RESUMEN
Notch signaling is essential to maintain skeletal muscle stem cells in quiescence. However, the precise roles of different Notch receptors are incompletely defined. Here, we demonstrate a role for Notch3 (N3) in the self-renewal of muscle stem cells. We found that N3 is active in quiescent C2C12 reserve cells (RCs), and N3 over-expression and knockdown studies in C2C12 and primary satellite cells reveal a role in self-renewal. The Notch ligand Delta-like 4 (Dll4) is expressed by newly formed myotubes and interaction with this ligand is sufficient to maintain N3 activity in quiescent C2C12 RCs to prevent activation and progression into the cell cycle. Thus, our data suggest a model whereby during regeneration, expression of Dll4 by nascent muscle fibers triggers N3 signaling in associated muscle stem cells to recruit them to quiescence, thereby renewing the stem cell pool. Stem Cells 2018;36:458-466.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Mioblastos Esqueléticos/metabolismo , Receptor Notch3/metabolismo , Animales , Ciclo Celular/genética , Ciclo Celular/fisiología , Línea Celular , Células Cultivadas , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Ratones , Fibras Musculares Esqueléticas/metabolismo , Mioblastos Esqueléticos/citología , Receptor Notch3/genética , Células Satélite del Músculo Esquelético/citología , Células Satélite del Músculo Esquelético/metabolismoRESUMEN
When unperturbed, somatic stem cells are poised to affect immediate tissue restoration upon trauma. Yet, little is known regarding the mechanistic basis controlling initial and homeostatic 'scaling' of stem cell pool sizes relative to their target tissues for effective regeneration. Here, we show that TEAD1-expressing skeletal muscle of transgenic mice features a dramatic hyperplasia of muscle stem cells (i.e. satellite cells, SCs) but surprisingly without affecting muscle tissue size. Super-numeral SCs attain a 'normal' quiescent state, accelerate regeneration, and maintain regenerative capacity over several injury-induced regeneration bouts. In dystrophic muscle, the TEAD1 transgene also ameliorated the pathology. We further demonstrate that hyperplastic SCs accumulate non-cell-autonomously via signal(s) from the TEAD1-expressing myofiber, suggesting that myofiber-specific TEAD1 overexpression activates a physiological signaling pathway(s) that determines initial and homeostatic SC pool size. We propose that TEAD1 and its downstream effectors are medically relevant targets for enhancing muscle regeneration and ameliorating muscle pathology.
Asunto(s)
Proliferación Celular , Proteínas de Unión al ADN/genética , Distrofina/deficiencia , Expresión Génica , Proteínas Nucleares/genética , Células Satélite del Músculo Esquelético/fisiología , Factores de Transcripción/genética , Animales , Ratones Endogámicos C57BL , Ratones Transgénicos , Factores de Transcripción de Dominio TEARESUMEN
BACKGROUND: Preclinical testing of potential therapies for Duchenne muscular dystrophy (DMD) is conducted predominantly of the mdx mouse. But lack of a detailed quantitative description of the pathology of this animal limits our ability to evaluate the effectiveness of putative therapies or their relevance to DMD. METHODS: Accordingly, we have measured the main cellular components of muscle growth and regeneration over the period of postnatal growth and early pathology in mdx and wild-type (WT) mice; phalloidin binding is used as a measure of fibre size, myonuclear counts and BrdU labelling as records of myogenic activity. RESULTS: We confirm a two-phase postnatal growth pattern in WT muscle: first, increase in myonuclear number over weeks 1 to 3, then expansion of myonuclear domain. Mdx muscle growth lags behind that of WT prior to overt signs of pathology. Fibres are smaller, with fewer myonuclei and smaller myonuclear domains. Moreover, satellite cells are more readily detached from mdx than WT muscle fibres. At 3 weeks, mdx muscles enter a phase of florid myonecrosis, accompanied by concurrent regeneration of an intensity that results in complete replacement of pre-existing muscle over the succeeding 3 to 4 weeks. Both WT and mdx muscles attain maximum size by 12 to 14 weeks, mdx muscle fibres being up to 50% larger than those of WT as they become increasingly branched. Mdx muscle fibres also become hypernucleated, containing twice as many myonuclei per sarcoplasmic volume, as those of WT, the excess corresponding to the number of centrally placed myonuclei. CONCLUSIONS: The best-known consequence of lack of dystrophin that is common to DMD and the mdx mouse is the conspicuous necrosis and regeneration of muscle fibres. We present protocols for measuring this in terms both of loss of muscle nuclei previously labelled with BrdU and of the intensity of myonuclear labelling with BrdU administered during the regeneration period. Both measurements can be used to assess the efficacy of putative antinecrotic agents. We also show that lack of dystrophin is associated with a number of previously unsuspected abnormalities of muscle fibre structure and function that do not appear to be directly associated with myonecrosis.
