RESUMEN
Because post-mortem human skeletal muscle is not viable, autologous muscle grafts are typically required in tissue reconstruction after muscle loss due to disease or injury. However, the use of autologous tissue often leads to donor-site morbidity. Here, we show that intraspecies and interspecies chimaeric pig embryos lacking native skeletal muscle can be produced by deleting the MYF5, MYOD and MYF6 genes in the embryos via CRISPR, followed by somatic-cell nuclear transfer and the delivery of exogenous cells (porcine blastomeres or human induced pluripotent stem cells) via blastocyst complementation. The generated intraspecies chimaeras were viable and displayed normal histology, morphology and function. Human:pig chimaeras generated with TP53-null human induced pluripotent stem cells led to higher chimaerism efficiency, with embryos collected at embryonic days 20 and 27 containing humanized muscle, as confirmed by immunohistochemical and molecular analyses. Human:pig chimaeras may facilitate the production of exogenic organs for research and xenotransplantation.
Asunto(s)
Animales Modificados Genéticamente/metabolismo , Músculo Esquelético/metabolismo , Proteína MioD/genética , Factor 5 Regulador Miogénico/genética , Factores Reguladores Miogénicos/genética , Animales , Blastómeros/citología , Blastómeros/metabolismo , Linaje de la Célula , Reprogramación Celular , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Embrión de Mamíferos/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Edición Génica , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Proteína MioD/metabolismo , Factor 5 Regulador Miogénico/deficiencia , Factores Reguladores Miogénicos/metabolismo , Porcinos , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genéticaRESUMEN
BACKGROUND: Secondary dystroglycanopathies are a subset of muscular dystrophy caused by abnormal glycosylation of α-dystroglycan (αDG). Loss of αDG functional glycosylation prevents it from binding to laminin and other extracellular matrix receptors, causing muscular dystrophy. Mutations in a number of genes, including FKTN (fukutin), disrupt αDG glycosylation. METHODS: We analyzed conditional Fktn knockout (Fktn KO) muscle for levels of mTOR signaling pathway proteins by Western blot. Two cohorts of Myf5-cre/Fktn KO mice were treated with the mammalian target of rapamycin (mTOR) inhibitor rapamycin (RAPA) for 4 weeks and evaluated for changes in functional and histopathological features. RESULTS: Muscle from 17- to 25-week-old fukutin-deficient mice has activated mTOR signaling. However, in tamoxifen-inducible Fktn KO mice, factors related to Akt/mTOR signaling were unchanged before the onset of dystrophic pathology, suggesting that Akt/mTOR signaling pathway abnormalities occur after the onset of disease pathology and are not causative in early dystroglycanopathy development. To determine any pharmacological benefit of targeting mTOR signaling, we administered RAPA daily for 4 weeks to Myf5/Fktn KO mice to inhibit mTORC1. RAPA treatment reduced fibrosis, inflammation, activity-induced damage, and central nucleation, and increased muscle fiber size in Myf5/Fktn KO mice compared to controls. RAPA-treated KO mice also produced significantly higher torque at the conclusion of dosing. CONCLUSIONS: These findings validate a misregulation of mTOR signaling in dystrophic dystroglycanopathy skeletal muscle and suggest that such signaling molecules may be relevant targets to delay and/or reduce disease burden in dystrophic patients.
