RESUMEN
Skeletal muscle regenerates through the activation of resident stem cells. Termed satellite cells, these normally quiescent cells are induced to proliferate by wound-derived signals1. Identifying the source and nature of these cues has been hampered by an inability to visualize the complex cell interactions that occur within the wound. Here we use muscle injury models in zebrafish to systematically capture the interactions between satellite cells and the innate immune system after injury, in real time, throughout the repair process. This analysis revealed that a specific subset of macrophages 'dwell' within the injury, establishing a transient but obligate niche for stem cell proliferation. Single-cell profiling identified proliferative signals that are secreted by dwelling macrophages, which include the cytokine nicotinamide phosphoribosyltransferase (Nampt, which is also known as visfatin or PBEF in humans). Nampt secretion from the macrophage niche is required for muscle regeneration, acting through the C-C motif chemokine receptor type 5 (Ccr5), which is expressed on muscle stem cells. This analysis shows that in addition to their ability to modulate the immune response, specific macrophage populations also provide a transient stem-cell-activating niche, directly supplying proliferation-inducing cues that govern the repair process that is mediated by muscle stem cells. This study demonstrates that macrophage-derived niche signals for muscle stem cells, such as NAMPT, can be applied as new therapeutic modalities for skeletal muscle injury and disease.
Asunto(s)
Macrófagos/metabolismo , Músculo Esquelético/citología , Músculo Esquelético/lesiones , Mioblastos/citología , Nicotinamida Fosforribosiltransferasa/metabolismo , Nicho de Células Madre , Pez Cebra/metabolismo , Animales , Proliferación Celular , Modelos Animales de Enfermedad , Humanos , Macrófagos/citología , Masculino , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Mioblastos/metabolismo , Nicotinamida Fosforribosiltransferasa/genética , Factor de Transcripción PAX7/metabolismo , RNA-Seq , Receptores CCR5/genética , Receptores CCR5/metabolismo , Regeneración/fisiología , Análisis de la Célula Individual , Pez Cebra/inmunologíaRESUMEN
The recently uncovered role of Fukutin-related protein (FKRP) in fibronectin glycosylation has challenged our understanding of the basis of disease pathogenesis in the muscular dystrophies. FKRP is a Golgi-resident glycosyltransferase implicated in a broad spectrum of muscular dystrophy (MD) pathologies that are not fully attributable to the well-described α-Dystroglycan hypoglycosylation. By revealing a new role for FKRP in the glycosylation of fibronectin, a modification critical for the development of the muscle basement membrane (MBM) and its associated muscle linkages, new possibilities for understanding clinical phenotype arise. This modification involves an interaction between FKRP and myosin-10, a protein involved in the Golgi organization and function. These observations suggest a FKRP nexus exists that controls two critical aspects to muscle fibre integrity, both fibre stability at the MBM and its elastic properties. This review explores the new potential disease axis in the context of our current knowledge of muscular dystrophies.
Asunto(s)
Fibronectinas , Distrofias Musculares , Distroglicanos/genética , Distroglicanos/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Glicosilación , Humanos , Músculo Esquelético , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , Distrofias Musculares/patología , Mutación , Pentosiltransferasa/genética , Pentosiltransferasa/metabolismoRESUMEN
Deficiency of muscle basement membrane (MBM) component laminin-α2 leads to muscular dystrophy congenital type 1A (MDC1A), a currently untreatable myopathy. Laminin--α2 has two main binding partners within the MBM, dystroglycan and integrin. Integrins coordinate both cell adhesion and signalling; however, there is little mechanistic insight into integrin's function at the MBM. In order to study integrin's role in basement membrane development and how this relates to the MBM's capacity to handle force, an itgß1.b-/- zebrafish line was created. Histological examination revealed increased extracellular matrix (ECM) deposition at the MBM in the itgß1.b-/- fish when compared with controls. Surprisingly, both laminin and collagen proteins were found to be increased in expression at the MBM of the itgß1.b-/- larvae when compared with controls. This increase in ECM components resulted in a decrease in myotomal elasticity as determined by novel passive force analyses. To determine if it was possible to control ECM deposition at the MBM by manipulating integrin activity, RGD peptide, a potent inhibitor of integrin-ß1, was injected into a zebrafish model of MDC1A. As postulated an increase in laminin and collagen was observed in the lama2-/- mutant MBM. Importantly, there was also an improvement in fibre stability at the MBM, judged by a reduction in fibre pathology. These results therefore show that blocking ITGß1 signalling increases ECM deposition at the MBM, a process that could be potentially exploited for treatment of MDC1A.