RESUMEN
We report the generation of five mouse strains with the tamoxifen-inducible Cre (Cre-ER(T) (2) ; CE) gene cassette knocked into the endogenous loci of Pax3, Myod1, Myog, Myf6, and Myl1, collectively as a resource for the skeletal muscle research community. We characterized these CE strains using the Cre reporter mice, R26R(L) (acZ) , during embryogenesis and show that they direct tightly controlled tamoxifen-inducible reporter expression within the expected cell lineage determined by each myogenic gene. We also examined a few selected adult skeletal muscle groups for tamoxifen-inducible reporter expression. None of these new CE alleles direct reporter expression in the cardiac muscle. All these alleles follow the same knock-in strategy by replacing the first exon of each gene with the CE cassette, rendering them null alleles of the endogenous gene. Advantages and disadvantages of this design are discussed. Although we describe potential immediate use of these strains, their utility likely extends beyond foreseeable questions in skeletal muscle biology.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Desarrollo de Músculos/genética , Músculo Esquelético/citología , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Tamoxifeno/farmacología , Alelos , Animales , Linaje de la Célula , Técnicas de Sustitución del Gen , Ratones , Músculo Esquelético/crecimiento & desarrolloRESUMEN
Striated muscles that enable mouth opening and swallowing during feeding are essential for efficient energy acquisition, and are likely to have played a fundamental role in the success of early jawed vertebrates. The developmental origins and genetic requirements of these muscles are uncertain. Here, we determine by indelible lineage tracing in mouse that fibres of sternohyoid muscle (SHM), which is essential for mouth opening during feeding, and oesophageal striated muscle (OSM), which is crucial for voluntary swallowing, arise from Pax3-expressing somite cells. In vivo Kaede lineage tracing in zebrafish reveals the migratory route of cells from the anteriormost somites to OSM and SHM destinations. Expression of pax3b, a zebrafish duplicate of Pax3, is restricted to the hypaxial region of anterior somites that generate migratory muscle precursors (MMPs), suggesting that Pax3b plays a role in generating OSM and SHM. Indeed, loss of pax3b function led to defective MMP migration and OSM formation, disorganised SHM differentiation, and inefficient ingestion and swallowing of microspheres. Together, our data demonstrate Pax3-expressing somite cells as a source of OSM and SHM fibres, and highlight a conserved role of Pax3 genes in the genesis of these feeding muscles of vertebrates.
Asunto(s)
Esófago/embriología , Maxilares/embriología , Desarrollo de Músculos , Músculo Estriado/embriología , Factores de Transcripción Paired Box/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Movimiento Celular , Deglución , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Esófago/citología , Feto/citología , Feto/metabolismo , Maxilares/citología , Ratones , Músculo Estriado/citología , Músculo Estriado/metabolismo , Proteína MioD/metabolismo , Factor 5 Regulador Miogénico/metabolismo , Factor de Transcripción PAX3 , Factores de Transcripción Paired Box/genética , Somitos/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
Recently in Developmental Cell, Bröhl et al. (2012) reported that Notch regulates muscle stem cell homing to its niche. Notch is required when myogenic cells cease producing new fibers and become sequestered between a newly forming basement membrane and the muscle fiber surface: the position that defines them as satellite cells.