Asunto(s)
Distroglicanos/metabolismo , Músculo Esquelético/efectos de los fármacos , Distrofia Muscular Animal/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas/metabolismo , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Fenómenos Biomecánicos , Modelos Animales de Enfermedad , Regulación hacia Abajo , Estimulación Eléctrica , Femenino , Predisposición Genética a la Enfermedad , Glicosilación , Masculino , Ratones Noqueados , Contracción Muscular , Fuerza Muscular , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patología , Distrofia Muscular Animal/fisiopatología , Factor 5 Regulador Miogénico/deficiencia , Factor 5 Regulador Miogénico/genética , Fenotipo , Procesamiento Proteico-Postraduccional , Proteínas/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Factores de Tiempo , Torque , TransferasasRESUMEN
Myf5, a myogenic regulatory factor, plays a key role in regulating muscle differentiation. However, it is not known if Myf5 has a regulatory role during early embryogenesis. Here, we used myf5-morpholino oligonucleotides [MO] to knock down myf5 expression and demonstrated a series of results pointing to the functional roles of Myf5 during early embryogenesis: (1) reduced head size resulting from abnormal morphology in the cranial skeleton; (2) decreased expressions of the cranial neural crest (CNC) markers foxd3, sox9a, dlx2, and col2a1; (3) defect in the chondrogenic neural crest similar to that of fgf3 morphants; (4) reduced fgf3/fgf8 transcripts in the cephalic mesoderm rescued by co-injection of myf5 wobble-mismatched mRNA together with myf5-MO1 during 12 h postfertilization; (5) abnormal patterns of axial and non-axial mesoderm causing expansion of the dorsal organizer, and (6) increased bmp4 gradient, but reduced fgf3/fgf8 marginal gradient, during gastrulation. Interestingly, overexpression of fgf3 could rescue the cranial cartilage defects caused by myf5-MO1, suggesting that Myf5 modulates craniofacial cartilage development through the fgf3 signaling pathway. Together, the loss of Myf5 function results in a cascade effect that begins with abnormal formation of the dorsal organizer during gastrulation, causing, in turn, defects in the CNC and cranial cartilage of myf5-knockdown embryos.
Asunto(s)
Región Branquial/embriología , Cartílago Articular/crecimiento & desarrollo , Desarrollo Embrionario , Factor 5 Regulador Miogénico/fisiología , Proteínas de Pez Cebra/fisiología , Pez Cebra/embriología , Animales , Condrogénesis , Técnicas de Silenciamiento del Gen , Cabeza/embriología , Músculos/embriología , Factor 5 Regulador Miogénico/deficiencia , Cresta Neural/embriología , Transducción de Señal/fisiología , Proteínas de Pez Cebra/deficienciaRESUMEN
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.
Asunto(s)
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
Galectin1, the prototype member of a family of carbohydrate binding proteins, is involved in muscle stem cell behavior and in tissue regeneration after muscle injury in adult mice. Here, we addressed the question of when this gene is first acting in the muscle lineage. We found that Galectin1 is an early marker of myogenesis as the transcripts and protein are initially confined to the somites, starting from day 9.0 of embryogenesis. We next investigated its relationship with the muscle determination factors, Myf5 and Myod. By comparing the spatio-temporal distribution of Galectin1 transcripts in control and Myf5 null mutant embryos, we were able to establish that it acts downstream of Myf5. However, early myogenesis does not seem affected in Galectin1 null mutant embryos indicating that, unlike in the adult, Galectin1 does not play a role in muscle fate acquisition during development.
Asunto(s)
Embrión de Mamíferos/metabolismo , Galectina 1/genética , Regulación del Desarrollo de la Expresión Génica , Desarrollo de Músculos , Animales , Biomarcadores , Linaje de la Célula , Desmina/metabolismo , Embrión de Mamíferos/citología , Galectina 1/metabolismo , Ratones , Ratones Noqueados , Factor 5 Regulador Miogénico/deficiencia , Factor 5 Regulador Miogénico/genética , Miogenina/metabolismoRESUMEN
Muscle regulatory factors activate myogenesis in all vertebrates, but their role has been studied in great detail only in the mouse embryo, where all but myogenin--Myod, Myf5 and Mrf4--are sufficient to activate (albeit not completely) skeletal myogenesis. In the zebrafish embryo, myod and myf5 are required for induction of myogenesis because their simultaneous ablation prevents muscle development. Here we show that mrf4 but not myog can fully rescue myogenesis in the myod/myf5 double morphant via a selective and robust activation of myod, in keeping with its chromatin-remodelling function in vitro. Rescue does not happen spontaneously, because the gene, unlike that in the mouse embryo, is expressed only at the onset of muscle differentiation, Moreover, because of the transient nature of morpholino inhibition, we were able to investigate how myogenesis occurs in the absence of a myotome. We report that in the complete absence of a myotome, subsequent myogenesis is abolished, whereas myogenesis does proceed, albeit abnormally, when the morpholino inhibition was not complete. Therefore our data also show that the early myotome is essential for subsequent skeletal muscle differentiation and patterning in the zebrafish.
