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1.
Methods Mol Biol ; 2640: 227-248, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36995599

RESUMO

Muscle regeneration models have revealed mechanisms of inflammation, wound clearance, and stem cell-directed repair of damage, thereby informing therapy. Whereas studies of muscle repair are most advanced in rodents, the zebrafish is emerging as an additional model organism with genetic and optical advantages. Various muscle wounding protocols (both chemical and physical) have been published. Here we describe simple, cheap, precise, adaptable, and effective wounding protocols and analysis methods for two stages of a larval zebrafish skeletal muscle regeneration model. We show examples of how muscle damage, ingression of muscle stem cells, immune cells, and regeneration of fibers can be monitored over an extended timecourse in individual larvae. Such analyses have the potential to greatly enhance understanding, by reducing the need to average regeneration responses across individuals subjected to an unavoidably variable wound stimulus.


Assuntos
Células Satélites de Músculo Esquelético , Peixe-Zebra , Animais , Peixe-Zebra/fisiologia , Fibras Musculares Esqueléticas , Células-Tronco , Células Satélites de Músculo Esquelético/fisiologia , Proliferação de Células , Músculo Esquelético/fisiologia
2.
J Vis Exp ; (184)2022 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-35781279

RESUMO

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish with fluorescently-marked plasma membranes can be scanned in a confocal laser scanning microscope in order to determine the volume of muscle tissue and the number of muscle fibers present. Efficient approaches for the measurement of cell number and size in live animals over time are described and validated against more arduous segmentation methods. Methods are described that permit the control of muscle electrical, and thus contractile, activity. Loss of skeletal muscle contractile activity greatly reduced muscle growth. In larvae, a protocol is described that allows reintroduction of patterned electrical-evoked contractile activity. The described methods minimize the effect of inter-individual variability and will permit analysis of the effect of electrical, genetic, drug, or environmental stimuli on a variety of cellular and physiological growth parameters in the context of the living organism. Long-term follow-up of the measured effects of a defined early-life intervention on individuals can subsequently be performed.


Assuntos
Fenômenos Fisiológicos do Sistema Nervoso , Peixe-Zebra , Animais , Larva , Contração Muscular , Fibras Musculares Esqueléticas
3.
Proc Natl Acad Sci U S A ; 117(49): 31208-31218, 2020 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-33229575

RESUMO

Muscle tissue shows diurnal variations in function, physiology, and metabolism. Whether such variations are dependent on the circadian clock per se or are secondary to circadian differences in physical activity and feeding pattern is unclear. By measuring muscle growth over 12-h periods in live prefeeding larval zebrafish, we show that muscle grows more during day than night. Expression of dominant negative CLOCK (ΔCLK), which inhibits molecular clock function, ablates circadian differences and reduces muscle growth. Inhibition of muscle contraction reduces growth in both day and night, but does not ablate the day/night difference. The circadian clock and physical activity are both required to promote higher muscle protein synthesis during the day compared to night, whereas markers of protein degradation, murf messenger RNAs, are higher at night. Proteasomal inhibitors increase muscle growth at night, irrespective of physical activity, but have no effect during the day. Although physical activity enhances TORC1 activity, and the TORC1 inhibitor rapamycin inhibits clock-driven daytime growth, no effect on muscle growth at night was detected. Importantly, day/night differences in 1) muscle growth, 2) protein synthesis, and 3) murf expression all persist in entrained larvae under free-running constant conditions, indicating circadian drive. Removal of circadian input by exposure to either permanent darkness or light leads to suboptimal muscle growth. We conclude that diurnal variations in muscle growth and metabolism are a circadian property that is independent of, but augmented by, physical activity, at least during development.


Assuntos
Relógios Circadianos/genética , Ritmo Circadiano/genética , Locomoção/genética , Músculo Esquelético/crescimento & desenvolvimento , Animais , Relógios Circadianos/efeitos dos fármacos , Ritmo Circadiano/efeitos dos fármacos , Larva/genética , Larva/crescimento & desenvolvimento , Luz , Locomoção/efeitos dos fármacos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Desenvolvimento Muscular/genética , Músculo Esquelético/metabolismo , Fotoperíodo , Sirolimo/farmacologia , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento
4.
Development ; 147(4)2020 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-32054660

RESUMO

La-related protein 6 (Larp6) is a conserved RNA-binding protein found across eukaryotes that has been suggested to regulate collagen biogenesis, muscle development, ciliogenesis, and various aspects of cell proliferation and migration. Zebrafish have two Larp6 family genes: larp6a and larp6b Viable and fertile single and double homozygous larp6a and larp6b zygotic mutants revealed no defects in muscle structure, and were indistinguishable from heterozygous or wild-type siblings. However, larp6a mutant females produced eggs with chorions that failed to elevate fully and were fragile. Eggs from larp6b single mutant females showed minor chorion defects, but chorions from eggs laid by larp6a;larp6b double mutant females were more defective than those from larp6a single mutants. Electron microscopy revealed defective chorionogenesis during oocyte development. Despite this, maternal zygotic single and double mutants were viable and fertile. Mass spectrometry analysis provided a description of chorion protein composition and revealed significant reductions in a subset of zona pellucida and lectin-type proteins between wild-type and mutant chorions that paralleled the severity of the phenotype. We conclude that Larp6 proteins are required for normal oocyte development, chorion formation and egg activation.


