RESUMEN
It is crucial to develop a long-term therapy that targets hemophilia A and B, including inhibitor-positive patients. We have developed an Adeno-associated virus (AAV) based strategy to integrate the bypass coagulation factor, activated FVII (murine, mFVIIa) gene into the Rosa26 locus using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 mediated gene-editing. AAV vectors designed for expression of guide RNA (AAV8-gRNA), Cas9 (AAV2 neddylation mutant-Cas9), and mFVIIa (AAV8-mFVIIa) flanked by homology arms of the target locus were validated in vitro. Hemophilia B mice were administered with AAV carrying gRNA, Cas9 (1 × 1011 vgs/mouse), and mFVIIa with homology arms (2 × 1011 vgs/mouse) with appropriate controls. Functional rescue was documented with suitable coagulation assays at various time points. The data from the T7 endonuclease assay revealed a cleavage efficiency of 20-42 %. Further, DNA sequencing confirmed the targeted integration of mFVIIa into the safe-harbor Rosa26 locus. The prothrombin time (PT) assay revealed a significant reduction in PT in mice that received the gene-editing vectors (22 %), and a 13 % decline in mice that received only the AAV-FVIIa when compared to mock treated mice, 8 weeks after vector administration. Furthermore, FVIIa activity in mice that received triple gene-editing vectors was higher (122.5mIU/mL vs 28.8mIU/mL) than the mock group up to 15 weeks post vector administration. A hemostatic challenge by tail clip assay revealed that hemophilia B mice injected with only FVIIa or the gene-editing vectors had significant reduction in blood loss. In conclusion, AAV based gene-editing facilitates sustained expression of coagulation FVIIa and phenotypic rescue in hemophilia B mice.
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Dependovirus , Modelos Animales de Enfermedad , Hemofilia B , Animales , Hemofilia B/terapia , Hemofilia B/genética , Dependovirus/genética , Ratones , Fenotipo , Edición Génica/métodos , Hemorragia/genética , Hemorragia/terapia , Factor VIIa , Humanos , Terapia Genética/métodos , Ratones Endogámicos C57BL , Vectores Genéticos , Sistemas CRISPR-Cas , Ingeniería Genética/métodosRESUMEN
Four SIX homeoproteins display a combinatorial expression throughout embryonic developmental myogenesis and they modulate the expression of the myogenic regulatory factors. Here, we provide a deep characterization of their role in distinct mouse developmental territories. We showed, at the hypaxial level, that the Six1:Six4 double knockout (dKO) somitic precursor cells adopt a smooth muscle fate and lose their myogenic identity. At the epaxial level, we demonstrated by the analysis of Six quadruple KO (qKO) embryos, that SIX are required for fetal myogenesis, and for the maintenance of PAX7+ progenitor cells, which differentiated prematurely and are lost by the end of fetal development in qKO embryos. Finally, we showed that Six1 and Six2 are required to establish craniofacial myogenesis by controlling the expression of Myf5. We have thus described an unknown role for SIX proteins in the control of myogenesis at different embryonic levels and refined their involvement in the genetic cascades operating at the head level and in the genesis of myogenic stem cells.
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Proteínas de Homeodominio , Somitos , Ratones , Animales , Proteínas de Homeodominio/metabolismo , Diferenciación Celular/genética , Somitos/metabolismo , Desarrollo de Músculos/genética , Regulación del Desarrollo de la Expresión Génica , Músculo Esquelético/metabolismoRESUMEN
BACKGROUND: Vertebrate cranial neural crest cells (CNCCs) are multipotent, proximal to the source CNCC form the cranial ganglia. Distally, in the pharyngeal arches, they give rise to the craniofacial skeleton and connective tissues. Fate choices are made as CNCC pattern into distinct destination compartments. In spite of this importance, the mechanism patterning CNCC is poorly defined. RESULTS: Here, we report that a novel ß-catenin-dependent regulation of N-Cadherin levels may drive CNCC patterning. In mouse embryos, at the first pharyngeal arch axial level, membrane ß-catenin levels correlate with the extent of N-cadherin-mediated adhesion and thus suggest the presence of collective and dispersed states of CNCC. Using in vitro human neural crest model and chemical modulators of ß-catenin levels, we show a requirement for down-modulating ß-catenin for regulating N-cadherin levels and cell-cell adhesion. Similarly, in ß-catenin gain-of-function mutant mouse embryos, CNCC fail to lower N-cadherin levels. This indicates a failure to reduce cell-cell adhesion, which may underlie the failure of mutant CNCC to populate first pharyngeal arch. CONCLUSION: We suggest that ß-catenin-mediated regulation of CNCC adhesion, a previously underappreciated mechanism, underlies the patterning of CNCC into fate-specific compartments.
