CCAAT/Enhancer Binding Proteins are a family of six bZIP transcription factors. C/EBPβ, the second member cloned, has been implicated in adipogenesis and osteogenesis, but the role of C/EBPβ in myogenesis remained undetermined. In adults, muscle-resident stem cells, called satellite cells (SCs), have the greatest propensity to regenerate the skeletal muscle. We found that C/EBPβ is expressed in SCs, and its expression progressively declines upon differentiation. Forcing the expression of C/EBPβ in myoblasts enhanced the expression of the SC marker Pax7, and repressed MyoD and the myogenic genes expression, resulting in the inhibition of myogenesis. Using a SC-specific conditional knockout (cKO) mouse model, we found that cKO myoblasts have decreased expression of Pax7, and we identified Pax7 as a direct target of C/EBPβ action. In vivo, excision of C/EBPβ resulted in muscle hypertrophy at the juvenile age, and adult cKO animals had enhanced muscle regeneration following BaCl2 muscle injury. Moreover, the number of Pax7+ cells in cKO animals decreased following BaCl2 injury. Upon performing a second injury into cKO animals, we demonstrate a decreased muscle fiber size and an exacerbation of the percentage number of SCs. While cKO animals repaired well a BaCl2 injury, regeneration failed in cKO animals following cardiotoxin (CTX) injury. We demonstrate that IL-1β expression is enhanced in muscle after CTX injury when compared to BaCl2, and we found that IL-1β can stimulate the expression of C/EBPβ in myoblasts. Ectopic C/EBPβ expression can protect myoblasts from apoptosis when triggered with thapsigargin, whereas cKO myoblasts are more sensitive to apoptosis. Using cancer cachexia as a model of chronic inflammation, we found that the expression of C/EBPβ is stimulated in the SCs of cachectic animals, and this correlated with a decrease in regenerative capacity. The severity of muscle wasting was not improved in cKO animals, but rather cKO SCs were lost to apoptosis. Together, this study establishes a protective role for C/EBPβ in muscle SCs in conditions of inflammation.

The post-natal regenerative capacity of skeletal muscle is attributed to myogenic satellite cells, which function as lineage-committed precursors to replace terminally differentiated muscle. The development and differentiation of the satellite cell lineage is regulated by Pax7 and the myogenic regulatory factors. While the expression of Pax7 is vital to the function of the satellite cell compartment, the paired domain alternative splicing events that regulate its DNA binding potential remain elusive. Interestingly, the generation of Pax7 splice variants differentially regulate Myf5 expression. We performed a global analysis of two Pax7 isoforms, which differ by a glycine-leucine dipeptide, to determine how paired domain splicing events modify the ability of Pax7 to regulate target genes. To this end, we observe that although the homeodomain is important for Pax7 binding, these isoform differences in the paired domain can regulate Pax7 targets during myogenesis. In addition to further examining the role of Pax7 during satellite cell proliferation and maintenance, it remains important to understand their downstream differentiation potential. Since activation of the canonical Wnt signalling pathway results in reduced regenerative efficiency in vivo, we undertook a global analysis of satellite cell derived myoblasts to examine their ability to respond to canonical Wnt signalling. We demonstrate that Wnt/β-Catenin signalling drives myogenic differentiation, via the myogenin-dependent control of follistatin expression, further fine-tuning the myogenic differentiation process. The effects of canonical Wnt signalling on myogenic differentiation complement our observations regarding Pax7 alternative splicing during myoblast proliferation and provide a greater comprehensive understanding of the molecular regulation of satellite cell development and differentiation during adult myogenesis.