Asunto(s)
Embrión no Mamífero/fisiología , Desarrollo de Músculos/fisiología , Factores Reguladores Miogénicos/metabolismo , Miogenina/metabolismo , Animales , Ensamble y Desensamble de Cromatina , Embrión no Mamífero/citología , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteína MioD/genética , Proteína MioD/metabolismo , Factor 5 Regulador Miogénico/deficiencia , Factor 5 Regulador Miogénico/genética , Factores Reguladores Miogénicos/genética , Miogenina/genética , Factores de Tiempo , Activación Transcripcional , Pez Cebra/embriología , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
MCAT elements and its cognate binding partners, the transcription enhancer factors (TEFs) play important roles in the regulation of expression of several muscle-specific genes. The biological effects of TEFs strongly depend on different co-factors, which might act as co-activators or anti-repressors to enable transcriptional activation of target genes by TEFs. Previously, we have cloned and characterized VITO-1, which acts as a skeletal muscle-specific transcriptional co-activator of TEFs. Here we describe the cloning and expression profile of a related gene, VITO-2 (also termed Vgl-3), which shares a high homology with VITO-1 in the SID domain responsible for interaction with TEFs. During early embryonic and fetal development VITO-2 is mainly expressed in the myogenic lineage with an onset of expression in the myotomes of somites VI at E9.5 slightly later than VITO-1. At later developmental stages VITO-2 is predominantly found in the nervous system. In adult mice VITO-2 was detected in different tissues, including skeletal muscle, heart, kidney, liver and brain, where it was found in cortical and cerebellar neurons as well as in Purkinje cells. The expression of VITO-2 in the mesoderm was repressed by the notch/delta pathway and activated by Myf-5 since Dll-1 mutant showed an aberrant expression of VITO-2 but not VITO-1 in the tail bud and in the caudal neural tube at E10.5 while Myf-5 mutant mice lack expression of VITO-1 and VITO-2 in somites until E10.5.
Asunto(s)
Desarrollo de Músculos/genética , Proteínas Musculares , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Animales , Secuencia de Bases , Encéfalo/metabolismo , Región Branquial/metabolismo , Proteínas de Unión al Calcio , Corteza Cerebral/metabolismo , Ganglios Espinales/embriología , Ganglios Espinales/metabolismo , Perfilación de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/deficiencia , Riñón/metabolismo , Hígado/metabolismo , Ratones , Datos de Secuencia Molecular , Proteínas Musculares/biosíntesis , Proteínas Musculares/genética , Músculo Esquelético/embriología , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Factor 5 Regulador Miogénico/deficiencia , Células Neuroepiteliales/metabolismo , Células de Purkinje/metabolismo , Somitos/metabolismoRESUMEN
In skeletal muscle development, the myogenic regulatory factors myf5 and myoD play redundant roles in the specification and maintenance of myoblasts, whereas myf6 has a downstream role in differentiating myocytes and myofibers. It is not clear whether the redundancy between myf5 and myoD is within the same cell lineage or between distinct lineages. Using lineage tracing and conditional cell ablation in mice, we demonstrate the existence of two distinct lineages in myogenesis: a myf5 lineage and a myf5-independent lineage. Ablating the myf5 lineage is compatible with myogenesis sustained by myf5-independent, myoD-expressing myoblasts, whereas ablation of the myf6 lineage leads to an absence of all differentiated myofibers, although early myogenesis appears to be unaffected. We also demonstrate here the existence of a significant myf5 lineage within ribs that has an important role in rib development, suggested by severe rib defects upon ablating the myf5 lineage.