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Tipificación del Cuerpo , Cresta Neural/embriología , Faringe/embriología , Cráneo/embriología , beta Catenina/metabolismo , Animales , Ratones , Ratones Transgénicos , Cresta Neural/citología , Faringe/citología , Cráneo/citología , beta Catenina/genéticaRESUMEN
During early development the vertebrate embryo elongates through a combination of tissue shape change, growth and progenitor cell expansion across multiple regions of the body axis. How these events are coordinated across the length of the embryo to generate a well-proportioned body axis is unknown. Understanding the multi-tissue interplay of morphogenesis, growth and cell fate specification is essential for us to gain a complete understanding how diverse body plans have evolved in a robust manner. Within the posterior region of the embryo, a population of bipotent neuromesodermal progenitors generate both spinal cord and paraxial mesoderm derivatives during the elongation of the vertebrate body. Here we summarize recent data comparing neuromesodermal lineage and their underlying gene-regulatory networks between species and through development. We find that the common characteristic underlying this population is a competence to generate posterior neural and paraxial mesoderm cells, with a conserved Wnt/FGF and Sox2/T/Tbx6 regulatory network. We propose the hypothesis that by maintaining a population of multi-germ layer competent progenitors at the posterior aspect of the embryo, a flexible pool of progenitors is maintained whose contribution to the elongating body axis varies as a consequence of the relative growth rates occurring within anterior and posterior regions of the body axis. We discuss how this capacity for variation in the proportions and rates of NM specification might have been important allowing for alterations in the timing of embryo growth during evolution.
RESUMEN
Vertebrate cranial mesoderm is a discrete developmental unit compared to the mesoderm below the developing neck. An extraordinary feature of the cranial mesoderm is that it includes a common progenitor pool contributing to the chambered heart and the craniofacial skeletal muscles. This striking developmental potential and the excitement it generated led to advances in our understanding of cranial mesoderm developmental mechanism. Remarkably, recent findings have begun to unravel the origin of its distinct developmental characteristics. Here, we take a detailed view of the ontogenetic trajectory of cranial mesoderm and its regulatory network. Based on the emerging evidence, we propose that cranial and posterior mesoderm diverge at the earliest step of the process that patterns the mesoderm germ layer along the anterior-posterior body axis. Further, we discuss the latest evidence and their impact on our current understanding of the evolutionary origin of cranial mesoderm. Overall, the review highlights the findings from contemporary research, which lays the foundation to probe the molecular basis of unique developmental potential and evolutionary origin of cranial mesoderm.
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Evolución Biológica , Mesodermo/crecimiento & desarrollo , Desarrollo de Músculos/genética , Cráneo/crecimiento & desarrollo , Animales , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Mesodermo/embriología , Músculo Esquelético/embriología , Músculo Esquelético/crecimiento & desarrollo , Cresta Neural/crecimiento & desarrollo , Cresta Neural/metabolismo , Cráneo/metabolismo , Vertebrados/embriología , Vertebrados/genéticaRESUMEN
Human pluripotent stem cells (hPSCs) acquire changes at the genomic level upon proliferation and differentiation (Peterson and Loring, 2014). Studies from International Stem Cell Initiative and independent laboratories identified a copy number variant (CNV) in hES cell lines displaying a normal karyotype, which provided a selective advantage to hES cells in culture. In our laboratory we have identified variant H9-hESC (derived from H9-hESC) with normal karyotype, pluripotency expression, differentiation profile but with altered traits of high cell survival and low E-CADHERIN expression.
