Gene Expression Profiling of Muscle Stem Cells Identifies Novel Regulators of Postnatal Myogenesis

International audienceSkeletal muscle growth and regeneration require a population of muscle stem cells, the satellite cells, located in close contact to the myofiber. These cells are specified during fetal and early postnatal development in mice from a Pax3/7 population of embryonic progenitor cells. As little is known about the genetic control of their formation and maintenance, we performed a genome-wide chronological expression profile identifying the dynamic transcriptomic changes involved in establishment of muscle stem cells through life, and acquisition of muscle stem cell properties. We have identified multiple genes and pathways associated with satellite cell formation, including set of genes specifically induced (EphA1, EphA2, EfnA1, EphB1, Zbtb4, Zbtb20) or inhibited (EphA3, EphA4, EphA7, EfnA2, EfnA3, EfnA4, EfnA5, EphB2, EphB3, EphB4, EfnBs, Zfp354c, Zcchc5, Hmga2) in adult stem cells. Ephrin receptors and ephrins ligands have been implicated in cell migration and guidance in many tissues including skeletal muscle. Here we show that Ephrin receptors and ephrins ligands are also involved in regulating the adult myogenic program. Strikingly, impairment of EPHB1 function in satellite cells leads to increased differentiation at the expense of self-renewal in isolated myofiber cultures. In addition, we identified new transcription factors, including several zinc finger proteins. ZFP354C and ZCCHC5 decreased self-renewal capacity when overexpressed, whereas ZBTB4 increased it, and ZBTB20 induced myogenic progression. The architectural and transcriptional regulator HMGA2 was involved in satellite cell activation. Together, our study shows that transcriptome profiling coupled with myofiber culture analysis, provides an efficient system to identify and validate candidate genes implicated in establishment/maintenance of muscle stem cells. Furthermore, tour de force transcriptomic profiling provides a wealth of data to inform for future stem cell-based muscle therapies

Pax3 and Pax7 have distinct and overlapping functions in adult muscle progenitor cells

The growth and repair of skeletal muscle after birth depends on satellite cells that are characterized by the expression of Pax7. We show that Pax3, the paralogue of Pax7, is also present in both quiescent and activated satellite cells in many skeletal muscles. Dominant-negative forms of both Pax3 and -7 repress MyoD, but do not interfere with the expression of the other myogenic determination factor, Myf5, which, together with Pax3/7, regulates the myogenic differentiation of these cells. In Pax7 mutants, satellite cells are progressively lost in both Pax3-expressing and -nonexpressing muscles. We show that this is caused by satellite cell death, with effects on the cell cycle. Manipulation of the dominant-negative forms of these factors in satellite cell cultures demonstrates that Pax3 cannot replace the antiapoptotic function of Pax7. These findings underline the importance of cell survival in controlling the stem cell populations of adult tissues and demonstrate a role for upstream factors in this context

Transcriptome analyses based on genetic screens for Pax3 myogenic targets in the mouse embryo