Chez les vertébrés, les muscles squelettiques du corps sont dérivés de la partie dorsale dessomites, le dermomyotome, structure transitoire mésodermique. Une première étape demyogenèse aboutit à la formation d’un muscle primitif, le myotome primaire, à partir desbordures du dermomyotome : ces cellules constituent les premières fibres musculaires, et formentl’architecture de base du futur muscle. Dans un second temps, une population de progéniteursmusculaires émerge de la région centrale du dermomyotome. Cette population est primordialedans la constitution du muscle. Elle prolifère, et une partie d’entre elle fusionne aux fibresexistantes pour donner les fibres multinucléées adultes. Finalement, une partie des progéniteursmusculaires reste indifférenciée jusqu’à l’âge adulte et compose la population de cellules souchesmusculaires, les cellules satellites. Ainsi, les progéniteurs musculaires contribuent audéveloppement musculaire tout au long du développement embryonnaire et foetal, mais égalementà la myogenèse post-natale avec les cellules satellites.Lors de ma thèse, je me suis intéressé à cette population de progéniteurs musculaires. Deux souspopulationsde progéniteurs musculaires ont précédemment été identifiées dans notre laboratoireau cours de l’embryogénèse précoce de poulet, l’une exprimant le facteur de transcription Pax7,l’autre co-exprimant Pax7 et le facteur de différenciation myogénique précoce Myf5. Face àl’absence de données concernant les progéniteurs musculaires, et à l’importance de cettepopulation pour la myogenèse, j’ai réalisé une étude systématique des progéniteurs musculairestout au long du développement embryonnaire et foetal de deux organismes modèles : le poulet etla souris. J’ai pu montrer que ces deux sous-populations coexistent tout au long dudéveloppement, depuis l’émergence des progéniteurs de la partie centrale du dermomyotome,jusqu’au moment où ces cellules deviennent des cellules satellites à la fin du développementfoetal. De manière très intéressante, j’ai pu montrer qu’au sein des progéniteurs musculaires, lapopulation principale co-exprime Pax7 et Myf5, et prolifère activement, alors que la populationPax7 est mineure et prolifère à un taux moins élevé. Cette dernière entre de manière importanteen quiescence à la fin du développement embryonnaire. Ces caractéristiques sont semblablesentre le poulet et la souris, et montrent que des stratégies cellulaires et moléculaires similairessont conservées au sein des amniotes. / Duringembryonicandfetallife,skeletalmusclegrowthisdependentupontheproliferationandthedifferentiationofapopulationofresidentmuscleprogenitors,fromwhichderivethemusclestemcellsof theadult,thesatellitecells.Underpoorlydefinedextrinsicandintrinsicinfluences,muscleprogenitorsproliferate,differentiateorenteraquiescentstatetobecomereservesatellitecells.Despitetheir primordialrole,surprisinglylittleisknownonthehomeostasisofresidentprogenitorsduringembryogenesis.Preliminarystudiesinchickandmousedescribingthekeyprogenitorpopulationscontributingtomusclegrowthduringembryogenesishaveledtodifferingresultsthatcouldbeduetotechnicalissuesortofundamentaldifferencesbetweenanimalmodels.Toaddressthisquestion,we haveundertakenacomprehensiveanalysisofthestateofdifferentiationandproliferationofmuscleprogenitorcellsfromthetimeoftheiremergencewithinthedermomyotomeuntillatefetallife,whenthey adoptasatellitecell-likepositionunderthebasallamina.Thiswasdonebyimmunostainingagainstkeyplayersofmyogenicdifferentiation,inmuscleschosenfromdifferentregionsofthebodyintwo modelorganisms,thechickandmouse.This studyidentifiedtwoco-existingpopulationsofprogenitorsduringembryonicandfetallifeinboth chickandmouse:aminor,slow-cyclingpoolofundifferentiatedresidentprogenitorswhichexpress Pax7,co-existingwithamajorfast-cyclingpopulationthatco-expressPax7andtheearlymyogenicdifferentiationmarkerMyf5.Wefoundthattheoverallproliferationrateofbothprogenitorsdrasticallydecreasedwithembryonicage,asanincreasinglylargeportionofslowandfast-cyclingprogenitorsenteredquiescenceduringdevelopment.Together,thisdatasuggeststhatthecellularstrategiesthatdrivemusclegrowthduringembryonicand fetallifeareremarkablyconservedinamniotesthroughoutevolution.Theyrelyonthetightregulationofproliferation,entryinquiescence,andmodulationofthecellcycle’slengthforbothoftheco-existingpopulationsofmuscleprogenitorstomaintainthehomeostasisofgrowingmusclesduringdevelopment.

