de régénération. L'altération de la b-oxidation peroxisomale et non mitochondriale induit une différentiation précoce des cellules satellites. L'inhibition pharmacologique des b-oxidations peroxisomale et mitochondriale après blessure aiguë montre différentes contributions de ces organelles à la régénération musculaire. Les transitions entre différents états des cellules satellites s'accompagnent de modifications drastiques de leurs besoins énergétiques et l'altération de vois métaboliques spécifiques peut altérer le destin des cellules myogéniques et la régénération musculaire.

International audience; The changes imposed on the nucleus, chromatin and its regulators during mitosis lead to the dismantlement of most gene regulatory processes. However, an increasing number of transcriptional regulators are being identified as capable of binding their genomic targets during mitosis. These so-called 'mitotic bookmarking factors' encompass transcription factors and chromatin modifiers that are believed to convey gene regulatory information from mother to daughter cells. In this Primer, we review mitotic bookmarking processes in development and stem cells and discuss the interest and potential importance of this concept with regard to epigenetic regulation and cell fate transitions involving cellular proliferation.

Adult skeletal muscles can regenerate after repeated trauma, yet our understanding of how adult muscle satellite (stem) cells (MuSCs) restore muscle integrity and homeostasis after regeneration is limited. In the adult mouse, MuSCs are quiescent and located between the basal lamina and the myofibre. After injury, they re-enter the cell cycle, proliferate, differentiate and fuse to restore the damaged fibre. A subpopulation of myogenic cells then self-renews and replenishes the stem cell pool for future repair. When MuSCs are removed from their niche, they rapidly express the commitment marker Myod and proliferate. The basal lamina that ensheaths MuSCs is rich in collagens, non-collagenous glycoproteins and proteoglycans. Whether these and other extracellular matrix (ECM) proteins constitute functional components of MuSCs niche remains unclear. Moreover, although signalling pathways that maintain MuSCs quiescence have been identified, how these regulate stem cell properties and niche composition remains largely unknown. Sustained, high activity of the Notch signalling pathway is critical for the maintenance of MuSCs in a quiescence state. Of interest, whole-genome ChIP for direct Notch/Rbpj transcriptional targets identified specific micro-RNAs and collagen genes in satellite cells. Using genetic tools to conditionally activate or abrogate Notch signalling, we demonstrate that the expression of these target genes is controlled by the Notch pathway in vitro and in vivo. Further, we propose that Collagen V and miR708 can contribute cell-autonomously to the generation of the MuSCs niche via a Notch signalling-regulated mechanism.; Le muscle squelettique adulte est capable de se régénérer à plusieurs reprises après blessure grâce à sa population de cellules souches résidentes: les cellules satellites. Cependant, les mécanismes impliquant les cellules satellite dans la recouvrement de l'homéostasie et de l'intégrité musculaire ne sont toujours pas clairs. Chez l'adulte, les cellules satellites sont quiescentes et localisées dans une niche entre la lame basale et la fibre musculaire. Après blessure, elles prolifèrent, se différencient et fusent afin de restaurer les fibres endommagées. Lorsque la niche des cellules satellite est altérée elles expriment rapidement le marqueur d'activation Myod puis prolifèrent. La lame basale des cellules souches est riche en collagène, glycoprotéines et de protéoglycan. Cependant, le mécanisme de fonction de ces protéines de la matrice extracellulaire (MEC) dans le maintien de la cellule satellite dans sa niche est toujours inconnu. De plus, l'interaction entre la MEC et des voies de signalisation cellulaire essentielles au maintien des cellules souches quiescentes sont toujours un mystère. Nous avons identifiés la voie Notch comme effecteur indispensable à la quiescence des cellules satellites. Un ChIP screening dans des cellules musculaires nous a permit d'identifier des microRNAs et collagènes spécifiques comme des gènes cibles de la voie Notch. L'utilisation d'outils génétiques permettant de moduler l'activité de la voie Notch démontrent que ces microRNAs et collagènes sont régulés transcriptionnellement par la voie Notch in vitro et in vivo. Nous proposons que le Collagène de type V et miR-708, induits par Notch, peuvent autoréguler la niche des cellules souches.