<p>Abstract</p> <p>Background</p> <p>Pax3 is a key upstream regulator of the onset of myogenesis, controlling progenitor cell survival and behaviour as well as entry into the myogenic programme. It functions in the dermomyotome of the somite from which skeletal muscle derives and in progenitor cell populations that migrate from the somite such as those of the limbs. Few Pax3 target genes have been identified. Identifying genes that lie genetically downstream of <it>Pax3 </it>is therefore an important endeavour in elucidating the myogenic gene regulatory network.</p> <p>Results</p> <p>We have undertaken a screen in the mouse embryo which employs a <it>Pax3^GFP</it>allele that permits isolation of Pax3 expressing cells by flow cytometry and a <it>Pax3^PAX3-FKHR</it>allele that encodes PAX3-FKHR in which the DNA binding domain of Pax3 is fused to the strong transcriptional activation domain of FKHR. This constitutes a gain of function allele that rescues the <it>Pax3 </it>mutant phenotype. Microarray comparisons were carried out between <it>Pax3^GFP/+</it>and <it>Pax3^{GFP/PAX3-FKHR}</it>preparations from the hypaxial dermomyotome of somites at E9.5 and forelimb buds at E10.5. A further transcriptome comparison between Pax3-GFP positive and negative cells identified sequences specific to myogenic progenitors in the forelimb buds. Potential Pax3 targets, based on changes in transcript levels on the gain of function genetic background, were validated by analysis on loss or partial loss of function <it>Pax3 </it>mutant backgrounds. Sequences that are up- or down-regulated in the presence of PAX3-FKHR are classified as somite only, somite and limb or limb only. The latter should not contain sequences from Pax3 positive neural crest cells which do not invade the limbs. Verification by whole mount <it>in situ </it>hybridisation distinguishes myogenic markers. Presentation of potential Pax3 target genes focuses on signalling pathways and on transcriptional regulation.</p> <p>Conclusions</p> <p>Pax3 orchestrates many of the signalling pathways implicated in the activation or repression of myogenesis by regulating effectors and also, notably, inhibitors of these pathways. Important transcriptional regulators of myogenesis are candidate Pax3 targets. Myogenic determination genes, such as <it>Myf5 </it>are controlled positively, whereas the effect of <it>Pax3 </it>on genes encoding inhibitors of myogenesis provides a potential brake on differentiation. In the progenitor cell population, <it>Pax7 </it>and also <it>Hdac5 </it>which is a potential repressor of <it>Foxc2</it>, are subject to positive control by <it>Pax3</it>.</p

La voie Notch

Les muscles du squelette des vertébrés dérivent de structures mésodermiques transitoires : les somites. Dans la partie dorsale des somites, le dermomyotome, on trouve des cellules progénitrices capables de contribuer à la formation de l’ensemble des muscles du squelette du tronc et des membres. L’engagement de ces progéniteurs dans le programme de différenciation musculaire est induit par l’activation des facteurs de régulation myogéniques. Les progéniteurs fournissent l’ensemble des cellules myogéniques pendant la croissance de l’embryon et du fœtus et adoptent une position en périphérie des fibres musculaires lors de la période post-natale. Ces cellules quiescentes, appelées cellules satellites, constituent un réservoir de cellules souches musculaires post-natales qui permettent la croissance et la régénération musculaires. Parmi les mécanismes qui contrôlent leur maintien à l’état de progéniteur, on trouve la voie Notch. Cette voie de signalisation est également essentielle à la formation des somites et au développement des muscles du squelette chez l’embryon. Au cours du vieillissement, l’activité de la voie Notch diminue, ce qui participe à la diminution des capacités régénératrices du muscle. Cette voie de signalisation est donc un régulateur majeur de la biologie du muscle

Identification de nouveaux acteurs de la myogenèse adulte chez la souris (rôle de la signalisation Ephrin dans les cellules satellites du muscle du squelette chez la souris)