Les homéoprotéines SIX sont codées par les gènes Sine oculis homeobox related genes Six1 à Six6 chez les vertébrés parmi lesquels Six1, Six2, Six4 et Six5 sont exprimés dans le lignage myogénique. Bien que Six1 et Six4 soient requis pour la myogenèse hypaxiale, les animaux doubles KO pour ces deux gènes (s1s4KO) forment leurs muscles épaxiaux et craniofaciaux. Nous avons caractérisé le phénotype de mutants composites des gènes Six et avons montré que l’absence de Six1 et Six2 empêchait la formation des muscles craniofaciaux et empirait les défauts de formation des muscles des membres observés chez les fœtus mutants pour Six1. Nous avons aussi observé que les fœtus dépourvus d’activité de SIX1, SIX2, SIX4 et SIX5 étaient toujours capables de former leurs muscles épaxiaux, mais que l’expression de Pax7 dans leurs progéniteurs myogéniques était fortement diminuée et mêlée à l’expression de Myogénine. Alors que les fœtus s1s4KO forment des muscles épaxiaux, leurs cellules PAX7+ ont un défaut de nichage entre la membrane plasmique des myofibres et la lame basale qui les entoure. Nos analyses transcriptomiques, nos expériences de transplantation et nos études in vitro nous ont permis de conclure que le nichage des cellules PAX7+ nécessitait un environnement adéquat combinant des propriétés des myofibres et des cellules PAX7+ ; environnement perturbé dans les muscles épaxiaux s1s4KO. Nos expériences de transplantation nous ont aussi permis de conclure que Six1 et Six4 étaient requis pour une bonne ré-innervation des myofibres après blessure et pour la mise en place du phénotype rapide de ces myofibres. De plus, les muscles transplantés avec des cellules PAX7+ fœtales s1s4KO après blessure se reforment d’un grand nombre de petites myofibres. Nous avons pu relier ce phénotype au comportement des cellules s1s4KO in vitro où elles montrent un défaut de fusion. Enfin, les homéoprotéines SIX ont besoin de co-facteurs pour induire l’expression de leurs gènes cibles, tels que les protéines EYA codées par les gènes Eya1 à Eya4 chez les vertébrés. Eya3 et Eya4 sont fortement exprimés dans les cellules satellite au cours de la régénération, cellules qui requièrent aussi Six1 pour une réparation musculaire efficace. Nous avons étudié la régénération musculaire en absence d’expression d’Eya3 et n’avons pas observé de défaut nous menant à la conclusion qu’Eya3 n’est pas requis pour la régénération musculaire adulte, mais que sa perte d’expression était peut-être compensée par un autre gène Eya chez les animaux mutants. Pour conclure, Six1 et Six2 sont indispensables à la formation des muscles craniofaciaux, et Six1 et Six4 sont requis pour la myogenèse hypaxiale, et pour l’établissement d’un environnement propice à la maturation des myofibres fœtales et au nichage des cellules PAX7+ au cours de la myogenèse épaxiale, et permettant la croissance des myofibres et leur ré-innervation après blessure. La collaboration des protéines SIX avec leurs co-facteurs EYA au cours de la myogenèse nécessite d’autres études pour mieux définir leurs fonctions. / SIX homeoproteins are encoded by the Sine oculis homeobox related genes Six1 to Six6 in vertebrates among which Six1, Six2, Six4 and Six5 are expressed in the muscle lineage. Whereas Six1 and Six4 are required for hypaxial myogenesis, double KO for those two genes (s1s4KO) still form their epaxial and craniofacial muscles. We further characterized the phenotype of compound Six mutant embryos and showed that the absence of Six1 and Six2 completely impairs craniofacial myogenesis and worsen muscle limb development observed in single Six1 mutants. We also showed that mouse fetuses devoid of SIX1, SIX2, SIX4 and SIX5 activity are still able to develop epaxial muscles, but that Pax7 expression in myogenic progenitors of these mutants is reduced and intermingled with Myogenin expression. While s1s4KO fetuses still develop epaxial muscles, their PAX7+ cells show a perturbed