Asunto(s)
Desarrollo de Músculos/fisiología , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/metabolismo , Factor 5 Regulador Miogénico/metabolismo , Alelos , Animales , Marcación de Gen , Ratones , Ratones Noqueados , Ratones Transgénicos , Desarrollo de Músculos/genética , Proteína MioD/genética , Proteína MioD/metabolismo , Mioblastos Esqueléticos/clasificación , Factor 5 Regulador Miogénico/deficiencia , Factor 5 Regulador Miogénico/genética , Factores Reguladores Miogénicos/genética , Factores Reguladores Miogénicos/metabolismo , Costillas/embriología , Costillas/metabolismoRESUMEN
We examined the effects of a single injection of exogenous NT-3, administered at embryonic day (E) 13.5, on the survival of two populations of motor neurons and two populations of sensory neurons. Both wild-type and double knockout, Myf5-/-:MyoD-/-, mutant embryos were examined to determine the effects of the aforementioned neurotrophin on motor and sensory neuron survival in the presence and absence, respectively, of skeletal muscle. We found that, although NT-3 rescues select populations of motor neurons in the absence of muscles, there is a lack of increase in neuron survival when skeletal muscle is present. Additionally, NT-3 was found to rescue a select population of proprioceptive sensory neurons in the absence of target tissue, while, at times, exacerbating neuron cell death when target tissues are present. Lastly, we found that neurons in the spinal cord and brainstem show both a regional and functional specificity in their response to the administration of NT-3 in utero. Our results indicate the possibility that different pathways are involved in the survival of neurons during naturally occurring programmed cell death and during excessively occurring programmed cell death.
Asunto(s)
Neuronas Motoras/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/inervación , Factores de Crecimiento Nervioso/farmacología , Neuronas Aferentes/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Tronco Encefálico/citología , Tronco Encefálico/efectos de los fármacos , Tronco Encefálico/fisiología , Supervivencia Celular/efectos de los fármacos , Femenino , Masculino , Ratones , Ratones Noqueados , Neuronas Motoras/citología , Neuronas Motoras/fisiología , Músculo Esquelético/embriología , Proteína MioD/genética , Proteína MioD/metabolismo , Factor 5 Regulador Miogénico/deficiencia , Factor 5 Regulador Miogénico/genética , Neuronas Aferentes/citología , Neuronas Aferentes/fisiología , Embarazo , Proteínas Recombinantes/farmacología , Médula Espinal/citología , Médula Espinal/efectos de los fármacos , Médula Espinal/fisiologíaRESUMEN
Fetal breathing-like movements (FBMs) are important in normal lung growth and pneumocyte differentiation. In amyogenic mouse embryos (designated as Myf5-/-:MyoD-/-, entirely lacking skeletal musculature and FBMs), type II pneumocytes fail to differentiate into type I pneumocytes, the cells responsible for gas exchange, and the fetuses die from asphyxia at birth. Using oligonucleotide microarrays, we compared gene expression in the lungs of Myf5-/-:MyoD-/- embryos to that in normal lungs at term. Nine genes were found to be up-regulated and 54 down-regulated at least 2-fold in the lungs of double-mutant embryos. Since many down-regulated genes are involved in lymphocyte function, immunohistochemistry was employed to study T- and B-cell maturity in the thymus and spleen. Our findings of normal lymphocyte maturity implied that the down-regulation was specific to the double-mutant lung phenotype and not to its immune system. Immunostaining also revealed altered distribution of transcription and growth factors (SATB1, c-Myb, CTGF) from down-regulated genes whose knockouts are now known to undergo embryonic or neonatal death secondary to respiratory failure. Together, it appears that microarray analysis has identified a profile of genes potentially involved in pneumocyte differentiation and therefore in the mechanisms that may be implicated in the mechanochemical signal transduction pathways underlying FBMs-dependent pulmonary hypoplasia.