Asunto(s)
Integrina beta1/metabolismo , Laminina/deficiencia , Oligopéptidos/farmacología , Animales , Membrana Basal/metabolismo , Biomarcadores , Colágeno/metabolismo , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Sitios Genéticos , Inmunohistoquímica , Integrina beta1/genética , Ratones Noqueados , Fibras Musculares Esqueléticas/metabolismo , Distrofias Musculares/etiología , Distrofias Musculares/metabolismo , Distrofias Musculares/patología , Fenotipo , Estabilidad Proteica/efectos de los fármacosRESUMEN
Fukutin and fukutin-related protein (FKRP) are involved in the glycosylation of α-dystroglycan, a key receptor for basement membrane proteins. Aberrant α-dystroglycan glycosylation leads to a broad spectrum of disorders, ranging from limb girdle muscular dystrophy to Walker-Warburg syndrome. This is the first study investigating a role of fukutin and FKRP-mediated glycosylation in angiogenesis. Transgenic zebrafish expressing enhanced green fluorescent protein in blood vessels were treated with morpholino antisense oligonucleotides that blocked the expression of fukutin, FKRP and dystroglycan. All morphant fish showed muscle damage and vascular abnormalities at day 1 post-fertilization. Intersegmental vessels of somites failed to reach the dorsal longitudinal anastomosis and in more severe phenotypes retracted further or were in some cases even completely missing. In contrast, the eye vasculature was distorted in both fukutin and FKRP morphants, but not in dystroglycan morphants or control fish. The eye size was also smaller in the fukutin and FKRP morphants when compared with dystroglycan knockdown fish and controls. In general, the fukutin morphant fish had the most severe skeletal muscle and eye phenotype. Our findings suggest that fukutin and FKRP have functions that affect ocular development in zebrafish independently of dystroglycan. Despite anecdotal reports about vascular abnormalities in patients affected by dystroglycanopathies, the clinical relevance of such lesions remains unclear and should be subject to further review and investigations.
Asunto(s)
Vasos Sanguíneos/anomalías , Vasos Sanguíneos/embriología , Glicosiltransferasas/deficiencia , Proteínas de Pez Cebra/deficiencia , Pez Cebra/embriología , Animales , Animales Modificados Genéticamente , Anticuerpos/inmunología , Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/patología , Distroglicanos/metabolismo , Embrión no Mamífero/anomalías , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/metabolismo , Embrión no Mamífero/patología , Ojo/irrigación sanguínea , Ojo/efectos de los fármacos , Ojo/patología , Glicosiltransferasas/metabolismo , Modelos Animales , Morfolinos/farmacología , Faloidina/metabolismo , Proteína Proto-Oncogénica c-fli-1 , Somitos/anomalías , Somitos/irrigación sanguínea , Somitos/efectos de los fármacos , Somitos/embriología , Coloración y Etiquetado , Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
The Nuclear receptor subfamily 4 group A member 2 (Nr4a2) is crucial for the formation or maintenance of dopaminergic neurons in the central nervous system including the retina, where dopaminergic amacrine cells contribute to visual function. Little is known about which cells express Nr4a2 at which developmental stage. Furthermore, whether Nr4a2 functions in combination with other genes is poorly understood. Thus, we generated a novel transgenic to visualize Nr4a2 expression in vivo during zebrafish retinogenesis. A 4.1 kb fragment of the nr4a2a promoter was used to drive green fluorescent protein expression in this Tg(nr4a2a:eGFP) line. In situ hybridization showed that transgene expression follows endogenous RNA expression at a cellular level. Temporal expression and lineages were quantified using in vivo time-lapse imaging in embryos. Nr4a2 expressing retinal subtypes were characterized immunohistochemically. Nr4a2a:eGFP labeled multiple neuron subtypes including 24.5% of all amacrine interneurons. Nr4a2a:eGFP labels all tyrosine hydroxylase labeled dopaminergic amacrine cells, and other nondopaminergic GABAergic amacrine populations. Nr4a2a:eGFP is confined to a specific progenitor lineage identified by sequential expression of the bhlh transcription factor Atonal7 (Atoh7) and Pancreas transcription factor 1a (Ptf1a), and labels postmitotic postmigratory amacrine cells. Thus, developmental Nr4a2a expression indicates a role during late differentiation of specific amacrine interneurons. Tg(nr4a2a:eGFP) is an early marker of distinct neurons including dopaminergic amacrine cells. It can be utilized to assess consequences of gene manipulations and understand whether Nr4a2 only carries out its role in the presence of specific coexpressed genes. This will allow Nr4a2 use to be refined for regenerative approaches.