homing process into their niche, between the plasma membrane of a myofibre and the basal lamina surrounding it. Transcriptomic analysis, transplantation experiments and in vitro studies allowed us to conclude that the homing of PAX7+ cells into their niche during fetal myogenesis requires an adequate environment combining properties of the myofibers and the PAX7+ cells; environment disturbed in s1s4KO epaxial muscles. Transplantation experiments also led us to conclude that Six1 and Six4 are required for proper myofiber re-inervation after injury and for the establishment of the fast phenotype of myofibers. Furthermore, muscles transplanted with s1s4KO fetal PAX7+ cells after injury are formed of numerous and tiny myofibers. We could link this phenotype to the behavior of s1s4KO cells in vitro where they showed perturbed fusion. Finally, SIX homeoproteins require co-factors to induce their target genes expression, as EYA proteins encoded by Eya1 to Eya4 in vertebrates. Eya3 and Eya4 are strongly expressed in satellite cells during regeneration, cells in which Six1 is also required for proper muscle repair. We investigated muscle regeneration in absence of Eya3 expression and observed no obvious phenotype. We concluded that Eya3 is not required for muscle regeneration but that other Eya genes might compensate its function in KO mouse. To conclude, Six1 and Six2 are required for craniofacial myogenesis and Six1 and Six4 for hypaxial myogenesis and for the establishment of a proper environment allowing myofibre maturation and PAX7+ cells homing during fetal epaxial myogenesis and enabling myofibre growth and re-innervation after injury. The role of the collaboration between SIX and EYA proteins during myogenesis still needs more investigation.

MLK3 est une ser/thr MAP3K qui active la voie de signalisation des MAPKs dans différents types cellulaires. GSK3β interagit et active MLK3 en la phosphorylant sur le residue ser 792. Cependant, le rôle de MLK3 ainsi que l’interaction entre MLK3 et GSK3β n’ont pas été précédemment étudiés dans le muscle squelettique. La croissance post-natale du muscle et la régénération musculaire chez l’adulte sont dépendantes de l’accrétion de myonoyaux, un processus médié par les cellules satellites qui prolifèrent, se différencient puis fusionnent aux fibres préexistantes. Durant ma thèse, j’ai démontré que GSK3β agit en amont de MLK3 pour induire la prolifération des cellules satellites, et cela par l’activation de la voie de signalisation MLK3-p38γ MAPK. In vivo, les muscles de souris déficientes injectés par la CTX montrent une diminution du nombre de cellules satellites prolifératrices Pax7+/ki67+, ainsi qu’une accélération du processus de régénération. En conclusion, mes résultats évoquent un nouveau rôle de MLK3 dans le muscle squelettique pouvant servir pour vaincre les dystrophies musculaire. / MLK3 is a Ser/Thr MAP3K, which activates MAPKs signalling pathways in different cell types. The Ser/Thr kinase GSK3-β directly phosphorylate Ser 792 residue and activate MLK3. Since neither the role of MLK3, nor GSK3-β -MLK3 interaction have been previously investigated in muscle, the aim of my thesis was to elucidate their contribution in the regulation of muscle mass and physiology.Skeletal muscle post-natal growth and adult regeneration relies on satellite cell-mediated myonuclear accretion, during which, activated satellite cells, proliferate, differentiate and fuse with preexisting myotubes.I have demonstrated that in skeletal muscle, GSK3-β acts upstream of MLK3 to induce satellite cells proliferation through the induction of MLK3-p38γ MAPK signalling. Similarly, in vivo CTX-induced TA damage in MLK3 KO mice resulted in decreased number of proliferating Pax7+/ki67+ satellite cells, with a rapid muscle regeneration ability.These data suggest provide a yet unknown role of MLK3 in skeletal muscle tissue that could help in curing age-related muscle dystrophies.