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Investigación Biomédica/estadística & datos numéricos , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Células Madre Embrionarias/citología , Células Madre Pluripotentes/citología , Teratoma/patología , Animales , Células Cultivadas , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCIDRESUMEN
BACKGROUND: Recombinant adeno-associated viruses (AAVs) are emerging as favoured transgene delivery vectors for both research applications and gene therapy. In this context, a thorough investigation of the potential of various AAV serotypes to transduce specific cell types is valuable. Here, we rigorously tested the infectivity of a number of AAV serotypes in murine testis by direct testicular injection. RESULTS: We report the tropism of serotypes AAV2, 5, 8, 9 and AAVrh10 in mouse testis. We reveal unique infectivity of AAV2 and AAV9, which preferentially target intertubular testosterone-producing Leydig cells. Remarkably, AAV2 TM, a mutant for capsid designed to increase transduction, displayed a dramatic alteration in tropism; it infiltrated seminiferous tubules unlike wildtype AAV2 and transduced Sertoli cells. However, none of the AAVs tested infected spermatogonial cells. CONCLUSIONS: In spite of direct testicular injection, none of the tested AAVs appeared to infect sperm progenitors as assayed by reporter expression. This lends support to the current view that AAVs are safe gene-therapy vehicles. However, testing the presence of rAAV genomic DNA in germ cells is necessary to assess the risk of individual serotypes.
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Dependovirus/fisiología , Terapia Genética/instrumentación , Vectores Genéticos/fisiología , Testículo/virología , Animales , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Dependovirus/clasificación , Dependovirus/genética , Terapia Genética/métodos , Vectores Genéticos/genética , Células Intersticiales del Testículo/virología , Masculino , Ratones , Serogrupo , Tropismo ViralRESUMEN
Head and trunk muscles have discrete embryological origins and are governed by distinct regulatory programmes. Whereas the developmental route of trunk muscles from mesoderm is well studied, that of head muscles is ill defined. Here, we show that, unlike the myogenic trunk paraxial mesoderm, head mesoderm development is independent of the T/Tbx6 network in mouse. We reveal that, in contrast to Wnt and FGF-driven trunk mesoderm, dual inhibition of Wnt/ß-catenin and Nodal specifies head mesoderm. Remarkably, the progenitors derived from embryonic stem cells by dual inhibition efficiently differentiate into cardiac and skeletal muscle cells. This twin potential is the defining feature of cardiopharyngeal mesoderm: the head subtype giving rise to heart and branchiomeric head muscles. Therefore, our findings provide compelling evidence that dual inhibition specifies head mesoderm and unravel the mechanism that diversifies head and trunk muscle programmes during early mesoderm fate commitment. Significantly, this is the first report of directed differentiation of pluripotent stem cells, without transgenes, into progenitors with muscle/heart dual potential. Ability to generate branchiomeric muscle in vitro could catalyse efforts in modelling myopathies that selectively involve head muscles.
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Cabeza/embriología , Mesodermo/embriología , Músculo Esquelético/citología , Músculo Esquelético/embriología , Células Madre Pluripotentes/citología , Animales , Diferenciación Celular/genética , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Proteína Nodal/metabolismo , Proteínas de Dominio T Box , Factores de Transcripción/genética , Proteínas Wnt/antagonistas & inhibidores , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMEN
After publication of this article [1], the authors noted that the legends for supplementary files Figures S3 and S4 were truncated in the production process, therefore lacking some information concerning these Figures. The complete legends are included in this Correction. The authors apologize for any inconvenience that this might have caused.