Asunto(s)
Diferenciación Celular , Regulación del Desarrollo de la Expresión Génica , Enfermedades Pulmonares/patología , Pulmón/patología , Proteína MioD/metabolismo , Factor 5 Regulador Miogénico/deficiencia , Análisis de Secuencia por Matrices de Oligonucleótidos , Animales , Diferenciación Celular/genética , Factor de Crecimiento del Tejido Conjuntivo , Perfilación de la Expresión Génica , Proteínas Inmediatas-Precoces/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Pulmón/embriología , Pulmón/metabolismo , Pulmón/fisiopatología , Enfermedades Pulmonares/embriología , Enfermedades Pulmonares/genética , Enfermedades Pulmonares/fisiopatología , Linfocitos/metabolismo , Linfocitos/patología , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Ratones , Ratones Noqueados , Proteína MioD/genética , Factor 5 Regulador Miogénico/genética , Proteínas Proto-Oncogénicas c-myb/metabolismo , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Mecánica Respiratoria , Músculos Respiratorios/embriología , Factores de TiempoRESUMEN
Congenital heart defects frequently involve a failure of outflow tract (OFT) formation during development. We analyzed the remodeling of the OFT, using the y96-Myf5-nlacZ-16 transgene, which marks a subpopulation of myocardial cells of the pulmonary trunk. Expression analyses of reporter transcript and protein suggest that the myocardial wall of the OFT rotates before and during the formation of the great arteries. Rotational movement was confirmed by Di-I injection experiments with cultured embryos. We subsequently examined the expression of the transgene in mouse models for OFT defects. In hearts with persistent truncus arteriosus (PTA), double outlet right ventricle (DORV), or transposition of the great arteries, rotation of the myocardial wall of the OFT is arrested or fails to initiate. This is observed in Splotch (Pax3) mutants with PTA or DORV and may be a result of defects in neural crest migration, known to affect OFT septation. However, in Pitx2deltac mutant embryos, where cardiac neural crest cells are present in the heart, PTA and DORV are again associated with a rotation defect. This is also seen in Pitx2deltac mutants, which have transposition of the great arteries. Because Pitx2c is involved in left-right signaling, these results suggest that embryonic laterality affects rotation of the myocardial wall during OFT maturation. We propose that failure of normal rotation of OFT myocardium may underlie major forms of congenital heart disease.
Asunto(s)
Aorta/anatomía & histología , Aorta/fisiología , Cardiopatías Congénitas/embriología , Corazón/anatomía & histología , Corazón/fisiología , Animales , Cartilla de ADN , Modelos Animales de Enfermedad , Cardiopatías Congénitas/patología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Factor 5 Regulador Miogénico/deficiencia , Factor 5 Regulador Miogénico/genética , Reacción en Cadena de la Polimerasa , RotaciónRESUMEN
The mechanical loading of striated muscle is thought to play an important role in shaping bones and joints. Here, we examine skeletogenesis in late embryogenesis (embryonic day 18.5) in Myf5-/-:MyoD-/- fetuses completely lacking striated muscle. The phenotype includes enlarged and fused cervical vertebrae and postural anomalies, some viscerocranial anomalies, long bone truncation and fusion, absent deltoid tuberosity of the humerus, scapular and clavicular hypoplasia, cleft palate, and cleft sternum. In contrast, neurocranial bone development was essentially normal. While the magnitude of individual effects varied throughout the skeletal system, the results are consistent with skeletal development depending on functional muscles. Novel abnormalities in the amyogenic fetuses relative to less severely paralyzed phenotypes extend our understanding of skeletogenic dependence on embryonic muscle contraction and static loading.
Asunto(s)
Huesos/embriología , Contracción Muscular/genética , Músculo Esquelético/embriología , Proteína MioD/genética , Factor 5 Regulador Miogénico/genética , Osteogénesis/genética , Animales , Huesos/anomalías , Feto , Ratones , Ratones Noqueados , Músculo Esquelético/fisiología , Proteína MioD/fisiología , Factor 5 Regulador Miogénico/deficiencia , Factor 5 Regulador Miogénico/fisiologíaRESUMEN
To determine which combination of skeletal muscle-derived neurotrophic factors may be important for the survival of specific subpopulations of developing spinal cord motor neurons, we used Myf5 and MyoD (myogenic regulatory factors) knockouts, containing differentially committed myogenic precursor cells (MPCc) and immunohistochemistry against several muscle-secreted neurotrophic factors. At the peak of motor neuron cell death, skeletal muscle development is delayed in the back and body wall muscles of Myf5-/- embryos and in the limb muscles of MyoD-/- embryos. We hypothesized that, if the skeletal muscle was indeed an important source of survival factors for motor neurons, the back, the abdominal wall, and the forelimb MPCs of Myf5-/- or MyoD-/- embryos should produce at least some neurotrophic factors necessary for the survival of motor neurons. In this report, we demonstrate that (1) different MPCs lacking Myf5, MyoD, or Myf5/MyoD have different capabilities in providing factors potentially required for the survival of motor neurons and intramuscular nerve branching, (2) MPCs in double-mutant embryos do not contain neurotrophic factors in the absence of myogenic specification, and (3) different subpopulations of MPCs contain different combinations of neurotrophic factors potentially required for the survival of the specific subpopulations of innervating motor neurons.