Les cellules souches jouent un rôle central pour le développement et le maintien des tissus chez l animal. Dans le tissu musculaire squelettique, les fibres différenciées ne peuvent plus se diviser. La production de cellules musculaires est assurée au cours du développement, et au cours de la régénération (après lésion) par une population de cellules souches qui ont la capacité de se diviser et différencier en nouvelle fibre. Au cours de l embryogenèse de la souris, les muscles squelettiques sont formés par des cellules appelées progéniteurs musculaires. Ces cellules sont prolifératives et désorganisées et vont donner autour de la naissance les cellules satellites associées aux fibres qui sont quiescentes, organisées le long des fibres et capables de s activer et se différencier pour former une nouvelle fibre mais également capables de s auto renouveler. Ces cellules satellites sont considérés comme étant les cellules souches du muscle adulte. Cette transition aux alentours de la naissance est caractérisée par l acquisition du caractère souche des cellules satellites. Une étude transcriptomique par biopuce a été réalisée au laboratoire pour identifier les gènes qui pouvaient être impliqués dans la transition des progéniteurs embryonnaire en cellules satellites, et donc jouant un rôle potentiel dans l acquisition du caractère souche . Ainsi nous avons comparé les transcriptôme entre le stade embryonnaire (noté E17.5) et le stade post-natal (noté P12). Parmi les gènes qui ont été identifiés, j ai choisi de caractériser 2 gènes appartenant à la famille des protéines à doigts de zinc (Zcchc5, Zfp354c) et 2 gènes appartenant à la famille des Ephrins (EphA4, EphB1). En effet, ces gènes présentent une forte variation d expression au cours de la transition et leur rôle est inconnu dans le muscle squelettique. Les deux protéines à doigts de zinc et EphA4 sont réprimés dans les cellules satellites, alors que EphB1 est induit dans celles-ci. Dans d autres tissus, les Ephrins sont impliqués dans les contacts entre cellules, la migration et la différenciation. Ces voies de signalisation pourraient donc jouer un rôle potentiel dans l interaction fibre/cellules satellites. Mon projet de thèse a permis de caractériser le rôle de ces nouveaux gènes candidats dans la myogenèse et l acquisition du caractère souche des cellules satellites. Pour cela, je me suis intéressée à leurs effets potentiels sur l activation, la prolifération, la différenciation et/ou l auto renouvellement des cellules satellites. La technique de culture sur fibres isolées flottantes a été utilisée. Après isolement des fibres isolées, elles ont été infectées afin de produire une surexpression (en gain de fonction pour Zcchc5, Zfp354c, EphA4 et avec un dominant négatif pour EphB1) et observer les conséquences sur l homéostasie des cellules satellites. Les résultats obtenus grâce à ce modèle suggèrent que les protéines à doigts de zinc pourraient agir sur la survie des cellules satellites et les Ephrins sont plutôt impliqués dans le contrôle de la prolifération/différenciation des cellules souches adultes. Il a été montré également que le gène EphB1 était impliqué dans le processus de régénération musculaire. La compréhension des signaux moléculaires permettant le contrôle et la régulation d une population de cellules souches musculaires sont essentiels afin de pouvoir ouvrir de nouvelles perspectives de thérapies des pathologies musculairesGrowth and regeneration of the skeletal muscle require a population of muscle stem cells, the satellite cells, located in close contact to the muscle myofiber. These cells develop from a population of embryonic progenitors and progressively enter quiescence. We have performed a screen to identify the genes that become express when the satellite cells are formed, in the early post-natal stages, and identified two zinc fingers (Zcch5 and Zfp354c) and two Ephrin receptors (EphA4 and EphB1). By C2C12 model and retroviral overexpressions, we show that two zinc fingers are involved in the fate of the satellite cells. EphA4 receptor is also involved in the proliferation / differentiation state of the muscular stem cells. And we decided to focus on EphB1, which is specifically expressed in the satellite cell population. Ephrins and Eph receptors have been previously implicated in many systems in cell migration and guidance, for instance in the nervous system. A previous report also implicated Ephrin signaling in muscle satellite cell motility and patterning, however, the possible involvement of this guidance signaling has not been linked with regulation of the myogenic programme. Here we show, using both C2C12 cell culture experiments and single fiber assays that EphB1 is involved in the control of adult myogenesis. Specific expression of a dominant negative form of EphB1 in activated satellite cells increase differentiation at the expense of self-renewal. We further show that EphB1 is specifically induced in regenerating muscle during satellite cells renewalPARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

Stress relief: emerging methods to mitigate dissociation-induced artefacts

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Master regulators of skeletal muscle lineage development and pluripotent stem cells differentiation

International audienceAbstract In vertebrates, the skeletal muscles of the body and their associated stem cells originate from muscle progenitor cells, during development. The specification of the muscles of the trunk, head and limbs, relies on the activity of distinct genetic hierarchies. The major regulators of trunk and limb muscle specification are the paired-homeobox transcription factors PAX3 and PAX7. Distinct gene regulatory networks drive the formation of the different muscles of the head. Despite the redeployment of diverse upstream regulators of muscle progenitor differentiation, the commitment towards the myogenic fate requires the expression of the early myogenic regulatory factors MYF5, MRF4, MYOD and the late differentiation marker MYOG. The expression of these genes is activated by muscle progenitors throughout development, in several waves of myogenic differentiation, constituting the embryonic, fetal and postnatal phases of muscle growth. In order to achieve myogenic cell commitment while maintaining an undifferentiated pool of muscle progenitors, several signaling pathways regulate the switch between proliferation and differentiation of myoblasts. The identification of the gene regulatory networks operating during myogenesis is crucial for the development of in vitro protocols to differentiate pluripotent stem cells into myoblasts required for regenerative medicine