RESUMEN
BACKGROUND: Skeletal muscle satellite (stem) cells are quiescent in adult mice and can undergo multiple rounds of proliferation and self-renewal following muscle injury. Several labs have profiled transcripts of myogenic cells during the developmental and adult myogenesis with the aim of identifying quiescent markers. Here, we focused on the quiescent cell state and generated new transcriptome profiles that include subfractionations of adult satellite cell populations, and an artificially induced prenatal quiescent state, to identify core signatures for quiescent and proliferating. METHODS: Comparison of available data offered challenges related to the inherent diversity of datasets and biological conditions. We developed a standardized workflow to homogenize the normalization, filtering, and quality control steps for the analysis of gene expression profiles allowing the identification up- and down-regulated genes and the subsequent gene set enrichment analysis. To share the analytical pipeline of this work, we developed Sherpa, an interactive Shiny server that allows multi-scale comparisons for extraction of desired gene sets from the analyzed datasets. This tool is adaptable to cell populations in other contexts and tissues. RESULTS: A multi-scale analysis comprising eight datasets of quiescent satellite cells had 207 and 542 genes commonly up- and down-regulated, respectively. Shared up-regulated gene sets include an over-representation of the TNFα pathway via NFKß signaling, Il6-Jak-Stat3 signaling, and the apical surface processes, while shared down-regulated gene sets exhibited an over-representation of Myc and E2F targets and genes associated to the G2M checkpoint and oxidative phosphorylation. However, virtually all datasets contained genes that are associated with activation or cell cycle entry, such as the immediate early stress response genes Fos and Jun. An empirical examination of fixed and isolated satellite cells showed that these and other genes were absent in vivo, but activated during procedural isolation of cells. CONCLUSIONS: Through the systematic comparison and individual analysis of diverse transcriptomic profiles, we identified genes that were consistently differentially expressed among the different datasets and shared underlying biological processes key to the quiescent cell state. Our findings provide impetus to define and distinguish transcripts associated with true in vivo quiescence from those that are first responding genes due to disruption of the stem cell niche.
Asunto(s)
Diferenciación Celular , Células Satélite del Músculo Esquelético/metabolismo , Transcriptoma , Animales , Bases de Datos Factuales , Femenino , Perfilación de la Expresión Génica , Masculino , RatonesRESUMEN
Elongation of the body axis is a key aspect of body plan development. Bipotential neuromesoderm progenitors (NMPs) ensure axial growth of embryos by contributing both to the spinal cord and mesoderm. The current model for the mechanism controlling NMP deployment invokes Tbx6, a T-box factor, to drive mesoderm differentiation of NMPs. Here, we identify a new population of Tbx6+ cells in a subdomain of the NMP niche in mouse embryos. Based on co-expression of a progenitor marker, Sox2, we identify this population as representing a transient cell state in the mesoderm-fated NMP lineage. Genetic lineage tracing confirms the presence of the Tbx6+ NMP cell state. Furthermore, we report a novel aspect of the documented Tbx6 mutant phenotype, namely an increase from two to four ectopic neural tubes, corresponding to the switch in NMP niche, thus highlighting the importance of Tbx6 function in NMP fate decision. This study emphasizes the function of Tbx6 as a bistable switch that turns mesoderm fate 'on' and progenitor state 'off', and thus has implications for the molecular mechanism driving NMP fate choice.
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Células Madre Embrionarias/citología , Mesodermo/citología , Tubo Neural/embriología , Factores de Transcripción SOXB1/biosíntesis , Médula Espinal/embriología , Factores de Transcripción/biosíntesis , Animales , Tipificación del Cuerpo/genética , Tipificación del Cuerpo/fisiología , Diferenciación Celular , Linaje de la Célula , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Transgénicos , Tubo Neural/citología , Factores de Transcripción SOXB1/genética , Proteínas de Dominio T Box , Factores de Transcripción/genéticaRESUMEN
The esophagus links the oral cavity to the stomach and facilitates the transfer of bolus. Using genetic tracing and mouse mutants, we demonstrate that esophagus striated muscles (ESMs) are not derived from somites but are of cranial origin. Tbx1 and Isl1 act as key regulators of ESMs, which we now identify as a third derivative of cardiopharyngeal mesoderm that contributes to second heart field derivatives and head muscles. Isl1-derived ESM progenitors colonize the mouse esophagus in an anterior-posterior direction but are absent in the developing chick esophagus, thus providing evolutionary insight into the lack of ESMs in avians. Strikingly, different from other myogenic regions, in which embryonic myogenesis establishes a scaffold for fetal fiber formation, ESMs are established directly by fetal myofibers. We propose that ESM progenitors use smooth muscle as a scaffold, thereby bypassing the embryonic program. These findings have important implications in understanding esophageal dysfunctions, including dysphagia, and congenital disorders, such as DiGeorge syndrome.