Pax3 protein is initially expressed in the dermomyotome of embryonic somites, which gives rise to skeletal muscle. Following myogenesis, Pax3 expression is mostly down-regulated and becomes restricted to a few satellite cells (SCs) of select mature muscles. SCs are activated to form new myonuclei during muscle hypertrophy, regeneration and repair. Intrafusal fibers of muscle spindles are thought to persist in a comparatively immature state as, unlike extrafusal fibers, they maintain small diameters, developmental myosins, Myf5 expression and high SC concentrations. This thesis tests the hypotheses that Pax3 expression is preferentially maintained in SCs of adult skeletal muscle spindles and can be augmented under conditions of SC activation. To study Pax3 through development, immunohistochemical techniques were used to identify SCs by their Pax7 expression, and analyze the proportion of SCs and myonuclei (MN) expressing Pax3 in chicken anterior latissimus dorsi (ALD) muscle excised at 9, 30, 62, and 145 days post-hatch. To induce SC activation, tenotomy was performed on the right ALD muscle of 138-day post-hatch chicks to induce compensatory hypertrophy of the ipsilateral synergistic posterior latissimus dorsi (PLD) muscle. The PLD was analyzed seven days after ALD tenotomy using similar immunohistochemical techniques. This is the first study to show Pax3 expressing SCs within intrafusal fibers of muscle spindles. This thesis demonstrates that throughout development there is a higher percentage of Pax3 expressing SCs within intrafusal fibers of muscle spindles than the surrounding extrafusal fibers that compose the bulk of the muscle. It is also revealed that the proportion of the SC population expressing Pax3 declines with age in both intrafusal and extrafusal fibers. Compensatory hypertrophy of the PLD resulted in a greater percentage of Pax3 expressing SCs in intrafusal and extrafusal fibers than under control conditions. The percentage of SCs expressing Pax3 after PLD overload was similar to that seen in young control muscle. The percentage of Pax3 expressing MN also increased after muscle overload to levels seen in young muscle. A disproportionate decrease in the proportion of SCs expressing Pax3 during development, and a disproportionate increase in the percentage of Pax3 positive SCs as a result of experimentally induced muscle hypertrophy, suggests that Pax3 expression in maturing muscle may be more than just a developmental vestige. Pax3 may be a factor in the activation and differentiation of SCs in maturing muscle.

Pax3 protein is initially expressed in the dermomyotome of embryonic somites, which gives rise to skeletal muscle. Following myogenesis, Pax3 expression is mostly down-regulated and becomes restricted to a few satellite cells (SCs) of select mature muscles. SCs are activated to form new myonuclei during muscle hypertrophy, regeneration and repair. Intrafusal fibers of muscle spindles are thought to persist in a comparatively immature state as, unlike extrafusal fibers, they maintain small diameters, developmental myosins, Myf5 expression and high SC concentrations. This thesis tests the hypotheses that Pax3 expression is preferentially maintained in SCs of adult skeletal muscle spindles and can be augmented under conditions of SC activation. To study Pax3 through development, immunohistochemical techniques were used to identify SCs by their Pax7 expression, and analyze the proportion of SCs and myonuclei (MN) expressing Pax3 in chicken anterior latissimus dorsi (ALD) muscle excised at 9, 30, 62, and 145 days post-hatch. To induce SC activation, tenotomy was performed on the right ALD muscle of 138-day post-hatch chicks to induce compensatory hypertrophy of the ipsilateral synergistic posterior latissimus dorsi (PLD) muscle. The PLD was analyzed seven days after ALD tenotomy using similar immunohistochemical techniques. This is the first study to show Pax3 expressing SCs within intrafusal fibers of muscle spindles. This thesis demonstrates that throughout development there is a higher percentage of Pax3 expressing SCs within intrafusal fibers of muscle spindles than the surrounding extrafusal fibers that compose the bulk of the muscle. It is also revealed that the proportion of the SC population expressing Pax3 declines with age in both intrafusal and extrafusal fibers. Compensatory hypertrophy of the PLD resulted in a greater percentage of Pax3 expressing SCs in intrafusal and extrafusal fibers than under control conditions. The percentage of SCs expressing Pax3 after PLD overload was similar to that seen in young control muscle. The percentage of Pax3 expressing MN also increased after muscle overload to levels seen in young muscle. A disproportionate decrease in the proportion of SCs expressing Pax3 during development, and a disproportionate increase in the percentage of Pax3 positive SCs as a result of experimentally induced muscle hypertrophy, suggests that Pax3 expression in maturing muscle may be more than just a developmental vestige. Pax3 may be a factor in the activation and differentiation of SCs in maturing muscle.