Adult skeletal muscles can regenerate after repeated trauma, yet our understanding of how adult muscle satellite (stem) cells (MuSCs) restore muscle integrity and homeostasis after regeneration is limited. In the adult mouse, MuSCs are quiescent and located between the basal lamina and the myofibre. After injury, they re-enter the cell cycle, proliferate, differentiate and fuse to restore the damaged fibre. A subpopulation of myogenic cells then self-renews and replenishes the stem cell pool for future repair. When MuSCs are removed from their niche, they rapidly express the commitment marker Myod and proliferate. The basal lamina that ensheaths MuSCs is rich in collagens, non-collagenous glycoproteins and proteoglycans. Whether these and other extracellular matrix (ECM) proteins constitute functional components of MuSCs niche remains unclear. Moreover, although signalling pathways that maintain MuSCs quiescence have been identified, how these regulate stem cell properties and niche composition remains largely unknown. Sustained, high activity of the Notch signalling pathway is critical for the maintenance of MuSCs in a quiescence state. Of interest, whole-genome ChIP for direct Notch/Rbpj transcriptional targets identified specific micro-RNAs and collagen genes in satellite cells. Using genetic tools to conditionally activate or abrogate Notch signalling, we demonstrate that the expression of these target genes is controlled by the Notch pathway in vitro and in vivo. Further, we propose that Collagen V and miR708 can contribute cell-autonomously to the generation of the MuSCs niche via a Notch signalling-regulated mechanism.; Le muscle squelettique adulte est capable de se régénérer à plusieurs reprises après blessure grâce à sa population de cellules souches résidentes: les cellules satellites. Cependant, les mécanismes impliquant les cellules satellite dans la recouvrement de l'homéostasie et de l'intégrité musculaire ne sont toujours pas clairs. Chez l'adulte, les cellules satellites sont quiescentes et localisées dans une niche entre la lame basale et la fibre musculaire. Après blessure, elles prolifèrent, se différencient et fusent afin de restaurer les fibres endommagées. Lorsque la niche des cellules satellite est altérée elles expriment rapidement le marqueur d'activation Myod puis prolifèrent. La lame basale des cellules souches est riche en collagène, glycoprotéines et de protéoglycan. Cependant, le mécanisme de fonction de ces protéines de la matrice extracellulaire (MEC) dans le maintien de la cellule satellite dans sa niche est toujours inconnu. De plus, l'interaction entre la MEC et des voies de signalisation cellulaire essentielles au maintien des cellules souches quiescentes sont toujours un mystère. Nous avons identifiés la voie Notch comme effecteur indispensable à la quiescence des cellules satellites. Un ChIP screening dans des cellules musculaires nous a permit d'identifier des microRNAs et collagènes spécifiques comme des gènes cibles de la voie Notch. L'utilisation d'outils génétiques permettant de moduler l'activité de la voie Notch démontrent que ces microRNAs et collagènes sont régulés transcriptionnellement par la voie Notch in vitro et in vivo. Nous proposons que le Collagène de type V et miR-708, induits par Notch, peuvent autoréguler la niche des cellules souches.

International audience; Vezatin, a protein associated to adherens junctions in epithelial cells, is already expressed in mouse oocytes and during pre-implantation development. Using a floxed strategy to generate a vezatin-null allele, we show that the lack of zygotic vezatin is embryonic lethal, indicating that vezatin is an essential gene. Homozygous null embryos are able to elicit a decidual response but as early as day 6.0 post-coitum mutant implantation sites are devoid of embryonic structures. Mutant blastocysts are morphologically normal, but only half of them are able to hatch upon in vitro culture and the blastocyst outgrowths formed after 3.5 days in culture exhibit severe abnormalities, in particular disrupted intercellular adhesion and clear signs of cellular degeneration. Notably, the junctional proteins E-cadherin and beta-catenin are delocalized and not observed at the plasma membrane anymore. These in vitro observations reinforce the idea that homozygous vezatin-null mutants die at the time of implantation because of a defect in intercellular adhesion. Together these results indicate that the absence of zygotic vezatin is deleterious for the implantation process, most likely because cadherin-dependent intercellular adhesion is impaired in late blastocysts when the maternal vezatin is lost.

International audience; While highly conserved through evolution, the cell cycle has been extensively modified to adapt to new developmental programs. Recently, analyses of mouse mutants revealed that several important cell cycle regulators are either dispensable for development or have a tissue- or cell-type-specific function, indicating that many aspects of cell cycle regulation during mammalian embryo development remain to be elucidated. Here, we report on the characterization of a new gene, Omcg1, which codes for a nuclear zinc finger protein. Embryos lacking Omcg1 die by the end of preimplantation development. In vitro cultured Omcg1-null blastocysts exhibit a dramatic reduction in the total cell number, a high mitotic index, and the presence of abnormal mitotic figures. Importantly, we found that Omcg1 disruption results in the lengthening of M phase rather than in a mitotic block. We show that the mitotic delay in Omcg1-/- embryos is associated with neither a dysfunction of the spindle checkpoint nor abnormal global histone modifications. Taken together, these results suggest that Omcg1 is an important regulator of the cell cycle in the preimplantation embryo.

International audience; Recent studies show that cell dispersal is a widespread phenomenon in the development of early vertebrate embryos. These cell movements coincide with major decisions for the spatial organization of the embryo, and they parallel genetic patterning events. For example, in the central nervous system, cell dispersal is first mainly anterior-posterior and subsequently dorsal-ventral. Thus, genes expressed in signaling centers of the embryo probably control cell movements, tightly linking cellular and genetic patterning. Cell dispersal might be important for the correct positioning of cells and tissues involved in intercellular signaling. The emergence of cell dispersal at the onset of vertebrate evolution indicates a shift from early, lineage-based cellular patterning in small embryos to late, movement-based cellular patterning of polyclones in large embryos. The conservation of the same basic body plan by invertebrate and vertebrate chordates suggests that evolution of the embryonic period preceding the phylotypic stage was by intercalary co-option of basic cell activities present in the ancestral metazoan cell.

Analysis of lacZ neuronal clones in the mouse cerebellum demonstrates genealogical independence of the primary and secondary germinal epithelia (PGE and SGE) from early development. PGE precursors and their neuronal descendants are organised into two polyclonal groups of similar sizes that exhibit parasagittal patterning and generally respect the midline. The relationship between these two groups cannot be traced back in time to less than 80 independent cells, which were probably recruited following a period of non-coherent growth that distributes unrelated cells into distinct territories of the neural tube. A lateromedial clonal organisation is observed in the mature cerebellum, suggesting the existence of many small parasagittal domains of clonal restriction and/or of cell dis- persion in the rostrocaudal but not in the mediolateral dimension. The organisation is orthogonal with respect to the cellular organisation in the neural tube as is the genetic organisation. Cellular and genetic patterning of the cer- ebellum therefore share similarities. A possible hypothesis is that distinct cell behaviours create the different clonal domains observed in this study and that the cellular and genetic organisation of the cerebellum are coordinated.