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Embrión de Mamíferos/citología , Esófago/embriología , Regulación del Desarrollo de la Expresión Génica , Mesodermo/embriología , Desarrollo de Músculos/fisiología , Músculo Estriado/embriología , Cráneo/embriología , Animales , Western Blotting , Diferenciación Celular , Células Cultivadas , Pollos , Embrión de Mamíferos/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Corazón/embriología , Técnicas para Inmunoenzimas , Proteínas con Homeodominio LIM/fisiología , Masculino , Ratones , Ratones Noqueados , Cresta Neural/citología , Factor de Transcripción PAX3 , Factores de Transcripción Paired Box/fisiología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Somitos/citología , Proteínas de Dominio T Box/fisiología , Factores de Transcripción/fisiologíaRESUMEN
Skeletal muscles in vertebrates have a phenomenal regenerative capacity. A muscle that has been crushed can regenerate fully both structurally and functionally within a month. Remarkably, efficient regeneration continues to occur following repeated injuries. Thousands of muscle precursor cells are needed to accomplish regeneration following acute injury. The differentiated muscle cells, the multinucleated contractile myofibers, are terminally withdrawn from mitosis. The source of the regenerative precursors is the skeletal muscle stem cells-the mononucleated cells closely associated with myofibers, which are known as satellite cells. Satellite cells are mitotically quiescent or slow-cycling, committed to myogenesis, but undifferentiated. Disruption of the niche after muscle damage results in their exit from quiescence and progression towards commitment. They eventually arrest proliferation, differentiate, and fuse to damaged myofibers or make de novo myofibers. Satellite cells are one of the well-studied adult tissue-specific stem cells and have served as an excellent model for investigating adult stem cells. They have also emerged as an important standard in the field of ageing and stem cells. Several recent reviews have highlighted the importance of these cells as a model to understand stem cell biology. This chapter begins with the discovery of satellite cells as skeletal muscle stem cells and their developmental origin. We discuss transcription factors and signalling cues governing stem cell function of satellite cells and heterogeneity in the satellite cell pool. Apart from satellite cells, a number of other stem cells have been shown to make muscle and are being considered as candidate stem cells for amelioration of muscle degenerative diseases. We discuss these "offbeat" muscle stem cells and their status as adult skeletal muscle stem cells vis-a-vis satellite cells. The ageing context is highlighted in the concluding section.
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Desarrollo de Músculos/genética , Músculo Esquelético/crecimiento & desarrollo , Células Satélite del Músculo Esquelético , Vertebrados/crecimiento & desarrollo , Envejecimiento/genética , Animales , Diferenciación Celular/genética , Linaje de la Célula , Humanos , Músculo Esquelético/lesiones , Regeneración , Células Madre/citología , Vertebrados/genéticaRESUMEN
The myogenic regulatory genes Myf5, Mrf4, Myod, and Myogenin likely arose by gene duplications during evolution, presumably to address the more demanding requirements of the vertebrate body plan. Two cell lineages were proposed to be regulated independently by Myf5 and Myod to safeguard against tissue failure. Here we report severe muscle loss following ablation of Myf5-expressing cells. Using both lineage-specific and ubiquitous reporter alleles, we show that the remaining muscles in Myf5(Cre)-DTA embryos arise mainly from Myf5(+) escaper cells. Elimination of Myf5(Cre)-DTA cells on a Myod null background did not result in the total absence of skeletal muscles, as would be expected if a Myod(+)/Myf5-independent cell population played a major role in this scenario. Therefore, these observations are incompatible with a previously proposed functional two-lineage model. These findings will have an impact on the interpretation of phenotypes obtained using similar strategies in other tissues.