La myogenèse fœtale repose sur les cellules progénitrices musculaires PAX7+ qui assurent la croissance musculaire au cours du développement et qui sont à l’origine des cellules satellites. Nous avons cherché à interpréter les signaux régulant la myogenèse fœtale et leur lien avec le cycle cellulaire. Nous avons effectué une analyse exhaustive du cycle cellulaire des cellules myogéniques au cours de la myogenèse fœtale. Nous avons aussi identifié que les cellules PAX7+ progressant dans le cycle cellulaire (phases S, G2, et M) sont régionalisées aux extrémités des muscles. Les voies de signalisation BMP et NOTCH régulent positivement le nombre de cellules PAX7+ pendant le développement fœtal mais ont un effet différent sur la différenciation musculaire. Nous avons montré que les voies de signalisation BMP ou NOTCH augmentent le nombre de cellules PAX7+ de manière indépendante. Nous avons aussi identifié des interactions antagonistes entre ces deux voies lors de la différenciation musculaire. Nous avons testé l'importance de la contraction musculaire pendant la myogenèse fœtale chez l’embryon de poulet. Le blocage des contractions musculaires mime un phénotype de perte de fonction NOTCH, à savoir une diminution du nombre de cellules progénitrices musculaires avec une tendance à la différenciation musculaire. Nous avons aussi montré que les forces mécaniques produites par les contractions musculaires sont détectées par le co-activateur transcriptionnel YAP1 qui régule l'expression d’un ligand de NOTCH au sein des fibres musculaires, qui à son tour va maintenir le pool de cellules progénitrices musculaires fœtaux. / Foetal myogenesis relies on PAX7+ muscle progenitors that provide the source of cells for muscle growth during development and for the generation of the satellite cell pool. We aimed to decipher the signals that regulate the balance between myogenic differentiation and proliferation. We performed an exhaustive analysis of the cell cycle phases of myogenic cells during foetal myogenesis. I defined that PAX7+ cells in the S/G2/M phases were enriched at the contact points to the tendons. BMP and NOTCH signals increase the number of PAX7+ cells during foetal development, but affect differentiation in a positive and negative manner, respectively. I revealed that BMP and NOTCH increase the number of PAX7+ cells independently of each other. However, they act antagonistically during differentiation. Thus, the interplay between NOTCH and BMP signalling differs in proliferation and differentiation. Because muscle is a mechanical tissue, we tested the importance of muscle contraction for foetal myogenesis in chick embryos. I found that the block of muscle contraction during foetal myogenesis mimicked a NOTCH loss-of-function, i.e. decreased the number of foetal muscle progenitors and shifted the balance between proliferation and differentiation towards a differentiation fate. Mechanical forces provided by muscle contractions are sensed in myonuclei by the transcriptional co-activator YAP1 that regulates expression of the NOTCH ligand JAGGED2 in muscle fibres. This JAGGED2 signal keeps the muscle progenitors in an undifferentiated state and suppresses differentiation.