Assuntos
Autoantígenos/genética , Autoantígenos/fisiologia , Córion/fisiologia , Oócitos/fisiologia , Ribonucleoproteínas/genética , Ribonucleoproteínas/fisiologia , Animais , Movimento Celular , Proliferação de Células , Colágeno/fisiologia , Proteínas do Ovo/fisiologia , Feminino , Edição de Genes , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Genótipo , Heterozigoto , Homozigoto , Lectinas/fisiologia , Masculino , Mutação , Oócitos/citologia , Oogênese/fisiologia , Fenótipo , Peixe-Zebra , Zona Pelúcida/fisiologia , Antígeno SS-B
5.
Dev Biol ; 431(2): 321-335, 2017 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-28887016

RESUMO

Balancing the number of stem cells and their progeny is crucial for tissue development and repair. Here we examine how cell numbers and overall muscle size are tightly regulated during zebrafish somitic muscle development. Muscle stem/precursor cell (MPCs) expressing Pax7 are initially located in the dermomyotome (DM) external cell layer, adopt a highly stereotypical distribution and thereafter a proportion of MPCs migrate into the myotome. Regional variations in the proliferation and terminal differentiation of MPCs contribute to growth of the myotome. To probe the robustness of muscle size control and spatiotemporal regulation of MPCs, we compared the behaviour of wild type (wt) MPCs with those in mutant zebrafish that lack the muscle regulatory factor Myod. Myodfh261 mutants form one third fewer multinucleate fast muscle fibres than wt and show a significant expansion of the Pax7+ MPC population in the DM. Subsequently, myodfh261 mutant fibres generate more cytoplasm per nucleus, leading to recovery of muscle bulk. In addition, relative to wt siblings, there is an increased number of MPCs in myodfh261 mutants and these migrate prematurely into the myotome, differentiate and contribute to the hypertrophy of existing fibres. Thus, homeostatic reduction of the excess MPCs returns their number to normal levels, but fibre numbers remain low. The GSK3 antagonist BIO prevents MPC migration into the deep myotome, suggesting that canonical Wnt pathway activation maintains the DM in zebrafish, as in amniotes. BIO does not, however, block recovery of the myodfh261 mutant myotome, indicating that homeostasis acts on fibre intrinsic growth to maintain muscle bulk. The findings suggest the existence of a critical window for early fast fibre formation followed by a period in which homeostatic mechanisms regulate myotome growth by controlling fibre size. The feedback controls we reveal in muscle help explain the extremely precise grading of myotome size along the body axis irrespective of fish size, nutrition and genetic variation and may form a paradigm for wider matching of organ size.


Assuntos
Desenvolvimento Muscular , Fibras Musculares Esqueléticas/metabolismo , Somitos/metabolismo , Peixe-Zebra/embriologia , Animais , Diferenciação Celular , Movimento Celular , Núcleo Celular/metabolismo , Proliferação de Células , Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Larva/metabolismo , Fibras Musculares Esqueléticas/citologia , Mutação/genética , Fator de Transcrição PAX7/metabolismo , Somitos/embriologia , Proteínas de Peixe-Zebra/metabolismo
6.
Dis Model Mech ; 9(6): 671-84, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-27149989

RESUMO

Heterogeneity of stem cells or their niches is likely to influence tissue regeneration. Here we reveal stem/precursor cell diversity during wound repair in larval zebrafish somitic body muscle using time-lapse 3D confocal microscopy on reporter lines. Skeletal muscle with incision wounds rapidly regenerates both slow and fast muscle fibre types. A swift immune response is followed by an increase in cells at the wound site, many of which express the muscle stem cell marker Pax7. Pax7(+) cells proliferate and then undergo terminal differentiation involving Myogenin accumulation and subsequent loss of Pax7 followed by elongation and fusion to repair fast muscle fibres. Analysis of pax7a and pax7b transgenic reporter fish reveals that cells expressing each of the duplicated pax7 genes are distinctly localised in uninjured larvae. Cells marked by pax7a only or by both pax7a and pax7b enter the wound rapidly and contribute to muscle wound repair, but each behaves differently. Low numbers of pax7a-only cells form nascent fibres. Time-lapse microscopy revealed that the more numerous pax7b-marked cells frequently fuse to pre-existing fibres, contributing more strongly than pax7a-only cells to repair of damaged fibres. pax7b-marked cells are more often present in rows of aligned cells that are observed to fuse into a single fibre, but more rarely contribute to nascent regenerated fibres. Ablation of a substantial portion of nitroreductase-expressing pax7b cells with metronidazole prior to wounding triggered rapid pax7a-only cell accumulation, but this neither inhibited nor augmented pax7a-only cell-derived myogenesis and thus altered the cellular repair dynamics during wound healing. Moreover, pax7a-only cells did not regenerate pax7b cells, suggesting a lineage distinction. We propose a modified founder cell and fusion-competent cell model in which pax7a-only cells initiate fibre formation and pax7b cells contribute to fibre growth. This newly discovered cellular complexity in muscle wound repair raises the possibility that distinct populations of myogenic cells contribute differentially to repair in other vertebrates.


Assuntos
Músculo Esquelético/patologia , Fator de Transcrição PAX2/metabolismo , Regeneração , Células-Tronco/metabolismo , Cicatrização , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Diferenciação Celular , Fusão Celular , Núcleo Celular/metabolismo , Proliferação de Células , Epiderme/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde/metabolismo , Larva/metabolismo , Leucócitos/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Somitos/metabolismo , Fatores de Tempo , Imagem com Lapso de Tempo , Transgenes
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