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Linaje de la Célula , Proteínas de Unión al ADN/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/citología , Factor 5 Regulador Miogénico/metabolismo , Animales , Diferenciación Celular/genética , Ratones , Ratones Transgénicos , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Proteína MioD/metabolismo , Factores Reguladores Miogénicos/metabolismo , Miogenina/metabolismoRESUMEN
Skeletal muscle satellite cells play a critical role during muscle growth, homoeostasis and regeneration. Selective induction of the muscle determination genes Myf5, Myod and Mrf4 during prenatal development can potentially impact on the reported functional heterogeneity of adult satellite cells. Accordingly, expression of Myf5 was reported to diminish the self-renewal potential of the majority of satellite cells. In contrast, virtually all adult satellite cells showed antecedence of Myod activity. Here we examine the priming of myogenic cells by Mrf4 throughout development. Using a Cre-lox based genetic strategy and novel highly sensitive Pax7 reporter alleles compared to the ubiquitous Rosa26-based reporters, we show that all adult satellite cells, independently of their anatomical location or embryonic origin, have been primed for Mrf4 expression. Given that Mrf4Cre and Mrf4nlacZ are active exclusively in progenitors during embryogenesis, whereas later expression is restricted to differentiated myogenic cells, our findings suggest that adult satellite cells emerge from embryonic founder cells in which the Mrf4 locus was activated. Therefore, this level of myogenic priming by induction of Mrf4, does not compromise the potential of the founder cells to assume an upstream muscle stem cell state. We propose that embryonic myogenic cells and the majority of adult muscle stem cells form a lineage continuum.
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Regulación del Desarrollo de la Expresión Génica , Factores Reguladores Miogénicos/metabolismo , Células Satélite del Músculo Esquelético/citología , Células Madre/citología , Alelos , Animales , Linaje de la Célula , Genes Reporteros , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Desarrollo de Músculos , Factor 5 Regulador Miogénico/metabolismo , Factores Reguladores Miogénicos/genética , Factor de Transcripción PAX7/metabolismo , Células Satélite del Músculo Esquelético/metabolismoRESUMEN
During organogenesis, a continuum of founder stem cells produces temporally distinct progeny until development is complete. Similarly, in skeletal myogenesis, phenotypically and functionally distinct myoblasts and differentiated cells are generated during development. How this occurs in muscle and other tissues in vertebrates remains largely unclear. We showed previously that committed cells are required for maintaining muscle stem cells. Here we show that active Notch signalling specifies a subpopulation of myogenic cells with high Pax7 expression. By genetically modulating Notch activity, we demonstrate that activated Notch (NICD) blocks terminal differentiation in an Rbpj-dependent manner that is sufficient to sustain stem/progenitor cells throughout embryogenesis, despite the absence of committed progeny. Although arrested in lineage progression, NICD-expressing cells of embryonic origin progressively mature and adopt characteristics of foetal myogenic cells, including expression of the foetal myogenesis regulator Nfix. siRNA-mediated silencing of NICD promotes the temporally appropriate foetal myogenic fate in spite of expression of markers for multiple cell types. We uncover a differential effect of Notch, whereby high Notch activity is associated with stem/progenitor cell expansion in the mouse embryo, yet it promotes reversible cell cycle exit in the foetus and the appearance of an adult muscle stem cell state. We propose that active Notch signalling is sufficient to sustain an upstream population of muscle founder stem cells while suppressing differentiation. Significantly, Notch does not override other signals that promote temporal myogenic cell fates during ontology where spatiotemporal developmental cues produce distinct phenotypic classes of myoblasts.