Mon travail de thèse a porté sur l'étude des mécanismes contrôlant la progression du cycle cellulaire et le devenir des cellules progénitrices dans différents tissus. Sortie du cycle cellulaire et différenciation cellulaire pendant la formation du muscle du membre Nous avons montré que la sortie du cycle cellulaire, régulée par les inhibiteurs de kinases cycline-dépendantes (CDKI) et la différentiation musculaire contrôlée par les facteurs myogéniques (MRF), peuvent être découplées génétiquement pendant la formation du muscle. Nous avons identifié une séquence régulant l'expression de CDKI, spécifique au muscle, activée par les MRF dans les myoblastes et réprimée par la voie Notch dans les progéniteurs, permettant de contrôler la balance entre amplification des progéniteurs et établissement du muscle squelettique. Contrôle de la croissance des dérivés de crête neurale craniale Bien que Pax3 et Pax7 soient essentiels pour la formation de la crête neurale, leurs rôles durant le développement craniofacial restent inconnus. À l'aide de mutants murins pour Pax3/7 présentant des fentes faciales, nous avons montré que ces défauts sont liés à la surexpression de la voie de signalisation régulée par le récepteur Aryl hydrocarbon (AhR, récepteur à la dioxine). L'augmentation de l'activité d'AhR pousse les cellules mésenchymateuses faciales hors du cycle cellulaire alors que son inhibition restaure la prolifération de ces cellules, permettant la fermeture de la face des mutants Pax3/7 et démontrant qu'une interaction entre une voie de signalisation impliquée dans la réponse au stress environnemental et les gènes régulés par Pax3/7 est nécessaire pendant le développement craniofacia. / This thesis aims to decipher how Pax3 and Pax7 transcription factors control cell cycle progression of progenitor cells in different tissues.Cell cycle regulation of Pax3+ myogenic progenitors during limb muscle developmentWe showed that cell cycle exit, mediated by the cyclin-dependent kinases inhibitors (CDKI), and muscle differentiation, controlled by the myogenic regulatory factors (MRF), can be genetically uncoupled during development. We dissected a functional interplay between Notch signalling and both MRF and CDKI activities, for maintaining the cycling status of the progenitor cells. Further, we identified a CDKI, muscle-specific DNA regulatory element, activated by the MRF in myoblasts but repressed by Notch signalling in progenitor cells, controlling the equilibrium between amplification of the progenitor pool and the establishment of functional muscle.Control of Pax3+ neural crest derivatives growth, and maintenance during craniofacial developmentAlthough studies showed Pax3 and Pax7 to be essential during early neural crest development, their role during craniofacial formation is unknown. Using Pax3/7 mutant mice displaying facial clefts, we uncovered that these defects are associated with an up-regulation of the Aryl hydrocarbon Receptor (AhR, the receptor to dioxin) signalling pathway. In Pax3/7 mutants, increased AhR activity drives facial mesenchymal cells out of the cell cycle, while inhibiting AhR rescues the cycling status of these cells and the facial closure of Pax3/7 mutants. Our results identify a molecular link between an environmental stress response pathway and a Pax3/7 downstream gene regulatory network during normal craniofacial development.

We studied patterning mechanisms and cell fates during limb regeneration in the axolotl. 1) It is crucial to understand the earliest events of patterning. Since it is technically challenging to study early events, we established single cell PCR. This new tool will allow us to obtain novel insight into the initial steps of limb patterning. 2)We have examined the roles of different tissues regarding their fates and features of proximo- distal patterning. Our strategy was to transplant GFP+ skin, skeleton, muscle and Schwann cells from transgenic donors to limbs of wild type hosts, amputate through the graft and analyze fluorescent progeny combined with the use of molecular markers. Our results revealed that different subpopulations of blastema cells exist regarding two aspects. First, we found that progeny of skin and skeleton have some tissue specific memory since they did not give rise to muscle lineages. However, cells of the skin contributed to other mesenchymal tissues like cartilage or tendons, while the majority of skeleton- derived cells undergoes self- renewal. Second, we performed one cellular and two molecular assays to investigate what tissues generate cells that exhibit features of proximo- distal patterning. Both assays revealed that Schwann cell- derived progeny do not display such features while progeny of skin, skeleton and muscle did. Therefore, we conclude that the blastema is a heterogeneous mix of cells regarding tissue lineages and features of proximo- distal patterning.