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Diferenciación Celular/genética , Músculo Esquelético/embriología , Mioblastos Esqueléticos/fisiología , Receptor Notch1/fisiología , Animales , División Celular/genética , Células Cultivadas , Replicación del ADN/genética , Embrión de Mamíferos , Desarrollo Embrionario/genética , Desarrollo Embrionario/fisiología , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/fisiología , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Transgénicos , Músculo Esquelético/metabolismo , Mioblastos Esqueléticos/metabolismo , Especificidad de Órganos/genética , Receptor Notch1/genética , Receptor Notch1/metabolismo , Factores de TiempoRESUMEN
Skeletal muscle stem cell fate in adult mice is regulated by crucial transcription factors, including the determination genes Myf5 and Myod. The precise role of Myf5 in regulating quiescent muscle stem cells has remained elusive. Here we show that most, but not all, quiescent satellite cells express Myf5 protein, but at varying levels, and that resident Myf5 heterozygous muscle stem cells are more primed for myogenic commitment compared with wild-type satellite cells. Paradoxically however, heterotypic transplantation of Myf5 heterozygous cells into regenerating muscles results in higher self-renewal capacity compared with wild-type stem cells, whereas myofibre regenerative capacity is not altered. By contrast, Pax7 haploinsufficiency does not show major modifications by transcriptome analysis. These observations provide a mechanism linking Myf5 levels to muscle stem cell heterogeneity and fate by exposing two distinct and opposing phenotypes associated with Myf5 haploinsufficiency. These findings have important implications for how stem cell fates can be modulated by crucial transcription factors while generating a pool of responsive heterogeneous cells.
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Células Madre Adultas/citología , Células Madre Adultas/metabolismo , Haploinsuficiencia/genética , Músculo Esquelético/metabolismo , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/metabolismo , Factor 5 Regulador Miogénico/genética , Animales , Linaje de la Célula , Ratones , Músculo Esquelético/citología , Factor 5 Regulador Miogénico/deficiencia , Factor 5 Regulador Miogénico/metabolismo , Factor de Transcripción PAX7/genética , Factor de Transcripción PAX7/metabolismo , FenotipoRESUMEN
Notch signaling plays a key role in virtually all tissues and organs in metazoans; however, limited examples are available for the regulatory role of this pathway in adult quiescent stem cells. We performed a temporal and ontological assessment of effectors of the Notch pathway that indicated highest activity in freshly isolated satellite cells and, unexpectedly, a sharp decline before the first mitosis, and subsequently in proliferating, satellite cell-derived myoblasts. Using genetic tools to conditionally abrogate canonical Notch signaling during homeostasis, we demonstrate that satellite cells differentiate spontaneously and contribute to myofibers, thereby resulting in a severe depletion of the stem cell pool. Furthermore, whereas loss of Rbpj function provokes some satellite cells to proliferate before fusing, strikingly, the majority of mutant cells terminally differentiate unusually from the quiescent state, without passing through S-phase. This study establishes Notch signaling pathway as the first regulator of cellular quiescence in adult muscle stem cells.
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Puntos de Control del Ciclo Celular , Músculo Esquelético/citología , Receptores Notch/metabolismo , Transducción de Señal , Células Madre/fisiología , Animales , Diferenciación Celular , Linaje de la Célula , Perfilación de la Expresión Génica , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Ratones , Ratones Noqueados , Desarrollo de Músculos , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Regeneración , Fase S , Células Satélite del Músculo Esquelético/metabolismo , Células Madre/metabolismoRESUMEN
Distinct cell populations with regenerative capacity have been reported to contribute to myofibres after skeletal muscle injury, including non-satellite cells as well as myogenic satellite cells. However, the relative contribution of these distinct cell types to skeletal muscle repair and homeostasis and the identity of adult muscle stem cells remain unknown. We generated a model for the conditional depletion of satellite cells by expressing a human diphtheria toxin receptor under control of the murine Pax7 locus. Intramuscular injection of diphtheria toxin during muscle homeostasis, or combined with muscle injury caused by myotoxins or exercise, led to a marked loss of muscle tissue and failure to regenerate skeletal muscle. Moreover, the muscle tissue became infiltrated by inflammatory cells and adipocytes. This localised loss of satellite cells was not compensated for endogenously by other cell types, but muscle regeneration was rescued after transplantation of adult Pax7(+) satellite cells alone. These findings indicate that other cell types with regenerative potential depend on the presence of the satellite cell population, and these observations have important implications for myopathic conditions and stem cell-based therapeutic approaches.